Structural Organization of the Mammalian Kidney

C H A P T E R

20 Structural Organization of the Mammalian Kidney Wilhelm Kriz1 and Brigitte Kaissling2 1

Department of Anatomy and Developmental Biology, Medical Faculty Mannheim, University of Heidelberg, Mannheim, Germany 2 Institute for Anatomy, University of Zu¨rich, Switzerland

KIDNEY TYPES AND RENAL PELVIS The mammalian kidney is multiform. The basic architecture is best understood in the unipapillary kidney, which is common in all small species. A coronal section of this kidney shows the main structural parts (Figure 20.1a). The renal cortex, as a whole, is cupshaped with inverted margins, and surrounds the renal medulla. The medulla can be roughly compared to a pyramid; its top portion, the papilla, projects into the renal pelvis. The pelvis is located within the renal sinus, which opens through the renal hilum to the medial surface of the kidney. The cortical parenchyma is divided into the cortical labyrinth and the medullary rays. The uppermost part of the cortex, a continuous layer that covers the tops of the medullary rays, is called the cortex corticis. The medulla is divided into an outer medulla (subdivided into outer and inner stripes) and an inner medulla. The innermost part of the inner medulla generally forms the papilla. The unipapillary kidney is the most simple kidney type; in comparative anatomy, such a kidney as a whole corresponds to a renculus. All other kidney types may be regarded as adaptations to larger body sizes. The crest kidney and the kidney with tubi maximi are magnifications of a one-reniculus unit. The multipapillary kidney (Figure 20.2) and the reniculus kidney multiply this unit.1,2,3 The human kidney is a multipapillary kidney; however, it is particular because a variable number of papillae are generally fused, forming compound papillae.4 Seldin and Giebisch’s The Kidney, Fifth Edition. DOI: http://dx.doi.org/10.1016/B978-0-12-381462-3.00020-3

The renal pelvis (Figures 20.1a and b) or the renal calyces (Figure 20.2) are anchored to the renal parenchyma by connective and smooth-muscle tissues that follow the intrarenal arteries. The cavity of the pelvis and calyx surrounds the renal papilla (or its equivalent in other kidney types). In many species the pelvic cavity forms different kinds of pelvic extensions (Figure 20.1b).5,6 Leaf-like extensions called “specialized fornices” accompany the large vessels for some distance along their entry into the renal parenchyma. Secondary pouches protrude toward the hilus, communicating with the primary pelvic cavity only above the free semilunar borders of the pelvic septa. These extensions increase the contact area between the pelvic cavity and the renal medulla, especially the outer medulla.7

RENAL VASCULATURE Close to the renal hilum and afterwards within the renal sinus the renal artery undergoes several divisions, finally establishing the interlobar arteries which then enter the renal tissue at the border between the cortex and medulla (Figures 20.1a and 20.2). From there they follow an arc-like course and are therefore called arcuate arteries. They give rise to the cortical radial arteries, which ascend radially within the cortical labyrinth. The cortex is very densely penetrated by arteries; in contrast, no arteries enter the medulla. The renal veins (cortical radial (interlobular) veins, arcuate veins) accompany the corresponding arteries. In some species (cat, dog, man) the venous blood from the outer

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a Cortical Labyrinth Medullary Ray

Outer Stripe Inner Stripe Inner Medulla

Pelvic Septum Secondary Pouch Pelvic Cavity Hilar Tunnel

b

Secondary Pouch Peripelvic Column

Pelvic Septum Pelvic Cavity Inner Medulla Inner Stripe

PA

Outer Medulla Cortex

FIGURE 20.1 Schematics of a coronal and two transverse (b) sections through the rabbit kidney. The inset in the right lower corner indicates the section plane. The general architecture of the kidney and the renal pelvis is demonstrated. (a) Arterial vessels, including glomeruli and descending vasa recta, are shown in black, venous vessels are gray, and lymphatics are hatched. (b) The section plane of the main drawing runs through the middle part of the inner medulla; a deeper section through the papilla (PA) is shown in the upper right quarter. The cross-sectioned pelvic septa are stippled. The leaf-like extensions of the pelvic cavity are marked by a star, the free semilunar edges of the main pelvic septa by an arrow (in a and b). (Adapted from Kaissling, B., and Kriz, W. (1979). Structured analysis of the rabbit kidney. Adv. Anat. Embryol. Cell Biol. 56, 1
123, with permission.)

cortex drains into veins on the renal surface (in man called “stellate veins”) which are connected by additional cortical radial veins (interlobular veins) to arcuate veins. Such additional veins are not accompanied by arteries.8

FIGURE 20.2 Schematic illustration of a compound multipapillary kidney (coronal section) similar to the human kidney. The renal cortex as a whole encloses several papillae; fused papillae typical for the human kidney are not shown. The central region, the renal sinus, contains the calyces and the pelvis (stippled), the pattern of branching arteries (black), and joining veins (white). The arcuate arteries, running at the cortico-medullary border, do not form true arches, but rather represent end-arteries. In contrast, the veins do form anastomoses at the level of the arcuate and interlobar veins. In the human kidney there are two types of cortical radial veins (interlobular veins); one group starting as stellate veins drains the most superficial cortex, the second group starts at deeper levels in the cortex; both drain into arcuate veins.

The microvasculature pattern of the kidney appears to be very similar among mamma*lian species; a basic pattern can be described (Figures 20.3 and 20.4a).9
11 The afferent arterioles arise from the cortical radial arteries (a minor portion from the arcuate arteries) and supply the glomerular tufts of the renal corpuscles. The efferent arterioles drain the glomeruli. Several types of efferent arterioles have been described.11,12 Basically, a distinction between superficial, midcortical, and juxtamedullary renal corpuscles is essential (Figures 20.3 and 20.5). The efferent arterioles of juxtamedullary glomeruli turn toward the medulla; they supply the medulla. Juxtamedullary glomeruli are best defined by this type of efferent arteriole. The superficial efferent arterioles extend to the kidney surface before dividing. Again, superficial glomeruli are best defined because of the typical pattern of their efferent arterioles. The efferent arterioles of midcortical nephrons (defined by exclusion) vary in length between those that branch abruptly near the glomerulus and others that extend to a medullary ray before splitting off into capillaries. All the efferent arterioles together (superficial, midcortical, and also small

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RENAL VASCULATURE

FIGURE 20.3 Schematic of the microvasculature of the rat kidney (C: cortex; OS: outer stripe; IS: inner stripe; IM: inner medulla). The left panel shows the arterial vessels and capillaries. An arcuate artery (arrow) gives rise to a cortical radial (interlobular) artery, from which afferent arterioles originate to supply the glomeruli. The efferent arterioles of the juxtamedullary glomeruli descend into the medulla and divide into the descending vasa recta, which, together with ascending vasa recta, form the vascular bundles of the renal medulla. At intervals, descending vasa recta leave the bundles to feed the adjacent capillaries. The right panel shows the venous vessels. The interlobular veins start in the superficial cortex. In the inner cortex they, together with the arcuate veins, receive the ascending vasa recta from the medulla. The vasa recta ascending from the inner medulla all traverse the inner stripe within the vascular bundles, whereas most of the vasa recta from the inner stripe ascend outside the bundles. Both of these types of ascending vasa recta traverse the outer stripe as wide, tortuous channels. (Adapted from Kriz, W., and Lever, A. F. (1969). Renal countercurrent mechanisms: Structure and function. Am. Heart J. 78(1), 101
118 and Rollhaeuser, H., and Kriz, W. (1964). The vascular system of the rat kidney. Z. Zellforsch. Mikrosk. Anat. 64, 381
403, with permission.)

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FIGURE 20.4 Microvasculature. (a) Rat kidney; silicon rubber (Microfil) filling of the arterial vessels. Cortex (C), outer stripe (OS), inner stripe (IS), and inner medulla (IM) are clearly distinguishable by their vessel patterns. The vascular bundles take shape along the OS and are best developed within the IS. Only a minor part of the descending vasa recta of each bundle enter the IM, where they gradually decrease in number toward the papilla. Note the scantiness of capillaries in the OS (38). (b) Rabbit kidney; silicon rubber filling of the venous vessels. The interlobular veins (IV) accept the cortical capillaries and a major part of the ascending vasa recta. Note the density of ascending vasa recta within the OS. In the IS, ascending vasa recta are found within the bundles (mostly originating from the IM) and between the vascular bundles (draining the interbundle regions of the IS) (AV: arcuate vein; 314). (In cooperation with L. Bankir.)

branches of the juxtamedullary efferent arterioles) supply the cortical peritubular capillaries. Direct aglomerular arterial supplies to the peritubular capillaries or to the medulla are sparse13 and have frequently been shown to be the result of degeneration of the corresponding glomeruli.14 Within the capillary network of the cortex (Figures 20.3 and 20.4), a differentiation between two parts is necessary: namely, the dense, round-meshed

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FIGURE 20.5

Arterial vessels after filling with silicone rubber; rabbit kidney. The broken lines show the renal surface and the cortico-medullary border. Arcuate arteries (AA) give rise to cortical radial arteries which split into the afferent arterioles. The efferent arterioles of superficial glomeruli (arrow) ascend unbranched to the kidney surface before splitting into capillaries. The efferent arterioles of juxtamedullary nephrons (arrowheads) descend into the outer stripe and devide into the descending vasa recta (380). (From ref. [5].)

capillary plexus of the cortical labyrinth (including the cortex corticis); and the less dense, long-meshed plexus of the medullary rays, both associated with the course of the tubules. Functionally these two plexuses are different with respect to their drainage. The blood from the medullary ray plexus has to pass the plexus of the cortical labyrinth to gain access to the interlobular veins. Therefore, the blood that has perfused the straight tubules within the medullary rays mixes with the blood that perfuses the convoluted tubules of the cortical labyrinth. The medulla (Figures 20.3, 20.4, and 20.5) is exclusively supplied by the efferent arterioles of the juxtamedullary glomeruli.5,8,10,11,15,16 These efferent arterioles descend through the outer stripe and divide into the descending vasa recta. In addition, the efferent

arteriole and its first divisions give rise to small side branches that supply the sparse capillary plexus of the outer stripe of the outer medulla. This plexus is continuous with the cortical capillary plexus above and the capillary plexus of the inner stripe below. The descending vasa recta then penetrate the inner stripe of the outer medulla in cone-shaped vascular bundles. At intervals, descending vasa recta leave the bundles to join the capillary plexus at the adjacent medullary level, most leaving the bundle within the inner stripe. Only a small portion of the descending vasa recta penetrate the inner medulla, and even fewer reach the tip of the papilla. The capillary plexuses of the renal medulla (Figures 20.3 and 20.4a) differ in the three regions. That of the outer stripe is sparse. In contrast, the capillary plexus of the inner stripe is very dense and characteristically round-meshed in appearance. In the inner medulla the capillary plexus is less dense and longmeshed. The ascending vasa recta are the draining vessels of the renal medulla (Figures 20.3 and 20.4b). In the inner medulla they arise at every level and ascend as unbranched vessels to the border between the inner and outer medulla. At this point, they join the vascular bundles and traverse the inner stripe of the outer medulla within the vascular bundles. The ascending vasa recta, which drain the inner stripe, behave differently. Those of the lowermost part of the inner stripe (and therefore probably a minor portion) join the bundles as they pass through this region. Those from the middle and upper part (and thus probably the majority) do not join the bundles, but ascend directly within the interbundle regions to the outer stripe. There are, however, interspecies differences; in the sand rat (Psammomys obesus), all ascending vasa recta that drain the inner stripe ascend directly to the outer stripe without joining the bundles.17 Within the outer stripe, the vasa recta ascending within the bundles spread out and, together with the directly ascending vasa recta, traverse the outer stripe as individual tortuous channels with wide lumina (Figure 20.4b). They contact the tubules like true capillaries, and because the true capillaries which are derived from direct branches of efferent arterioles are few in the outer stripe (Figure 20.3a), they mainly affect the blood supply to the tubules in this region. At the corticomedullary border, the ascending venous vessels of the medulla empty into the arcuate veins or into the basal parts of interlobular veins. In some species, such as rat, guinea pig, and especially the sand rat (Psammomys obesus) some of the venous medullary vessels continue to ascend within the medullary rays of the cortex and finally empty into middle or even upper parts of interlobular veins.

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RENAL VASCULATURE

Wall Structure of Intrarenal Vessels The intrarenal arteries and the proximal portions of the afferent arterioles appear to be similar to arteries and arterioles of the same size elsewhere in the body. The terminal portions of the afferent arterioles are unique because of the occurrence of granular cells (renin producing cells) which replace ordinary smooth muscle cells in their wall.18 It is generally agreed that granular cells are modified smooth muscle cells. Compared to proper smooth muscle cells, granular cells contain less myofilaments; thus, the contractile capacity of the very last portion of the afferent arteriole appears to be considerably decreased.19 The endocrine function of granular cells will be considered later in the context of the juxtaglomerular apparatus. The glomerular capillaries will be described together with the glomerulus. Efferent arterioles are already established inside the glomerular tuft. Thus, in contrast to afferent arterioles, efferent arterioles have an intraglomerular segment which passes through the glomerular stalk20,21 (Figure 20.6a). After this, efferent arterioles have a segment which is narrowly associated with the extraglomerular mesangium (details will be given later in the context of the glomerulus). Thereafter, the efferent arterioles are established as arterioles with a proper media made up of smooth muscle cells.

a

c

b

d

Efferent arterioles from juxtamedullary glomeruli differ considerably from those of cortical (midcortical and superficial) glomeruli (compare Figures 20.6b and 20.6c). Juxtamedullary efferent arterioles are larger in diameter than cortical efferent arterioles; their size even exceeds that of their corresponding afferent arterioles. In the rabbit, the diameters of afferent arterioles throughout the cortex average approximately 20 μm; juxtamedullary efferent arterioles average 28 μm, and cortical efferent arterioles average only 12 μm.22 Similar differences have been found in dog,12 rat,23 and human24 kidneys. Cortical efferent arterioles (Figure 20.6b) are only sparsely equipped with smooth muscle cells (generally not more than one layer). A striking feature of efferent arterioles (including those from juxtamedullary glomeruli) is the thick, irregular basement membrane. In contrast to the usual appearance of a basement membrane, basement membrane-like material fills the wide and irregular spaces between the endothelium and the muscle layer. The juxtamedullary efferent arterioles (Figure 20.6c) are surrounded by two to four layers of smooth muscle cells. Their endothelium is composed of a strikingly large number of longitudinally arranged cells; up to 30 individual cells may be found in crosssections.23,25 In the descending vasa recta (Figure 20.6d) the smooth muscle cells are gradually replaced by

FIGURE 20.6 Efferent arteriole. (a) Intraglomerular segment of the efferent arteriole. Between the basement membrane (GBM) and the endothelium a mesangial layer is interposed. Note the intimate relationships of the afferent arteriole (AA) to the mesangium. PO: podocyte (Rat; TEM 3B4600). (b) Efferent arteriole of a superficial glomerulus. Note the irregular basement membrane-like material beneath the endothelium (*). One to two layers of smooth muscle cells (SM) are encountered (Rabbit; TEM 3B3400). (c) Efferent arteriole of a juxtamedullary glomerulus. Note the many profiles of endothelial cells (*); the tight junctions between them are shallow (Rat; TEM 3B2450). (d) Descending (DV) and ascending (AV) vasa recta of a vascular bundle are shown. The continuous endothelium of the descending vas rectum is surrounded by a pericyte (P). The endothelium of the ascending vas rectum is highly fenestrated (arrows) (Rabbit; TEM 3B2400).

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pericytes, which form an incomplete layer around the vessel trunk. Pericytes should be regarded as contractile cells. The pattern of these cells, which encircle the endothelial tube-like hoops, and their dense assemblys of microfilaments strongly imply that they have a contractile function. In contrast to smooth muscle cells, they are not contacted by nerve terminals. The descending vasa recta finally lose their pericytes, and the concurrent appearance of endothelial fenestrations marks their gradual transformation into medullary capillaries. The ultrastructure of the capillaries in the kidney is similar in both the cortex and the medulla (with the exception of glomerular capillaries; vide infra). The capillaries of the kidney are of the fenestrated type (Figure 20.7). The capillary wall consists of an extremely flat endothelium surrounded by a thin basement membrane. In non-nuclear regions the endothelial cells contain densely and regularly arranged fenestrations that (in contrast to the glomerular capillaries) are bridged by a thin diaphragm. An estimated 50% of the capillary circumference is composed of these fenestration-bearing areas.23 The fenestrations themselves are of rather complex structure. In normal TEM sections the diaphragm appears as a very thin (5
6 nm) singlelayered proteinaceous membrane provided with a central knob. Deep-etch freezing techniques have revealed

FIGURE 20.7 Freeze-fracture electron micrograph demonstrating the dense arrangement of fenestrations within the wall of a peritubular capillary (rabbit). Pinocytotic vesicles (arrows) are found within areas of thicker cytoplasm, which connect the perikaryon and the more voluminous areas along the cell borders (not shown) (37600). (In cooperation with A. Schiller and R. Taugner.)

a composition of radial fibrils converging to the central knob.26 So far only one protein, PV1, a caveolar transmembrane protein, has been attributed to the diaphragm.27 The diaphragm is considered to be permeable to water and small water-soluble substances. The wall structure of the ascending vasa recta (Figure 20.6d) is similar to that of the capillaries. These draining vessels, with wide lumina, are bound for their entire length by an extremely flat endothelium with extensive fenestrations. The same structure is found in the large veins of the cortex and at the corticomedullary border (Figures 20.8 and 20.15). The interlobular and arcuate veins are not veins in the classic sense, but they have a wall structure fundamentally the same as that of the renal capillaries.9,23 This wall consists solely of an extremely flattened, partly fenestrated endothelium that rests on a basement membrane.

NEPHRONS AND COLLECTING DUCT SYSTEM The specific structural units of the kidney are the nephrons. In the rat, each kidney contains 30,000 to 35,000 nephrons28; each human kidney has an estimated 1 million,29 but great interindividual differences exist.30,31 The nephron consists of a renal corpuscle connected to a complicated and twisted tube that finally drains into a collecting duct. Based on the location of the renal corpuscles within the cortex, three types of nephrons are distinguished: superficial; midcortical; and juxtamedullary nephrons. Exact definitions of these types, grounded on more than arbitrary decisions, can be based on the different patterns of the efferent arterioles (vide supra). The tubular part of the nephron consists of a proximal and a distal portion connected by a loop of Henle. For details of subdivisions, see Figures 20.9 and 20.10. According to the lengths of the loops of Henle, two types of nephrons are distinguished (Figure 20.9): those with long loops and those with short loops (including those with cortical loops). Short loops turn back in the outer medulla. In many species (rat, rabbit), the bends of the short loops are all located roughly at the same level of the inner stripe, namely, near the junction to the inner medulla. In other species (pig and human), short loops may form their bends at any level of the outer medulla, and even in the cortex (cortical loops). The long loops turn back at successive levels of the inner medulla, many at its start; others reach intermediate levels, and only a few reach the tip of the papilla. Thus, the number of loops is successively reduced along the inner medulla toward the papilla. This decrease is paralleled by a decrease in collecting ducts

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NEPHRONS AND COLLECTING DUCT SYSTEM

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FIGURE 20.8 Scanning electron micrographs of the inner surface of an arcuate artery and vein (Rat). (a) The tubules underneath the venous wall are clearly discernible through the endothelium, which covers the tubules as a thin coat. (b) Higher magnification of the venous endothelium. The openings in the endothelial wall (arrows) mark the positions where venous vasa recta and capillaries empty into the vein. (a) 3B120; (b) 3B1900. (From Frank, M., and Kriz, W. (1988). The luminal aspect of intrarenal arteries and veins in the rat as revealed by scanning electron microscopy. Anat. Embryol. (Berl) 177(4), 371
376, with permission.)

and vasa recta, leading to the characteristic form of the inner medulla, which in all species tapers from a broad basis to a papilla (or crest). The division of nephrons according to the position of their corpuscles in the cortex does not coincide with the division based on the length of their loops. Among species, all three types of renal corpuscles may be attached to both short and long loops. However, within a given species (with short and long loops), the long loops always belong to the deeper renal corpuscles (i.e., juxtamedullary and deep midcortical) and the short loops to the more superficially situated corpuscles. The number of short and long loops varies among species. Some species have only short loops (mountain beaver, muskrat), and consequently lack an inner medulla, which results in a poor ability to concentrate urine.6 Only two species, cat and dog, are known to have just long loops. In comparison with other species, their urine concentrating ability is considered to be average. In the cat, however, many long loops penetrate into the inner medulla for a very short distance (less than 0.5 μm. Defining a loop by ultrastructural criteria (vide infra), a feline kidney does contain many loops resembling the short loops in other species. The formerly held presumption that rodent species with the most powerful ability to concentrate urine, like Psammomys or Meriones, have only long loops has been proved incorrect.32 Most species have short and long

loops whose ratio varies from species to species. A correlation between the ratio of short and long loops and urine concentrating ability is not obvious. Most rodent species that have a high urine concentrating ability (rat, mouse, golden hamster, Psammomys, Meriones) have more short loops than long loops.32
34 The collecting ducts are formed in the renal cortex by the joining of several nephrons (Figures 20.9 and 20.10). The location of the exact border between a nephron and a collecting duct is disputed. According to cytological criteria, a connecting tubule is interposed between a nephron and a cortical collecting duct. Whether this connecting tubule derives from the nephrogenic blastema, and therefore must be considered as a part of the nephron, or from the ureteral bud, and therefore is part of the collecting ducts, remains an open question. Microanatomically, the connecting tubules of deep and superficial nephrons differ (Figure 20.9). The connecting tubules of deep nephrons generally join to form an arcade before draining into a collecting duct; superficial nephrons drain via an individual connecting tubule. The numerical ratio between nephrons draining through an arcade and those draining individually varies greatly among species. In rat, rabbit, and pig, the majority of nephrons drain via arcades; as Sperber3 observed, some arcades probably exist in all mammalian kidneys. An arcade ascends within the cortical labyrinth before

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9

9*

8

1

7

2

Cortex

8

1

10

7

2

3

6

Outer Medulla

Outer 3 Stripe

INTERSTITIUM 6 11

Inner Stripe

Inner Medulla

traverse the outer medulla (outer medullary collecting ducts). On entering the inner medulla (inner medullary collecting ducts), they fuse successively. In the human kidney, an average of eight fusions has been found,36 a number that may also be a good approximation for other species.37 Because a cortical collecting duct in the human kidney accepts 11 nephrons on average, it can be calculated that a papillary duct (opening into the renal pelvis) drains a total of 2750 nephrons. In the rabbit kidney, which has only 6 nephron tributaries to a cortical collecting duct,5 approximately 1000 nephrons are drained by a terminal collecting duct. It must be emphasized that an inner medullary collecting duct is not a single unbranched tube, but rather is a system of tubules that fuse successively.

4

12

4 5

FIGURE 20.9

Schematic of nephrons and collecting duct. This scheme depicts a short-looped and a long-looped nephron, together with the collecting system. Not drawn to scale. Within the cortex a medullary ray is delineated by a dashed line. (From Kriz, W., and Bankir, L. (1988). A standard nomenclature for structures of the kidney. The Renal Commission of the International Union of Physiological Sciences (IUPS). Kidney Int, 33, 1
7, with permission.) 1: Renal corpuscle including Bowman’s capsule and the glomerulus (glomerular tuft); 2: Proximal convoluted tubule; 3: Proximal straight tubule; 4: Descending thin limb; 5: Ascending thin limb; 6: Distal straight tubule (thick ascending limb); 7: Macula densa located within the final portion of the thick ascending limb; 8: Distal convoluted tubule; 9: Connecting tubule; 9*: Connecting tubule of the juxtamedullary nephron that forms an arcade; 10: Cortical collecting duct; 11: Outer medullary collecting duct; 12: Inner medullary collecting duct.

draining into a cortical collecting duct (Figure 20.9). Functionally, an arcade appears to serve as a device that prevents the addition of dilute distal urine to collecting ducts at the corticomedullary junction.35 The cortical collecting ducts descend within the medullary rays of the cortex and then, as unbranched tubes,

Definition, Volume Fraction The space between the basement membranes of the renal epithelia and the peritubular capillaries (Figure 20.11) is called the “interstitial space.” Cells and extracellular matrix within this space constitute the “interstitium.” The fractional volume of the interstitium in the cortex in healthy kidneys has been estimated between 4 to 9%,38
41 in the outer stripe of the outer medulla and in the vascular bundle compartment B3
5%. In the interbundle compartment of the inner stripe the fractional volume amounts to 10% in rat,42,41 and in the inner zone the relative interstitial volume continuously increases from the base (10
15% fractional volume in rat, 20
25% in rabbit) to the tip of the papilla (B30% in rat; more than 40% in rabbit42,43). Reabsorption and secretion of fluid and solutes, as well as the transport for many regulatory substances from their site of production to their target site, implies a transit across the interstitial compartment.44 In the cortex only about 26%39 or 42%40 of the total outer tubular surfaces are directly apposed to capillaries.

Cellular Constituents The majority of cells in the interstitium of healthy kidneys are interstitial fibroblasts and dendritic cells (Figure 20.12). Other cell types (macrophages and lymphocytes) are scarce in healthy kidneys,45 but they invade the interstitial spaces under inflammatory conditions.46

Interstitial Fibroblasts Interstitial fibroblasts provide the scaffolding of the tissue, take part in the modeling of the extracellular matrix, and play a role in the production of regulatory

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FIGURE 20.10 Segmentation of the renal tubule. This table summarizes the nomenclature of segments and cells of the renal tubule. A continuous serpentine arrow means that the transition between the two structures is gradual. An interrupted serpentine arrow means that the transition is gradual in some species, abrupt in others. Abbreviations marked by a star were introduced by Morel and co-workers: Functional segmentation of the rabbit distal tubule by microdetermination of hormone-dependent adenylate cyclase activity. Kidney Int. 1976; 9, 264
277 (DCTa: Distal convoluted tubule, initial portion; DCTb: Distal convoluted tubule, bright portion; DCTg: Distal convoluted tubule, granular portion; DCTl: Distal convoluted tubule, light portion; CCTg: Cortical collecting tubule, granular portion; CCTl: Cortical collecting tubule, light portion). (From ref. [542].)

604

FIGURE 20.11

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Peritubular interstitium of the renal cortex with narrow (arrows) and wide (stars) portions. Interstitial cells are resident fibroblasts (1) and temporarily sojourning dendritic/mononuclear cells (2) (rat kidney; TEM 3 B720).

FIGURE 20.12 Schematic representation of cortical interstitial fibroblast (F) and dendritic cell (D) in the cortical interstitial space of a healthy kidney. The dark outline of fibroblasts indicates the factin layer under the plasma membrane (except for nuclei no cell organelles are shown); the fibroblasts are affixed to tubules and capillaries (C); the arrow heads indicate interconnection of fibroblasts by adhering junctions; the extensions of dendritic cells are narrowly intermingled with fibroblast cell processes.

FIGURE 20.13 Interstitial cells, labeled by immunogold staining for ecto-50 -nucleotidase (a,b) and MHC class II (c,d) on consecutive (a,c) cryostat sections. P: Proximal tubule; D: Distal convoluted tubule; G: Glomerulus; C: Capillary; Arrowheads: Fibroblasts; Arrows: Dendritic cells. (a) 50 NT labeling highlights the abundance of interstitial fibroblasts (arrow) and the brush border of proximal tubules. Insert: Higher magnification of a fibroblast, labeled for 50 NT by enzyme-histochemistry, demonstrating the far extending processes within the interstitial space. (b) Fibroblasts (arrowheads) bridge the interstitial space between the basement membranes of tubules and capillaries. Insert: Detail of the attachment of a fibroblast process to a tubular basement membrane. (c) Dendritic cells (arrow) labeled for MHC class II, share the interstitial space with fibroblasts. (d) and insert: Differential interference contrast shows the narrow contact of dendritic cells and their extensions (arrow) to fibroblasts (arrowhead) (a,c 3 B340; b,d 3 B1200; Bars a,c B50 μm; b,d B10 μm).

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INTERSTITIUM

FIGURE 20.14 (a) Fibroblast with sharply outlined pericaryon in the cortical interstitium of a rat kidney; a filiform processes (1) is interconnected with another fibroblast by intermediate junctions (Insert 1); pedicle-like processes of the same fibroblast adhere to the basement membrane of a capillary (c) (2; Insert 2) and of a proximal tubule (PT) (3; Insert 3; Star: Extracellular matrix) with pedicle-like processes that reveal dense stress-fiber-like F-actin filaments; (Insert 4): collagen fibrils (asterisk) closely associated with a fibroblast extension (F) which encloses part of a dendritic cell (D); (Insert 5) the broad cytoplasmic extensions show abundant cisterns of rough endoplasmic reticulum (TEM 3B11,800; Inserts: 1 3B50,000; 2,3 3B23,000; 4 3B11,800; 5 3B23,000). (b) Fibroblast (F) in focal peritubular inflammation, caused by a lesion in a distal tubule; the fibroblast bridges the space between a healthy proximal tubule (PT), and a diseased distal tubule (DT), extends with thin processes closely along the basement membrane of the DT, partially encloses a profile of a peritubular capillary (C), and has close contact to migrating cells of the immune system (L: lymphocyte; D: dendritic cell; Insert: Higher magnification of the contact (“kiss”) of the extension of the dendritic cell and a lymphocyte; TEM 3B6000; Insert 3B23,000).

substances. Interstitial fibroblasts bridge the interstitial space (Figures 20.12, 20.13 and 20.14). They are physically affixed to the basement membranes of tubules, renal corpuscles, and peritubular capillaries, they are interconnected by adhering junctions44,45,47
49 (Figures 20.12 and 20.14), and narrowly contact all types of migrating cells within the interstitial space (Figures 20.12 and 20.13).

Cortical Interstitial Fibroblasts In transmission electron microscopic (TEM) images (Figure 20.14), cortical interstitial fibroblasts display a heterochromatin-rich angular nucleus which is surrounded by a narrow organelle-free cytoplasmic rim.

605

Filiform and leaf-like, perforated and filiform processes spread from the cell body, traverse the interstitial space and are affixed to the basement membranes of tubules, enveloping glomerular arterioles and cells of the immune system.50 Characteristic for fibroblasts is the dense layer of f-actin filaments immediately under the plasmalemma (Figures 20.12 and 20.14). In the filiform processes and the pedicle- or spine-like attachments to the basement membranes, the f-actin is markedly dense48 (Figures 20.12 and 20.14). The anchorage of fibroblasts to tubular and capillary basement membranes and their interconnections suggests the possibility that each configurational change of tubules or capillaries (e.g., related with tubular/capillary growth, tubular/capillary dilatation or collapse) exerts mechanical forces on the f-actin frame of the fibroblasts and induces signaling pathways.51,52 In concert with chemokines and other factors53 the mechanical forces might be essential components in the cross-talk between fibroblasts and epi- or endothelia, The production of extracellular matrix is another distinguishing characteristic of fibroblasts. The morphological correlate for matrix production is the prominent apparatus for protein synthesis, i.e., abundant large profiles of rough endoplasmic reticulum filled with flocculent, rather electron-dense material, as well as several sets of Golgi-fields. These organelles, including mitochondria, are predominantly located in the peripheral thicker parts of the leaf-like processes, close to the sites of release of matrix and collagen fibrils into the interstitial space (Figure 20.14).47,48 The extracellular matrix of the interstitium is composed of a network of fibers, proteoglycans, glycoproteins, and interstitial fluid.44,54 Several types of fibers are found, among them typical interstitial collagen fibers (type 1, type 3, and type 6).55 Microfibrils (collagen type 1) are found throughout the renal interstitium. Type 3 fibers correspond to the reticular fibers which form a network enveloping individual tubules. Proteoglycans are an important component of the interstitial matrix in the kidney.56 As elsewhere in the body, various glycoproteins (fibronectin, laminin, and others) are found associated with tubular basement membranes, as well as with fibrillar structures. All these substances contribute to the scaffolding function of the interstitium. Furthermore, they are important substrates for migrating immune cells in the interstitial space. Fibroblasts can accumulate lipid droplets. These are not common in cortical fibroblasts (in contrast to medullary fibroblasts, see below) of healthy kidneys; yet, they may also appear in cortical fibroblasts under specific functional conditions (e.g., anemia38). Lysosomal bodies are rarely observed under control conditions. Cortical interstitial fibroblasts play important roles in the adaptive response to local and systemic hypoxia.

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The cleavage of AMP by ecto-50 -nucleotidase (50 NT) on the plasmalemma of cortical fibroblasts generates extracellular adenosine (ADO) in the cortical interstitium.57 ADO has been widely implicated in adaptive responses to local hypoxia58 and in regulating local hemodynamics.59 The particularly narrow sheathing of glomerular arterioles by 50 NT-positive fibroblasts48,60 suggests a role of ADO in the regulation of glomerular blood flow and glomerular filtration rate. Indeed, purinergic receptors in afferent and efferent glomerular arterioles are implicated in the regulation of renal functions and hypertension.61 Studies on 50 NT-deficient mice have confirmed that ADO mediates the vascular response elicited by changes in NaCl concentration at the macula densa.62 Cortical fibroblasts also exhibit soluble guanylyl cyclase (sGC),63 and a b-type cytochrome 558.64 Interstitial fibroblasts in the deep cortex are the source of renal erythropoietin.65
67 The hypoxia-inducible factor (HIF Hif-2), which has also been located to 50 NT-positve fibroblasts,68,69 mediates regulation of transcription of erythropoietin following changes in oxygen supply.70 In conditions of anemia38 and hypoxia, cortical interstitial fibroblasts from all cortical regions are rapidly recruited for EPO-synthesis.71 Extensive phenotypical modulations of cortical peritubular fibroblasts in vivo occur under the concerted action of inflammatory cytokines and growth factors.72
74 Under these conditions, the interstitial fibroblasts proliferate and transform into myofibroblasts.75 Morphologically myofibroblasts differ from interstitial fibroblasts of the healthy renal interstitium, having rounded, euchromatin-rich nuclei and large irregularly-shaped cellular extensions containing dramatically expanded cisterns of rER,75 and by increased junctional coupling.76 Differing from healthy interstitial fibroblasts, myofibroblasts express αSMA and vimentin, and 50 NT is internalized from the plasma membrane into the cytoplasm. Functionally, myofibroblasts have an increased capacity for quantitatively and qualitatively different matrix production,72
74 and a reduced potential for erythropoietin gene expression.70

Medullary Interstitial Fibroblasts The phenotype of fibroblasts in the medulla is basically the same as in the cortex, yet their three-dimensional configuration50 changes in correlation with the change in tubular arrangement, from convolutions in the cortex to the strictly parallel course of tubules and vessels in the inner zone. The longitudinal axis of the pericaryon of inner medullary fibroblasts is oriented perpendicularly to the longitudinal axis of tubules and vessels (Figure 20.15), and in two-dimensional microscopic images they appear like “rungs of a ladder”.44

FIGURE 20.15

Interstitial fibroblasts of the inner medulla, demonstrated in longitudinal sections. The fibroblasts (asterisks) are arranged like the rungs of a ladder between parallel running tubules or vessels. The fibroblasts contain numerous lipid granules (black) of different sizes (visible in a). (L: loop limb; V: vessel; (a) Psammomys; TEM 3B1350; (b) Rat; SEM 3B3400). (In cooperation with J. M. Barrett.)

One noticeable change in the ultrastructure of medullary fibroblasts is the progressive increase in cytoskeletal elements towards the deep inner zone; actin filaments form a very prominent layer under the plasma membrane of the pericaryon (Figure 20.16) and the processes. The latter may be interconnected by a composite type of intercellular junction.49 The increase in cytoskeletal elements in the cells in the inner zone most probably contributes to withstanding the increasing osmotic pressure towards the papillary tip. Furthermore, the occurrence of lipid granules increases progressively from the outer medulla towards the inner medulla where they may be so prominent that the cells were designated as “lipid-laden cells.” However, inner medullary fibroblasts may also lack lipid droplets.44 In vitro studies have revealed that the

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osmolarity. Glycosaminoglycans are particularly abundant in the inner medullary interstitium,78 and condensed hyaluronate-proteoglycan aggregates are associated with basement membranes, with collagen fibers, as well as with diffuse reticular structures. The inner medulla has the greatest capacity for renal prostaglandin (PG) synthesis.79,80 Cyclooxygenase (COX) isoforms, rate-limiting enzymes in PG biosynthesis, are expressed at substantially higher levels in the inner medulla than in the renal cortex.81
84 COX-2 is found predominantly in inner medullary interstitial fibroblasts, and its expression increases under chronic salt loading.84

Dendritic Cells

FIGURE 20.16 Medullary fibroblasts, (a, b, d) human fibroblasts from a renal biopsy; (c) fibroblast from a perfusion-fixed rat kidney. (a) Human fibroblast at the cortico-medullary border; stellate pericaryon and microfilament-rich cell processes (small arrows), extending between immune cells. (b) Part of a human fibroblast in the inner stripe of the outer medulla; the cisterns of the rough endoplasmic reticulum are widened and filled with flocculent material (Asterisk: Dilated perinuclear cistern; Small arrows: Accumulations of microfilaments along the plasma membrane). (c) Rat fibroblast from the inner medulla showing dilated perinuclear and endoplasmic reticulum cisterns (asterisk); infoldings of the ER into the cistern (arrow head) (L: Lipid droplets). (d) Fibroblast in the outer stripe of the outer medulla; a profile of rough endoplasmic reticulum in direct contact with the plasma membrane (arrow) (TEM (a): 3B4760; (b): 3B8500; (c) 3B37,400; (d) 3B33,150; Bars: (a,b): B2 mm; (c,d): B0.2 mm). (From ref. [47].)

occurrence and amount of lipid droplets in the inner medullary fibroblasts depends on the specific environment of the cells, conditioned by the presence of inner medullary collecting duct cells, and that inner medullary fibroblasts can transform to myofibroblasts with upregulation of alpha smooth muscle actin and desmin.77 In medullary fibroblasts the cisterns of rough endoplasmic reticulum, including the perinuclear cistern, are often strikingly enlarged and they may narrowly enclose mitochondria (Figure 20.16). Occasionally the rER membranes are in direct contact with the plasma membrane (Figure 20.16). The functional interpretation of these particular features, only rarely observed in cortical fibroblasts, is still to be resolved. The medullary fibroblasts do not display 50 NT or mRNA for EPO. A role of medullary interstitial cells has been proposed in the regulation of urinary

Dendritic cells (DC) belong to the mononuclear phagocyte system and constitute the major antigenpresenting cell population in the healthy kidney.85 Interstitial DCs continually probe the surrounding environment through dendrite extensions, and readily respond to insults to the parenchyma.86
88 DCs have been recognized by their expression of MHC class II (Figures 20.12 and 20.13) and CD11c. In the healthy kidney DCs are present in their immature phenotype with comparatively low levels of MHC class II and of co-stimulatory proteins,89 but with a high capacity for uptake of antigens.86 Similar to fibroblasts, DCs form an organ-spanning network,88 located in very close contact with fibroblasts (Figures 20.12, 20.13 and 20.14).45,47 DCs are constantly moving. Unlike fibroblasts, the pericaryon of DCs is large and confines the often rounded or elongated nucleus together with most cell organelles. The rER profiles of dendritic cells are narrow and less abundant than in fibroblasts. The intermediate filament protein vimentin is regularly present in the pericaryon of DCs, whereas it is absent in fibroblasts in the healthy renal cortex. Dendritic cells have, in comparison to macrophages and lymphocytes, more mitochondria, more rER, and a large Golgi apparatus. Lysosomes are less apparent than in macrophages. The so-called Birbeck granules, which are characteristic for dendritic cells, are a special formation of the endocytotic compartment serving as a loading compartment and/or reservoir of antigens before DC maturation.90 The ramified “veil-like” and perforated cellular extensions (Figure 20.12) lack the prominent stuffing with f-actin filaments, and are largely devoid of cell organelles, in marked contrast to fibroblasts. The long filiform processes of DCs have approximately the same diameter as the filiform fibroblast processes, but due to the lack of f-actin filaments are much less electrondense (Figure 20.14). In contrast to fibroblasts, DCs

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have no junctional connections among each other or with tubules or vessels. However, frequently the plasma membranes of dendritic cells and of fibroblasts or dendritic cells and lymphocytes form points of membrane adhesion, so-called “kisses” (Figure 20.14). Parts of dendritic cells are often nestled into the hollows of the pericaryon or the processes of fibroblasts. The narrow intermingling of both cell types (Figures 20.12, 20.13 and 20.14) suggests the possibility of extensive cross-talk between them. Dendritic cells are abundant in the inner stripe in the outer medulla and, as in the cortex, are narrowly associated with fibroblasts. Accumulations of dendritic cells are particularly striking around collecting ducts and thick ascending limbs. In the inner medulla the pericaryon of dendritic cells is often situated in the spaces between the “ladder rungs” formed by the fibroblasts, and their processes may extend over several “ladder rungs.” In the lower two-thirds of the inner medulla bone marrow-derived cells are not detected in the healthy kidney.45,47

Macrophages and Lymphocytes Macrophages and lymphocytes are rarely found in the healthy renal interstitium, but they massively invade the interstitial spaces under inflammatory conditions.46 A large proportion of the invading mononuclear cells display the established “marker proteins” (CD 45, CD3, CD4, CD 8; ED1, ED2, CD44, etc.) and the protein S100A4.35,91 Neutrophil granulocytes are found occasionally, basophil and eosinophil granulocytes and plasma cells are rare in the healthy cortical renal interstitium.

PERIARTERIAL CONNECTIVE TISSUE AND LYMPHATICS The periarterial tissue is a sheath of loose connective tissue, surrounding the intrarenal arteries (arcuate arteries, cortical radial arteries). The considerable thickness of the periarterial sheath is apparent in quick-frozen specimens,92 and in perfusion fixed tissue. It attenuates towards the end of the cortical radial arteries and terminates along the afferent arteriole at the vascular pole of the glomerulus (Figures 20.17, 20.18 and 20.19). The periarterial sheath is continuous with the peritubular interstitium and with the connective tissue underlying the epithelium of the renal pelvis and ureter at all sites. The renal veins are apposed to the periarterial sheath.92 The periarterial sheath constitutes wide meshes of the extremely attenuated processes of 50 NT-negative, but weakly alpha-smooth muscle actin- and vimentinpositive fibroblasts.47 The meshes are filled with thick

FIGURE 20.17 Cross-section through the deep cortex (rat). The cortical radial artery (A) is surrounded by a layer of loose connective tissue that contains the lymphatics (LY). The cortical radial vein is only partly shown; its lumen and those of direct tributaries have been highlighted by a dotted pattern. Note the intimate relationships of the artery, vein, and lymphatic, mediated by the periarterial loose connective tisue (TEM: 3B360).

bundles of collageneous fibers and interstitial fluid and regularly confine some macrophages and dendritic cells. The periarterial connective tissue sheath provides the path for renal nerves (see below) and for renal lymphatics.92 Lymphatics start in the vicinity of the glomerular vascular pole25 or at a more proximal level of the afferent arteriole, depending on the species, and travel along the branches of the renal arteries towards the renal hilum (Figures 20.17 and 20.18). Their recognition and distinction from blood capillaries at light microscopic levels is facilitated by their specific expression of podoplanin.93 Also, 50 NT labels in rat and mice lymphatics, but not in blood vessels. Lymphatic endothelial cells secrete chemokines that attract dendritic cells. An increase in lymphatic microvessels has been observed, e.g., in tubulointerstitial fibrosis and progression to end-stage renal failure in remnant kidney.93 Regulatory substances that are released into the peritubular interstitium might access the systemic blood circulation via the lymphatics in the periarterial sheath. This suggestion has been made for renin,92 and it may

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b

a

1 A Interlobular artery + vein

2 3

V

Ly

N

Arcuate artery + vein

Vascular bundle

FIGURE 20.18 Schematics to show (a) the distribution, and (b) the topographical relationships of the periarterial connective tissue sheath. Not drawn to scale. (a) The periarterial sheath is schematically indicated as a wide “stocking” drawn over the intrarenal arteries and lymphatics. In reality this stocking has no limiting tissue that separates the interior from the surroundings. An arcuate artery transforms into a cortical radial artery, which gives rise to afferent arterioles. These segments are surrounded by the periarterial connective tissue sheath. The efferent arterioles, as well as the veins (drawn in black), are not included. The lymphatics (stippled) originate and travel within the periarterial sheath. Note there are no lymphatics coming up from the medulla. Within the cortex, medullary rays are indicated by a broken line. In (b) a transverse section through an cortical radial artery (A) shows the relationships of the periarterial sheath and the possibilities for functional exchanges (double-headed arrows) with surrounding structures: (1) with the peritubular interstitium; (2) with the accompanying vein (V); and ( 3 ) with lymphatics (Ly). The single-headed arrows indicate the flow of the respective fluid. Note the nerves (N) traveling through the periarterial tissue. In addition, two neighboring tubules, including an arcade and a proximal tubule, together with peritubular capillaries, are drawn. (From ref. [92], with permission.)

FIGURE 20.19 The renal nerves accompany (a) the intrarenal arteries. A cortical radial artery (arrow) is seen which is associated with three nerve bundles, identified by staining with a monoclonal antibody against protein gene product 9.5 (PGP 9.5) which is a universal marker for vertebrate neurons. Among the tubules no nerves are found. A medullary ray is delineated by a hatched line (Rat; LM 3B240). (In cooperation with M. Siry, W. Kummer and S. Bachmann). (b) A large terminal nerve accompanying an afferent arteriole. Several axons (arrows) and a varicosity (star) with synaptic vesicles are seen (SM: Smooth muscle cell; CO: Collagen; C: Capillary; Rabbit kidney: TEM 3B12,000). II. STRUCTURAL ORGANIZATION OF THE MAMMALIAN KIDNEY

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apply to other protein hormones such as erythropoietin. The periarterial tissue sheaths have been interpreted as a mixing chamber for a variety of vasoactive substances, ultimately determining the contractile status of the renal resistance vessels. In addition to the lymphatics, the periarterial tissue itself constitutes a pathway for interstitial fluid drainage. Indeed, a tracer injected under the renal capsule can be followed within the periarterial tissue, as well as within the lymphatics. While a fraction of cortical interstitial fluid gains access to the periarterial tissue, and eventually to lymphatics, there is no direct pathway for regulatory substances released in the cortical interstitium to reach medullary targets. It has been proposed that the intimate relationships between cortical radial arteries and veins permits countercurrent exchange of O2, being responsible
at least in part
for the low partial pressure of O2 in the superficial cortex.94,95

NERVES The efferent nerves of the kidney are composed of sympathetic nerves and terminal axons, which accompany the intrarenal arteries, and the afferent and efferent arterioles (Figures 20.18 and 20.19).96 The nerve fibers are monoaminergic. Norepinephrine97
99 and dopamine have been identified.100 In addition, several neuropeptides are co-localized with norepinephrine in renal nerves.101 The presence of acetylcholinesterase in renal nerves cannot be taken to indicate cholinergic nerves, but rather that monoaminergic nerves obviously possess acetylcholinesterase activity.97 The nerve fibers run in the loose connective tissue around the arteries and arterioles. The descending vasa recta within the medulla are also innervated by adrenergic nerve terminals as far as they are enveloped by smooth muscle cells.102,103 A dense assembly of nerves and terminal axons is found around the juxtaglomerular apparatus99 which is described in more detail along with the JGA. Tubules have direct relationships to terminal axons only when they are located around the arteries or arterioles.96,99 Tubules adjacent to the juxtaglomerular apparatus (terminal portion of the cortical thick ascending limb) are more frequently touched by terminal axons than at other sites.99 The density of nerve contacts to convoluted proximal tubules (located in the cortical labyrinth) is low104; nerve contacts to straight proximal tubules (located in the medullary rays and the outer stripe) have never been encountered. The overwhelming majority of tubular portions have no direct relationships to nerve terminals. Consequently, morphologists are left with the question of how the neuronal influence on tubular

function105 is mediated. In addition to a systemic distribution of catecholamines, a more specific, but also indirect, mode seems possible for certain tubular segments.5 Because nerve fibers do pass along the vascular pole of each glomerulus from afferent to efferent arterioles, the distribution of nerves in the renal cortex is dense. Catecholamines (and other transmitters) released from nerve terminals at the vascular poles and at the efferent arterioles may gain access to peritubular capillaries, and in this way may perfuse the convoluted tubules of the cortical labyrinth. Tubules arranged around the cortical radial arteries would be reached most easily by transmitters released from periarterially located nerve terminals. This may be of relevance with respect to arcades (connecting tubules), which have been shown to be sensitive to isoproterenol.106 Exposure of the straight tubules within the medullary rays of the cortex to neural transmitters reaching them directly by diffusion from nerve terminal is improbable. Tubules in the outer medulla may only be reached by neurotransmitters if they are either situated adjacent to vascular bundles (a minority of tubules) or secondarily, by a capillary distribution of the transmitters from nerve terminals accompanying the vascular bundles. The tubules of the inner medulla cannot be reached by neurotransmitters directly delivered from nerve terminals in the medulla. Little is known about the afferent nerves of the kidney; they are commonly believed to be sparse, but the issue remains unresolved.102,105,107
110

TOPOGRAPHICAL RELATIONSHIPS Cortex The architectural pattern of the renal cortex is best understood when viewing a cross-section through the midcortex (Figures 20.20 and 20.21). Two portions within the cortical parenchyma, the labyrinth and the medullary rays, are distinguishable. Within the cortical labyrinth, the vascular axes, which consist of the cortical radial (interlobular) artery, vein, and a lymphatic, are regularly distributed. The renal corpuscles and the corresponding convoluted tubules (proximal and distal) are situated around each vascular axis. Barriers separating the population of renal corpuscles and convoluted tubules belonging to another vascular axis are not discernible. Thus, the cortical labyrinth is a continuous parenchymal layer that contains the vascular axes of the cortex and the medullary rays in a regular pattern. The straight tubules (proximal and distal), together with the collecting ducts, are located within the medullary rays. Because the number of straight tubules increases

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FIGURE 20.20

Schematics of histotopography. Histotopography of the kidney as revealed by four successive cross-sections through: (a) cortex; (b) outer stripe; (c) inner stripe; and (d) inner medulla. The simple type of the medulla (rabbit, man) is shown. (From Koushanpour, E., and Kriz, W. (1986). In “Renal Physiology. Principles, Structure and Function.” Springer-Verlag, New York, with permission.) In the cortex the cortical labyrinth (shaded area) and the medullary rays (white) are shown. The labyrinth contains the cortical radial (interlobular) blood vessels, glomeruli, and the convoluted tubular portions (the latter are not shown). The arcades accompany the interlobular vessels. The medullary rays contain the collecting ducts and the straight proximal and distal tubules. Note the typical grouping of collecting ducts within a medullary ray. In the outer stripe, vascular bundles replace the interlobular vessel axis of the cortex. The continuations of the medullary rays are surrounded by hatched lines. Within these areas the collecting ducts and the loop limbs of superficial and midcortical nephrons are found. The loop limbs of juxtamedullary nephrons are situated around the vascular bundles. In the inner stripe, the vascular bundles are fully developed. Like in the outer stripe, the loop limbs of juxtamedullary nephrons are situated near the bundles, and those from superficial and midcortical nephrons together with the collecting ducts are located distant from the bundles. Note the heterogeneity of the thin limbs: Those of juxtamedullary nephrons lie near the bundles and are thicker in diameter, whereas those of the superficial nephrons lie distant from the bundles. Inner medulla. The area defined by the dashed rectangle corresponds to the entire area shown in section (c). This reduction in size is because short loops and many vasa recta have turned back in the inner stripe. Note the grouping of collecting ducts reflecting their medullary ray arrangement in the cortex. Thin limbs (both descending and ascending) are associated with vasa recta or collecting ducts.

toward the corticomedullary border, the medullary rays increase in width toward the outer stripe. A regular pattern of the convoluted tubules within the labyrinth is not apparent. Proximal and distal convoluted tubules (the latter constitute only a minor portion of profiles in comparison with proximal tubules) are equally embedded in the dense capillary plexus of this region. A strikingly constant position is occupied by the arcades (if they are present). They ascend within the cortical labyrinth and are grouped immediately around the vascular axes. The topographical

relationships within the juxtaglomerular apparatus will be described later. Within the medullary rays the straight tubules of superficial nephrons (proximal and distal) generally occupy a central position, and those of midcortical nephrons occupy a peripheral position. The collecting ducts are situated between the two groups, and therefore are situated neither in the center nor at the very border of a medullary ray. Efferent arterioles do not enter the medullary rays, but frequently break off into capillaries just at the border between the labyrinth and

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FIGURE 20.21 Renal cortex of the rat; 1 µm cross-section. The architectural pattern of the cortex is demonstrated. A medullary ray is delineated by a dashed line. It contains the straight proximal tubules (P), the straight distal tubules (thick ascending limb; asterisks), and the collecting ducts (C). The cortical radial vessels (A: Artery; V: Vein), glomeruli (G), and convoluted proximal (Pc) and distal (D) tubules establish the cortical labyrinth. Arcades (stars) ascend close to the cortical radial vessels (3B200).

the medullary rays. As a result, the blood supply of the medullary ray tubules is as direct as that of the tubules within the cortical labyrinth. However, blood that perfuses the straight tubules of the medullary rays mixes afterwards with the blood that perfuses the convoluted tubules (vide supra).

Medulla The three regions of the medulla contain different populations of nephron segments. The outer stripe contains straight parts of the proximal tubule (S3 segments), straight parts of the distal tubule (thick ascending limbs), and collecting ducts. The inner stripe is composed of descending thin limbs, ascending thick limbs (distal straight tubules), and collecting ducts. The inner medulla contains thin descending and ascending limbs, and collecting ducts. The architectural organization of the medulla can best be described by considering the vascular bundles as central axes and studying how the tubules are arranged around them.33,112 A “simple” and a “complex” type of renal medulla are distinguished (Figure 20.22); the differences between both are mainly found in the inner stripe.

The vascular bundles develop in the outer stripe (Figures 20.20 and 20.23). At the very beginning of the bundles the straight proximal and distal tubules of juxtamedullary nephrons are grouped immediately around the bundles. In the continuation of the medullary rays, the tubules of superficial and midcortical nephrons, together with the collecting ducts, fill the spaces between the bundles and their adjacent juxtamedullary tubules. In the outer stripe straight proximal tubules and straight distal tubules (thick ascending limbs) should theoretically be present in equal numbers; however, a cross-section through the outer stripe shows that proximal tubular profiles are much more numerous than distal tubules. In the rat, proximal tubules occupy roughly 68% of the space in the outer stripe, in contrast to approximately 13% by the thick ascending limbs, and 5% by the collecting ducts.41 The dominance of the proximal tubules is rooted in the fact that the straight proximal tubules of juxtamedullary nephrons are not straight (as their name indicates), but rather take a tortuous course when descending through the outer stripe; this holds true for the mouse kidney.113 In addition, straight proximal tubules are much thicker in diameter than the straight distal tubules, and proximal tubules of juxtamedullary nephrons are even thicker than those of the midcortical and superficial nephrons.5 The tubules of the outer stripe are perfused by a specific “capillary” plexus. “True” capillaries, derived from direct branches of efferent arterioles, are few; the dominating “capillary” vessels in the outer stripe are the ascending vasa recta (Figures 20.3 and 20.4b). They traverse the outer stripe as wide tortuous channels closely contacting the tubules like proper capillaries. Because these vessels carry the entire venous blood from the medulla, the outer stripe tubules are mainly supplied by venous blood from deeper parts of the medulla. The outer stripe varies considerably in thickness among species; in the rat33 and mouse,34 it is very welldeveloped and constitutes approximately one-third of the outer medulla. In contrast, in Psammomys,32 cat,37 dog,114 and humans,37 the outer stripe is very thin. The inner stripe (Figures 20.20 and 20.24) of the outer medulla is the most constant part of the renal medulla, consisting of the regularly distributed vascular bundles (VB) leaving between them the interbundle region (IBR). Two types of vascular bundles can be distinguished, which form the basis for the discrimination of a “simple” and a “complex” type of medulla. In most species5,115 vascular bundles of the simple type are present, which exclusively contain descending and ascending vasa recta. The tubules are found in the IBR and are arranged around the bundles in a pattern similar to that found in the outer stripe. The loops of

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FIGURE 20.22 Schematic to demonstrate the difference between the simple (a and a1) and complex (b and b1) types of medulla. In the simple medulla (a), loops of Henle surround the vascular bundle according to the pattern established in the cortex. The long loops lie nearest to the bundle, the short loops of superficial nephrons farthest away. The collecting ducts are situated at a distance from the bundle. The bundle itself (a1) contains only descending (black) and ascending (white) vasa recta. In the complex medulla (b), the descending thin limbs of short loops descend within the vascular bundles. The complex bundle (b1) contains, in addition to descending (black) and ascending (white) vasa recta, the descending thin limbs of short loops (hatched). (From ref. [133], with permission.) (c
c3): Schematics of cross-sections through vascular bundles to show different degrees of bundle fusing and loop integration in the complex type of renal medulla (c1
c3) compared with the simple type (c) (Large circle: Vascular bundle; Small hatched circles: Descending thin limbs of short loops). In the simple type of medulla (c), the descending thin limbs of short loops are all located outside the bundle. Complex bundles (c1
c3) may be established in different degrees. In rat (c1), descending thin limbs of short loops are arranged in the periphery of the bundles; bundles generally do not fuse. In mouse (c2), bundles frequently fuse; descending thin limbs of short loops have penetrated deeper into the bundle. In Psammomys or Meriones (c3), bundle fusing has produced giant bundles; descending thin limbs of short loops are distributed over the entire bundle area. (From ref. [115], with permission.)

Henle, originating from juxtamedullary nephrons (generally the longest long loops), lie nearest to the bundles, whereas the loops derived from superficial and midcortical nephrons (in most species, short loops) lie distant from the bundles. The collecting ducts are generally arranged in distant rings around the bundles, and are intermingled with loops derived from superficial and midcortical nephrons. Altogether, they are perfused by the dense capillary plexus of the IBR. The complex type of vascular bundle (Figure 20.25) is present in several rodent species with a high urine concentrating ability, including rat,33 mouse,16,34 Meriones,116 and Psammomys.32 It differs from the

simple type in that the descending thin limbs of short loops (only of short loops!) descend within the vascular bundle (Figure 20.26). Consequently, the bundles within the inner stripe change from the classic countercurrent arrangement of a rete mirabile, consisting of DVRs and AVRs, to a system in which one ascending tube (AVRs) is closely packed together with two descending tubes (DVRs and SDTLs). In addition, the vascular bundles in the complex type of medulla tend to fuse and to form larger bundles, up to giant bundles (Psammomys). These complex bundles are developed at the transition from the outer stripe to the inner stripe, and are maintained only throughout the inner stripe.

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FIGURE 20.23 Outer stripe of rat kidney; 1 µm cross-section. The vascular bundle (VB) is surrounded by the straight proximal tubules of juxtamedullary nephrons (asterisk), which are larger in diameter than those of superficial and midcortical nephrons (P), which lie distant from the bundles (Asterisks: thick ascending limb; C: collecting duct). Interstitial spaces are sparsely developed (3B220).

FIGURE 20.25 Inner stripe of Meriones shawii kidney (complex medulla); paraffin cross-section. The large vascular bundle originates by fusing of primary bundles. In addition to descending (D) and ascending (A) vasa recta, complex vascular bundles contain descending thin limbs of short loops (L). Collecting ducts (some are marked by asterisks) are situated distant from the bundles (3B190).

FIGURE 20.24

Inner stripe of rabbit kidney (simple medulla); 1 µm cross-section. The vascular bundles (VB) are regularly distributed. The collecting ducts (C) lie distant from the bundles. Descending thin limbs (asterisks) and thick ascending limbs (stars) are situated within the interbundle regions (3B200).

FIGURE 20.26 Longitudinal section through a vascular bundle in the inner stripe of a gerbil kidney (complex medulla); paraffin section. One superficial nephron has been injected with microfill; at the transition from the outer (OS) to the inner stripe (IS) the proximal tubule transforms to the intermediate tubule (thin descending limb; left arrow) which descends within the vascular bundle; the tubule leaves the bundle at the border between the inner stripe and the inner zone (IZ), and ascends as thick ascending limb (right arrows) within the interbundle region (RP: Extension of the renal pelvis; 3B200).

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At the border to the inner medulla, the SDTLs leave the bundles, and the fused bundles split into the original number of bundles. The characteristics of the complex type are developed to different degrees in the species so far investigated. A somewhat “gradual” transition from the rat, via the mouse, to Meriones and Psammomys is observed. The tubular pattern around these complex bundles is different from that of the simple type. At the border between the outer stripe and inner stripe, the SDTLs leave their position distant from the bundles, then turn toward a bundle and descend within the bundle. Their TALs maintain a position distant from the bundles and near to a collecting duct throughout the outer medulla. As observed in the simple type, the tubules of the interbundle regions are embedded in the dense capillary plexus of the IBR. In contrast to what is observed in the simple type, it is worthwhile to stress the fact that in the complex type only the LDTLs, scattered among the TALs of short and long loops, traverse IBR. Specific variations of this pattern in mice117 and Psammomys17,32 are described elsewhere. To understand the possible functional implications of the inner stripe architecture, as well as the differences between the simple type and the complex type, we have to consider precisely the composition of the vascular bundles. The vascular bundles of the simple type contain all descending and all ascending vasa recta servicing the inner medulla. Furthermore, they contain most of the descending vasa recta, which service the inner stripe, but only few of the ascending vasa recta, which drain the inner stripe. The numerical relationship between descending and ascending vasa recta is about 1 to 1 (at the level of the inner stripe). Thus, in the simple type of vascular bundle, the venous blood from the inner medulla contacts the arterial blood that supplies both the inner medulla and the inner stripe of the outer medulla in a countercurrent arrangement. Therefore, inner medullary venous blood may exchange not only with the arterial blood that is predetermined for the inner medulla, but also with blood predetermined for the inner stripe. Substances originating from the inner medulla could be trapped by countercurrent exchange to the inner medulla, but could also be shifted to the inner stripe capillary plexus, and thereby be offered to inner stripe tubules (see below). In the complex type of medulla, the vascular bundles incorporate the descending thin limbs of short loops. In Psammomys, at the level of the inner stripe, the bundles consist of approximately 10% descending vasa recta, 45% ascending vasa recta, and 45% descending thin limbs,32 with the descending thin limbs being completely surrounded by ascending vasa recta. The difference between the simple and the complex types of bundles is even more pronounced when it is

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realized that the bundles in Psammomys no longer contain any vasa recta servicing the inner stripe. All vasa recta present in the giant bundles of Psammomys either descend to the inner medulla or ascend from the inner medulla. The vasa recta servicing the inner stripe in Psammomys descend or ascend, respectively, independent of the bundles. Thus, the giant bundles of the inner stripe in Psammomys appear to form a countercurrent trap for the inner medullary blood that is located in the inner stripe. In other species with complex bundles (rat, mouse), vasa recta servicing the inner stripe are not as strictly excluded from the bundles as in Psammomys; even in these species the vascular bundles appear to be a countercurrent trap for mainly the inner medullary circulation. The inner medulla develops very differently among species. Species with only short loops of Henle6 do not have an inner medulla; their urine concentrating ability is poor. All species with high urine concentrating ability have a well-developed inner medulla.3,118 It is characteristic for the inner medulla to taper from a broad basis to a papilla (or crest). The mass of the inner medulla is therefore unevenly distributed along the longitudinal axis. A study in the rat2,119 has shown that the decrease in the mass of the inner medulla along the longitudinal axis follows an exponential function. The upper half of the inner medulla accounts for roughly 80% of the total inner medullary volume, and consequently only 20% are left for the papillary half. With regard to the ratio between Henle’s loops and collecting ducts along the inner medulla, considerable differences are found when comparing the base with the tip of the inner medulla, as well as notable interspecies differences.42 In the rat, the ratio is about 2.5 (2.5 loops per one collecting duct) at the beginning of the inner medulla; this ratio rapidly decreases to about 1 toward the papilla. In the rabbit, the ratio increases from 3 at the beginning of the inner medulla to 9 within the papilla, then later decreases to 5 in the papillary tip. These data all await functional interpretation, thus indicating the limitation of our knowledge concerning structure
function correlations in the inner medulla. An architectural pattern within the inner medulla is less apparent than in the outer medulla.5,34,120 Constant histotopographical relationships between certain structures or spatial separations of others do not seem to be as important to the function of the inner medulla compared to the outer medulla. When entering the inner medulla, the vascular bundles already contain a drastically decreased number of vasa recta. Towards the papilla, this number continues to decrease; finally single descending vasa recta enter the tip of the papilla. Ascending vasa recta in the inner medulla generally ascend independent of the bundles, which they finally

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without joining with ascending vasa recta from the inner stripe. The blood flow of the inner stripe and that of the inner medulla are apparently distinct from each other. In the outer stripe, however, venous vasa recta coming up from the inner medulla and those from the inner stripe finally take a similar route. Both traverse the outer stripe as wide capillary channels representing the major capillary supply of the outer stripe tubules.

GLOMERULUS (RENAL CORPUSCLE)

Inner medulla of the rabbit kidney; 1 µm crosssection through the upper part. The collecting ducts are still arranged in groups, reflecting the pattern in the medullary rays of the cortex. Vascular bundles (dashed circles) are poorly delineated. Thin loop limbs (asterisks) lie near collecting ducts (C), as well as near vasa recta of the vascular bundles (3B240).

FIGURE 20.27

join at the border of the inner stripe. Thus, in the inner medulla, the vasa recta are never as closely packed to bundles as they are in the inner stripe. As far as vascular bundles are discernible, the collecting ducts are generally distanced from them. At the very beginning of the inner medulla, collecting ducts are still arranged in groups that reflect their grouping within the medullary rays of the cortex (Figure 20.27). Joining of collecting ducts first occurs among the ducts of one group. Descending and ascending thin loop limbs, together with individually running vasa recta and capillaries, fill the spaces between the bundle centers and the collecting ducts. DTLs in general tend to be more distant from CDs, whereas ATLs tend to be positioned more closely to CDs120,121; a thin limb of Henle, regardless of whether descending or ascending, may be associated with both collecting ducts and/or vasa recta. Obviously, the interactions of the structures in the inner medulla are mediated through the wide interstitial spaces. With regard to the functional connections of the inner medulla with the outer medulla, it is notable that all descending vasa recta servicing the inner medulla have already been established as individual vessels in the outer stripe and traverse the inner stripe within the bundles. All ascending vasa recta from the inner medulla traverse the inner stripe within the bundles

The renal corpuscle consists of a tuft of specialized capillaries that protrudes into Bowman’s space (urinary space) surrounded by Bowman’s capsule (BC). The tuft consists of specialized capillaries held together by the mesangium and covered
as a whole
by the glomerular basement membrane (GBM), followed by a layer of unique epithelial cells, the podocytes. Traditionally, this layer is called the visceral epithelium of BC, which
at the vascular pole of the glomerular tuft
reflects into the parietal epithelium of BC. Nowadays, the term Bowman’s capsule is generally used only for this parietal cell layer which
together with its basement membrane (parietal, BM, PBM)
forms the outer wall of a glomerulus. At the urinary pole, BC transforms into the proximal tubule epithelium, Bowman’s space opens into the tubular lumen (Figures 20.28 and 20.29). The diameters of the
more or less
spherical renal corpuscles in different species range from approximately 100 μm (mouse) up to 300 μm (elephant), in humans they are approximately 200 μm, in rat 120 μm, and in rabbit 150 μm.29,36,37 In many species (rodents) the diameter of juxtamedullary renal corpuscles may exceed that of midcortical and superficial nephrons by up to 5022,29,34,37; this does not hold true for the human kidney.122

Architecture of the Glomerulus The reflection of the parietal epithelium of Bowman’s capsule into the visceral epithelium creates an oval opening in the glomerulus, which is called the glomerular hilum. Actually, it is the reflection of the GBM into the PBM (i.e., the basement membrane of the parietal epithelium of Bowman’s capsule) that borders the opening. Through it the glomerular arterioles, together with the glomerular mesangium, enter the inner space of the GBM, which forms a complex folded sack. Inside this sack the glomerular capillaries pursue a tortuous course around centrally located mesangial axes. Together, capillaries and mesangium totally fill the labyrinthine spaces inside the GBM. The outer

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FIGURE 20.28

Diagram of a longitudinal section through a glomerulus and its juxtaglomerular apparatus (JGA). The capillary tuft consists of a network of specialized capillaries, which are outlined by a fenestrated endothelium. (From Cunningham, R. et al. (2005). Am. J. Physiol. Renal Physiol. 289(4), F933
938.) Defective PTH regulation of sodium-dependent phosphate transport in NHERF12/2 renal proximal tubule cells and wild-type cells adapted to low-phosphate media. At the vascular pole, the afferent arteriole (AA) branches into capillaries immediately after its entrance; the efferent arteriole (EA) is established inside the tuft and passes through the glomerular stalk before leaving at the vascular pole. The capillary network, together with the mesangium, is enclosed in a common compartment bounded by the glomerular basement membrane (GBM). Note that there is no basement membrane at the interface between the capillary endothelium and the mesangium. The glomerular visceral epithelium consists of highly-branched podocytes (PO) which, in a typical interdigitating pattern, cover the outer aspect of the GBM. At the vascular pole, the visceral epithelium and the GBM are reflected into the parietal epithelium (PE) of Bowman’s capsule, which passes over into the epithelium of the proximal tubule (PT) at the urinary pole. At the vascular pole, the glomerular mesangium is continuous with the extraglomerular mesangium (EGM) consisting of extraglomerular mesangial cells and an extraglomerular mesangial matrix. The extraglomerular mesangium, together with the granular cells (G) of the afferent arteriole and the macula densa (MD), establish the JGA. All cells which are suggested to be of smooth muscle origin are shown in black (F: foot processes; N: sympathetic nerve terminals; US: urinary space). (Adapted from Kriz, W., and Sakai, T. et al. (1988). Morphological aspects of glomerular function. In “Nephrology,” A. M. Davison, Vol. 1, Proceedings of the X International Congress of Nephrology,” 3
23. Bailliere Tindall, London.)

aspect of the GBM is covered by the visceral epithelium, i.e., by the podocytes. The glomerular tuft therefore consists of the glomerular capillaries and the mesangium inside the sack of the GBM (frequently called the “endocapillary compartment”), and the

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FIGURE 20.29 Longitudinal section through a glomerulus (rat). At the vascular pole the afferent arteriole (AA), the efferent arteriole (EA), the extraglomerular mesangium (EGM), and the macula densa (MD) are seen. At the urinary pole the beginning of the proximal tubule is seen (P) (PE: Parietal epithelial of Bowman’s capsule; US: Urinary space; LM 3B490).

podocytes covering this sack from outside (“exocapillary compartment”). The glomerular capillaries are derived from the afferent arteriole which
strictly at the entrance level
divides into several (two to five) primary capillary branches.20,123 Each of these branches gives rise to an anastomosing capillary network which runs toward the urinary pole and then turns back, running toward the vascular pole. Thereby, the glomerular tuft is subdivided into several (2
5) lobules, each of which contains an afferent and efferent capillary portion. The lobules are not strictly separated from each other; some anastomoses between lobules occur. The efferent portions of all lobules together establish the efferent domain of the capillary network out of which the efferent arteriole develops. In contrast to the afferent arteriole, the efferent arteriole is already established inside the glomerular tuft; thus, the efferent arteriole has a significant intraglomerular segment which runs through the glomerular stalk (Figures 20.30, 20.31 and 20.32).20 At this site, the efferent arteriole has close spatial relationships to the first branching of the afferent arteriole. After leaving the tuft, the efferent arteriole has a segment which is narrowly associated with the extraglomerular mesangium (see below). The intraglomerular segment is

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FIGURE 20.30

Schematic to show the branching pattern of the glomerular tuft. Immediately after its entrance into the tuft, the afferent arteriole splits into large superficially located capillaries which are the supplying vessels of glomerular lobules (three are shown). The capillaries run toward the urinary pole. After turning back they unite to establish the efferent arteriole still inside the glomerular tuft. Thus, in contrast to the afferent arteriole, the efferent arteriole has an intraglomerular segment (stippled). An afferent and an efferent capillary domain are distinguished. The efferent capillary domain occupies roughly a quarter sector of the tuft; it is partly covered by the afferent domain. (From Winkler, D., and Elger, M. et al. (1991). Branching and confluence pattern of glomerular arterioles in the rat. Kidney Int. Suppl 32, S2
8, with permission.)

made up by a continuous endothelium which is fully separated from the GBM by a “mesangial layer” consisting of mesangial cell processes and matrix. Thus, this initial segment of the EA is fully embedded into the mesangium. Along the course through the extraglomerular mesangium, the mesangial and/or extraglomerular mesangial cells in its wall are gradually replaced by smooth muscle cells.20 Thereafter, the efferent vessel is established as a proper arteriole. Glomerular capillaries are a specific type of blood vessel whose wall is made up of an endothelial tube only. A small strip of the outer circumference of this tube is in contact with the mesangium, the major part bulges toward the urinary space and is covered by the GBM, followed by the layer of podocyte foot processes. Taken together, these peripheral portions of the capillary wall represent the filtration area. The small juxtamesangial portion of the capillary wall is not underlain by a basement membrane, but directly abuts the mesangium.124 The glomerular mesangium constitutes the axis of a glomerular lobule, to which the glomerular capillaries are attached by their juxtamesangial portion. Apart from this attachment site, the mesangium is

FIGURE 20.31

Scanning electron micrograph of a vascular cast of a dog glomerulus with afferent (A) and efferent (E) arterioles. Note the superficially located branching pattern of the afferent arteriole, out of which the afferent capillary domain is supplied. The efferent arteriole emerges from inside of the glomerular tuft.

bounded by the perimesangial part of the GBM. Like the peripheral GBM, it is covered at its outer aspect by podocyte processes. At the turning points of the GBM the opposing parts of the GBM are interconnected by podocyte processes that are strongly armed with actin filaments.

The Glomerular Basement Membrane (GBM) The glomerular basement membrane represents the skeletal backbone of the glomerular tuft. Topographically, the GBM consists of a peripheral (pericapillary) and a perimesangial part. At the border between both parts, the GBM changes from a convex pericapillary into a concave perimesangial course; the turning points are called mesangial angles.124 During development, the GBM originates from the fusion of an endothelial and a podocytic basement membrane. In the adult, the collagen component of the GBM is solely derived from podocytes, whereas the laminin component originates from both podocytes and endothelial cells.125 The GBM is a remarkably stable structure; the in vivo loss of protein radioactivity suggests a half-life of more than 100 days.126 Nevertheless, a continuous turnover occurs,127,128 but few details are known about where and how new

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FIGURE 20.32

(a) Narrow association between the afferent arteriole (AA) and the intraglomerular segment of the efferent arteriole (*, EA) as seen in a section approximately 15 μm inside a glomerulus. The afferent arteriole (AA) splits into primary branches. The branching point of the AA has a narrow spatial relationship to the inraglomerular segment of the EA (asterisk), which is located in the center of the tuft. The intraglomerular segment of the EA is enclosed
together with the AA
in a common compartment bordered by the GBM. (b) Higher magnification of the intraglomerular segment in a subsequent section with several conspicuous features: the lumen is narrow; the continuous endothelium consists of four cell bodies that bulge into the lumen; the endothelium is surrounded by a mesangial envelope made up of mesangial cells (MC) and matrix; a few smooth muscle cell processes (SM) are interspersed. AA and EA are separated only by mesangial tissue (M); there is no basement membrane separating the AA and EA. P, cell body of a podocyte attached to the GBM surrounding the EA. (c) Schematic of a cross-section through the glomerular vascular pole, showing the spatial relationships of the AA and EA within the glomerular stalk corresponding to the situation in (a). Immediately after its entry into the glomerulus, the AA splits into wide capillary branches with open endothelial pores. The branching point of the AA has a narrow spatial association with the outflow segment of the EA. The outflow segment is enclosed, together with the AA, in a common compartment bordered by the GBM. The EA is completely surrounded by a layer of mesangial tissue (shown in gray), and is separated from the AA only by this layer; there is no basement membrane between AA and EA. Broken arrows represent blood flow from afferent branches through the capillary network to the outflow segment (TEMs: (a) 3B1500; (b) 3B4300). (From ref. [20], with permission.)

components are added, and others removed and degraded. Several extracellular matrix degrading enzymes have been found to be produced by podocytes and mesangial cells129
131; however, the relevance of these enzymes to the turnover of the GBM remains to be established. The GBM varies in width among species. In humans the thickness ranges between 240 and 370 nm, in rat and other experimental animals it is between 110 and 190 nm. In electron micrographs of traditionally fixed tissue the GBM appears as a trilaminar structure made up of a lamina densa bounded by two less dense

layers
the lamina rara interna and externa. Recent studies using freeze techniques reveal only one dense layer directly attached to the bases of the epithelium and endothelium.132 The major components of the mature GBM include type IV collagen, type II laminin (5 laminin 521), heparan sulphate proteoglycans (agrin, perlecan), and the glycoproteins entactin/nidogen133,134; type V and VI collagen have also been demonstrated.135 The mature GBM is established during the development of a glomerulus from the S-shaped body to the capillary loop stage. During this transition, the collagen

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IV α1 and α2 chains are replaced by α3, α4, and α5 chains, and the laminin α1 and β1 chains are replaced by α5 and β2 chains, the γ1 chain remains preserved, together forming laminin 521.136,137 The components of the mature GBM are all synthesized by the podocytes. The functional importance of this specific composition of the GBM compared to basement membranes elsewhere in the body becomes evident when looking at their involvement in glomerular diseases: the various forms of Alport syndrome are caused by mutations in the genes encoding the α3, α4, and α5 chains of collagen type IV; Goodpasture syndrome is mediated by antibodies against the α3 collagen IV chain.138 Current models depict the basic structure of the basement membrane as a three-dimensional network of collagen type IV.139 Monomers of type IV collagen consist of a triple helix of α3, α4, and α5 chains measuring 400 nm in length which, at its carboxy-terminal end, has a large non-collagenous globular domain, called NC1. At the amino-terminus the helix possesses a triple helical rod 60 nm in length, the 7S domain. Interactions between the 7S domains of two triple helices or the NC1 domains of four triple helices allow collagen type IV monomers to form dimers and tetramers. In addition, triple helical strands interconnect by lateral associations via binding of NC1 domains to sites along the collagenous region. Fibronectin, laminin, and entactin are the glycoproteins of the GBM140; the major one is laminin 521. Laminin forms a second network that is superimposed onto the collagenous network. Laminin is a noncollagenous glycoprotein consisting of three polypeptide chains, two of which are glycolylated and cross-linked by disulfide bridges.137 Laminin, via entactin, binds to specific sites on the polymerized network of type IV collagen, as well as to integrin and dystroglycan surface receptors of the podocytes and endothelial cells (see later). This combined network of type IV collagen and laminin is considered to provide mechanical strength to the basement membrane, and to serve as a scaffold for alignment of other matrix components. The proteoglycans of the GBM consist of core proteins and covalently bound glycosaminoclycans which are concentrated in the laminae rarae internae and externae. The electronegative charge of the GBM is mainly due to these polyanionic proteoglycans.141 The major proteoglycans of the GBM are heparan sulfate proteoglycans; most prominent is agrin but perlecan is also present.142,143 Proteoglycan molecules aggregate to form a meshwork that is kept highly hydrated by water molecules trapped in the interstices of the matrix. Within the GBM heparan sulfate proteoglycans may act as an anticlogging agent to prevent hydrogen bonding and adsorption of anionic plasma proteins and maintain an efficient flow of water through the membrane.

The Cells of the Glomerular Tuft Within the glomerular tuft three cell types (Figure 20.33) are found which all contact the GBM: (1) mesangial cells; (2) endothelial cells; and (3) podocytes (visceral epithelial cells). Mesangial cells, together with the mesangial matrix, establish the glomerular mesangium (Figure 20.34). Mesangial cells are quite irregular in shape, with many processes extending from the cell body towards the GBM.144,145 In these processes (to a lesser extent also in cell bodies) dense assemblies of microfilaments are found which have been shown to contain actin, myosin, and α-actinin.146 The processes of mesangial cells run towards the GBM, to which they are attached either directly or mediated by the interposition of microfibrils (see below). The GBM represents the effector structure of mesangial contractility.124,147 Mesangial
cell
GBM connections are

FIGURE 20.33 Schematic to show the arrangement of the structures in the glomerular tuft. Part of a glomerular lobule is shown with three glomerular capillaries (two are only partly shown) attached to a mesangial center. The glomerular capillary is made up of a fenestrated endothelium. The peripheral part of the endothelial tube is surrounded by the GBM which, at the mesangial angles (arrow), deviates from a pericapillary course and covers the mesangium. The interdigitated pattern of the podocyte (PO) foot processes form the external layer of the filtration barrier. Note the subcell body space (star). Podocyte foot processes are also found covering the paramesangial GBM. In the center a mesangial cell (M) is shown. Its many processes contain microfilament bundles and run towards the GBM, to which they are connected. The mesangial matrix (MM) contains an interwoven network of microfilaments. (From Venkatachalam, M. A., and Kriz, W. (1992). In “Anatomy of the Kidney. Pathology of the Kidney,” 1
92, Heptinstall, R. Little, Brown and Company, Boston, with permission.)

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FIGURE 20.34 Section through a glomerular lobule (rat). The relationships of glomerular capillaries to the mesangium in the lobule center are seen. Glomerular capillaries (C) and the glomerular mesangium occupy a common compartment enclosed by the glomerular basement membrane (GBM). The mesangial cell body (M) gives rise to several processes (some are marked by stars) which extend toward the peripherally located capillaries. Note the abundant mesangial matrix (triangles). The layer of podocytes (PO) covers the outer aspect of the GBM. Thus, neither the GBM, nor the podocyte layer encircle the capillaries completely; both together form a common surface cover around the entire lobule. Therefore, two subdomains of the GBM (as well as of the podocyte layer) can be delineated: the pericapillary (peripheral) GBM (cGBM; faced by podocytes and the endothelium); and the perimesangial GBM (mGBM) bordered by podocytes and the mesangium. The peripheral part of the capillary wall establishes the filtration barrier. Note the mesangial cell body (M) giving rise to many cell processes (some are marked by stars) which are embedded in the mesangial matrix (triangles) (US: Urinary space; TEM: 3B5500).

especially prominent alongside the capillaries. At these sites mesangial cell processes (densely stuffed with microfilament bundles) extend underneath the capillary endothelium towards the mesangial angles of the GBM where they are anchored. Generally, these processes interconnect the GBM from two opposing mesangial angles (Figure 20.35b). Functionally, the microfilament bundles bridge the entire distance between both mesangial angles. In the axial mesangial region as well, numerous microfilament bundles extending through mesangial cell bodies and processes bridge opposing parts of the GBM. The connection of mesangial cell processes to the GBM is mediated by the integrin α3β1 and the Lutheran glycoprotein, which both adhere to the laminin α5 chain.148

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The mesangial matrix fills the highly irregular spaces between the mesangial cells and the perimesangial GBM (for review see 147,149). A large number of common extracellular matrix proteins have been demonstrated within the mesangial matrix, including several types of collagen (III, IV, V, and VI), heparin sulfate proteoglycans (including the small proteoglycans biglycan and decorin),150 fibronectin, laminin, and entactin, as well as fibrillin 1 and other specific elastic fiber proteins.140,151
153 Among these components, fibronectin is the most abundant, and has been shown to be associated with microfibrils.151,154 The basic ultrastructural organization of the matrix is a network of microfibrils. In specimens prepared for TEM by routine methods a fine filamentous network is seen, which possibly corresponds to collagenous filaments. In specimens prepared by a technique that avoids osmium tetroxide and uses tannic acid for staining, the mesangial matrix is seen to contain abundant elastic microfibrils.124,155 Microfibrils are unbranched, noncollagenous tubular structures that have an indefinite length and are about 15 nm in diameter. They form a dense three-dimensional network establishing a functionally continuous medium anchoring the mesangial cells to the GBM.147,153 Distinct bundles of microfibrils may be regarded as “microtendons” that allow the transmission of contractile force of mesangial cells to specific sites of the GBM, predominantly to the mesangial angles.124,155 α-8 integrin serves as a specific matrix receptor in the mesangium.156 Glomerular endothelial cells (Figures 20.33, 20.34 and 20.35) are large flat cells consisting of a cell body (which contains all the usual cell organelles) and densely perforated peripheral parts. These regions are extremely attenuated and characterized by round to oval pores varying in diameter between 50 and 100 nm. Unlike fenestrae (unfortunately, these pores are frequently also called “fenestrae”), the pores of glomerular endothelial cells lack a diaphragm, they are virtually open26; (Figures 20.36b and 20.37b). Fenestrae bridged by diaphragms in glomerular capillaries are only found along the intraglomerular segment of the efferent arteriole and its tributaries.20 In rat, about 60% of the capillary surface is covered by the porous regions; the total area of pores occupies about 13% of the capillary surface.157 Micropinocytotic vesicles are very rare in glomerular endothelial cells, corroborating the fact that the open pores make transcytotic processes unnecessary. Glomerular endothelial cells contain the usual inventory of cytoplasmic organelles, generally located within the cell body cytoplasm. The endothelial skeleton comprises intermediate filaments and microtubules; individual pores are lined by clusters of microfilaments.158 The luminal membrane of endothelial cells is highly negatively charged, due to a cell coat that also fills the

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FIGURE 20.35 (a) Overview of a glomerular capillary (mouse). Within the mesangium, a mesangial cell (MC) is seen whose processes extend toward the peripherally located capillary (C). Microprojections (arrowheads) originating from the primary process extend toward the GBM. Note that the GBM (as well as the podocyte layer) deviates from its pericapillary course at the two mesangial angles (marked by arrows), continuing as a cover of the mesangium. Thus, the juxtamesangial part of the glomerular capillary lacks a basement membrane; at this site the endothelium is directly exposed to the mesangium. The capillary endothelium is thin and fenestrated. The podocyte layer consists of interdigitating foot processes (FP) which abut the GBM on its outside surface (TEM: 3B13,500). (b) Capillary
mesangium interface (rat). At this site a basement membrane is not developed. Beneath the endothelium, tongue-like mesangial cell processes (MP) are found which run toward both opposing mesangial angles. They contain microfilament bundles, which obviously interconnect the GBM of both mesangial angles (marked by arrows) (CL: capillary lumen; US: urinary space; MM: mesangial matrix; FP: foot processes; TEM: 3B23,000).

pores like “sieve plugs”.159 It consists of several polyanionic glycoproteins including a sialoprotein called podocalyxin, which is considered as the major surface polyanion of glomerular endothelial as well as epithelial cells.160 Endothelial cells are active participants in the processes controlling coagulation, inflammation and immune processes. Glomerular endothelial cells synthesize and release endothelin-1, endothelium-derived relaxing factor (EDRF),161 and PDGF B.162 Glomerular endothelial cells have receptors for VEGF A and angiopoetin that are produced by podocytes.163,164 The continuous stimulation of glomerular endothelial cells by podocyte-derived VEGF A has major relevance for the maintenance of glomerular capillaries and the formation of pores instead of fenestrae.165 Within the conspicuously narrow portion of the efferent arteriole (outflow segment) the endothelial cells are arranged in an eye-catching pattern: their cell bodies bulge into the lumen being longitudinally stretched, suggesting a specific shear stress receptor of glomerular capillaries.20,166 Mature podocytes are highly-differentiated cells. In the developing glomerulus at the S-shaped body stage, podocytes are a simple polygonal shape connected by

apical tight junctions. At the transition to the capillary loop stage the mitotic activity of the cells is completed, the interdigitating foot process pattern with basally located slit membranes instead of apical tight junctions is established, and the final number of podocytes is determined. In rat this point is reached soon after birth, in man it is established during prenatal life. Differentiated podocytes are unavailable for regenerative cell replication167; thus in the adult, lost podocytes cannot be replaced by division of the remaining cells. The only way to replace the function of lost podocytes is the hypertrophy of the remaining podocytes. Podocytes have a voluminous smooth surfaced cell body (Figures 20.36a and 20.37a), which floats within the urinary space; it appears to adapt in shape to the surrounding flow conditions created by the filtrate. The cells give rise to long primary processes (frequently branching another time) that extend towards the capillaries, finally splitting apart into terminal processes, called foot processes, which affix to the GBM (Figures 20.36 and 20.37a). The foot processes of neighbouring podocytes regularly interdigitate with each other, leaving meandering slits (filtration slits) between them, which are bridged by an extracellular structure,

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FIGURE 20.36 (a) Podocyte (rat). The cell body contains a large nucleus with indentations. The cytoplasm contains a well-developed Golgi apparatus (arrows), and a conspicuous lamellated inclusion body (arrowhead). The cell processes run toward the GBM forming the interdigitating pattern of foot processes (FP) there. Note the subcellbody space (stars) (C: capillary; TEM: 3 B7600). (b) Filtration barrier (rat). The peripheral part of the glomerular capillary wall comprises three layers: the endothelium with large open pores; the basement membrane (GBM); and the layer of interdigitating podocyte foot processes. The GBM consists of a lamina densa, a lamina rara interna toward the endothelium, and a lamina rara externa toward the epithelium. Note the slit diaphrams bridging the floor of the filtrations slits (arrows) (CL: capillary lumen; CB: cell body of a podocyte; TEM: 3B57,000).

the so-called slit diaphragm. Podocytes are polarized epithelial cells with a luminal and a basal cell membrane domain; the latter corresponds to the sole plates of the foot processes which are embedded into the GBM to a depth of 40 to 60 nm. The border between basal and luminal membrane is represented by the insertion of the slit diaphragm.167

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FIGURE 20.37 (a) Outer surface of glomerular capillaries (rat). Processes (P) of podocytes run from the cellbody (CB) toward the capillaries where they ultimately split into foot processes. By interdigitation, foot processes from neighbouring cells create the filtration slits (SEM: 3B3400). (b) Inner surface of a glomerular capillary (rat). The open fenestrations (not bridged by a diaphragm) are shown (SEM: 3B16,000).

The cell body contains a prominent nucleus, a welldeveloped Golgi system (Figure 20.36a), abundant rough and smooth endoplasmic reticulum, prominent lysosomes (including abundant multivesicular bodies), and many mitochondria. In contrast to the cell body, the cell processes contain only a few organelles (except from multivesicular bodies). The density of organelles in the cell body indicates a high level of anabolic, as well as catabolic, activity. In addition to the work necessary to sustain the structural integrity of these specialized cells, all components of the GBM are synthesized by podocytes.133,143 A well-developed cytoskeleton accounts for the complex shape of the cells. In the cell body and the primary processes, microtubules and intermediate filaments (vimentin, desmin) dominate, whereas

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α-actinin, and synaptopodin168,174,175; synaptopodin, a novel podocyte-specific actin-associated protein interacts with α-actinin inducing the formation of long unbranched parallel bundles of microfilaments.176 Peripherally, the actin bundles anchor in the dense cytoplasm associated with the basal cell membrane of podocytes, i.e., the sole plates of foot processes.167 Anchoring of the sole plates to the GBM is achieved by specific transmembrane receptors; two systems are so far known (Figure 20.38). First, a specific integrin heterodimer, consisting of α3β1 integrins, which bind within the GBM to collagen type IV, fibronectin, and laminin 521.177
179 Second, a dystroglycan complex connects the intracellular molecule utrophin to laminin 521, agrin, and perlecan in the GBM.180,181 Both integrins and dystroglycans are coupled via adapter molecules (paxillin, vinculin, α-actinin) to the podocyte cytoskeleton, allowing outside-in and inside-out signaling, as well as transmission of mechanical force in both directions. A major role in this issue is played by the integrin-linked kinase.182 A huge body of data has been accumulated in recent years concerning the inventory of receptors and signaling processes starting from podocytes. cGMP signaling

microfilaments are densely accumulated in the foot processes. In addition, in the cell body and the primary processes, microfilaments are seen as a thin layer underlying the cell membrane.168,169 The prominent bundles of microtubules in the large processes are associated with microtubule-associated proteins, including MAP3/MAP4 and tau.170 Moreover, like in neuronal dendrites, the microtubules of the podocyte foot processes are non-uniformly arranged with peripheral plus- and minus-end microtubules associated with the specific protein CHO1/ MKLP1.171 In addition, the large processes contain the intermediate type filament protein vimentin.168 In the foot processes a complete microfilament-based contractile apparatus is present. The microfilaments form loop-shaped bundles, with their limbs running in the longitudinal axis of the foot processes. The bends of these loops are located centrally at the transition to the primary processes, and are probably connected to the microtubules by “tau” which is concentrated at those sites.172 Tau is known from other places to mediate connections between microtubules and microfilaments.173 The microfilament bundles contain actin, myosin II,

–

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TRPC6 Podocin

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FIGURE 20.38 Glomerular filtration barrier. (Modified from Endlich, K. H., Kriz, W., and Witzgall, R. (2001). Update in podocyte biology. Curr. Opin. Nephrol. Hypertens. 10, 331
340.) Two podocyte foot processes bridged by the slit membrane, the GBM, and the porous capillary endothelium are shown. The surfaces of podocytes and of the endothelium are covered by a negatively-charged glycocalyx containing the sialoprotein podocalyxin (PC). The GBM is mainly composed of collagen IV (α3, α4, and α5), of laminin 11 (α5, β2, and γ1 chains) and the heparan sulphate proteoglycan agrin. The slit membrane represents a porous proteinaceous membrane composed of (as far as is known) Nephrin, Neph1, 2, and 3, P-cadherin, and FAT1. The actin-based cytoskeleton of the foot processes connects to both the GBM and the slit membrane. With regard to the GBM, β1/α3 integrin dimers specifically interconnect the TVP complex (talin, paxillin, vinculin) to laminin 11; the β and α dystroglycans interconnect utrophin to agrin. The slit membrane proteins are joined to the cytoskeleton via various adaptor proteins, including Podocin, Zonula occludens protein 1 (ZO-1; Z), CD2-associated protein (CD), and catenins (Cat). TRPC6 associates with podocin (and nephrin; not shown) at the slit membrane. Among the many surface receptors only the angiotensin II (ANG II) type 1 receptor (AT1) is shown (Additional abbreviations: Cas: P130Cas; Ez: ezrin; FAK: focal adhesion kinase; ILK: integrin-linked kinase; M: myosin; N: NHERF2 (Na1-H1 exchanger regulatory factor); NSCC: Non-selelctive cation channel; S: Synaptopodin). II. STRUCTURAL ORGANIZATION OF THE MAMMALIAN KIDNEY

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(stimulated by ANP, BNP, and CNP, as well as by NO), cAMP signaling (stimulated by prostaglandin E2, dopamine, isoproterenol, PTH/PTHrP), and Ca21 signaling (stimulated by a huge number of ligands including angiotensin II, acetylcholine, PGF2, AVP, ATP, endothelin, histamine) have been identified. Among the cation channels, TRPC6, a nonselective Ca21 channel, has recently received attention, since mutations in the respective gene lead to hereditary FSGS.183,184 The major target of this signaling orchestra is the cytoskeleton, the concrete effects, however, are poorly-understood. Other receptors, such as for C3b,185 TGFß,186,187 FGF2,188 and various other cytokines and chemokines have been shown to be involved in the development of podocyte diseases (for details see 189). Megalin, a multi-ligand endocytotic receptor, is associated with coated bits190
192; it represents the major antigen of rat Heymann nephritis.193 The filtration slits are the site of convective fluid flow through the visceral epithelium. They have a width of 30 to 40 nm and are bridged by the slit membrane. The structure and molecular composition of this proteinaceous membrane is insufficiently understood. Chemically fixed and tannic acid treated tissue reveals a zipper-like structure with a row of “pores” approximately 4 3 14 nm on either side of a central bar.194 According to its dimension and its components (as far as is known) the slit diaphragm may be considered as a specific adherens-like intercellular junction. Intensive research in recent years has uncovered several transmembrane proteins that participate in the formation of the slit membrane, including nephrin,195 Neph1,196 P-cadherin,197 and FAT198 (Figure 20.38). Other molecules, such as ZO1,199 Podocin,200 CD2AP,201 and catenins mediate the connection to the actin cytoskeleton (see below). Nephrin is a member of the immunoglobin superfamily (IgCAM); its gene NPHS1 has been identified as the gene whose mutations cause congenital nephritic syndrome of the Finnish type.195 In addition to its role as a structural component, nephrin acts as a signaling molecule that can activate MAP kinase cascades.202 Neph1 is considered as a ligand for nephrin. Podocin belongs to the raft associated stomatin family, whose gene NPHS2 is mutated in a subgroup of patients with autosomol-recessive stereoid-resistent nephrotic syndrome.200 These patients show disease onset in early childhood and rapid progression to end-stage renal failure. Podocin interacts with nephrin and CD2AP.203 FAT is a novel member of the cadherin superfamily, with 34 tandem cadherin-like extracellular repeats and a molecular weight of 516 kDa.204 Because FAT has a huge extracellular domain, it is speculated that it dominates the molecular structure of the slit membrane198; the FAT mutant mouse fails to develop a slit membrane.205 P-cadherin197 is thought to mediate the linkage to ß- and γ-catenin with its intracellular domain, a complex which then connects to the actin cytoskeleton via α-catenin and

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α-actinin. Taken together, many components of the slit membrane are known, but an integrative model of its substructure including all components is so far lacking. The luminal membrane and the slit diaphragm are covered by a thick surface coat which is rich in sialoglycoproteins (including podocalyxin, podoendin, and others) that are responsible for the high negative surface charge of the podocytes.206,207 Podocalyxin is anchored to the actin cytoskeleton beneath the cell membrane via the linker protein NHERF 2 (Na1/H1 exchanger regulatory factor 2) and ezrin.208,209 The surface charge of podocytes contributes to the maintenance of the interdigitating pattern of the foot processes. In response to neutralization of the surface charge by cationic substances (e.g., protamine sulfate), the foot processes retract, resulting in what is called “foot process effacement”.210

Filtration Barrier The walls of glomerular capillaries represent a specific barrier which is very permeable to water, and yet able to prevent all but very minute losses of serum albumin and other major plasma proteins from the circulation. The glomerular capillary wall consists of three distinct layers (Figures 20.36b and 20.37). Starting at the capillary lumen, there is the porous endothelium, followed by the GBM, and the layer of interdigitating foot processes with the filtration slits in between. The high hydraulic permeability of this barrier suggests that the filtrate pathway is entirely extracellular, passing through the endothelial fenestrae, across the GBM, and through the slit diaphragms of the filtration slits. According to a calculation by Drumond and Deen,211 the hydraulic resistance of the endothelium is negligible. The GBM and the filtration slits each make up roughly one half of the total hydraulic resistance of the filtration barrier. Charge, size, and shape determine the specific permeability of a macromolecule. It is now generally accepted that the charge barrier plays an important part in preventing polyanionic macromolecules such as albumin from passing through the glomerular filter. All components of the glomerular filter are heavily laden with negative charges.212 Recent investigation213,214 suggests that the negative residues of the endothelium play the major role in establishing a negative charge field which considerably decreases the entry of polyanionic macromolecules, i.e., albumin, into the filter. With regard to the size selectivity, direct experimental findings,211,215,216 as well as recent findings about the molecular composition of the slit membrane (see above) and the consequences of genetic mutations in these components, suggest that it is for the major part the slit membrane which is responsible for the size selectivity; it appears to be the main barrier for uncharged large molecules.

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There is another major unresolved problem in glomerular physiology, namely the regulation of the ultrafiltration coefficient Kf. Kf is the product of the local hydraulic permeability and the filtration area. There has been a widespread belief that Kf is regulated through changes in the filtration area due to an action of the mesangium.217 However, the structural arrangement of the mesangium,124 as well as several morphometric studies,218,219 do not support such an assumption. Dimensional changes in just the slit membrane area have also been regarded as a reasonable and, theoretically, very effective site to change Kf.220 In pathological conditions, e.g., in membranous nephropathy,221 the decrease in Kf correlates perfectly with the decrease in total slit length. With respect to acute regulatory mechanisms under physiological conditions, however, no convincing morphometric data have been published showing that changes in Kf are correlated with corresponding dimensional changes in the slit membrane. Thus, the question of where and how Kf is regulated remains an open problem.

Stability of the Glomerular Tuft The glomerular tuft is constantly exposed to comparably high intraglomerular pressures within glomerular capillaries and mesangium. The high intraglomerular pressures challenge not only the glomerular capillaries themselves, but also the folding pattern of the glomerular tuft. Increased pressures lead to loss of the folding pattern, and to dilation of the glomerular capillaries. Therefore, we have to ask what are the specific structures and mechanisms that counteract the expansile forces in the glomerular tuft. To answer this question we have to distinguish between the structures and mechanisms maintaining: (1) the folding pattern of the glomerular tuft; and those maintaining (2) the width of glomerular capillaries (Figure 20.39). The folding pattern of the glomerular tuft is primarily sustained by the mesangium.124,147,222 Mesangial cells are connected to the GBM by their contractile

processes (see above); by centripetal contractions they maintain the infoldings of the GBM, thereby allowing the capillaries to arrange in the peripheral expansion of the GBM. This supporting role of mesangial cells is best illustrated under circumstances with loss of mesangial cells, such as Thy-1 nephritis.223 Under those circumstances the folding pattern of the GBM is progressively lost, finally resulting in mesangial aneurysms. Podocytes clearly contribute to the maintenance of the folding pattern by specific cell processes that interconnect opposing parts of the GBM from outside within the niches of the infoldings. This function is again clearly illustrated in Thy-1 nephritis under circumstances with loss of mesangial support: podocytes are capable of maintaining a high degree of the GBM folding pattern for 2
4 days, after which they fail and mesangial aneurysms become prominent.223 The width of glomerular capillaries, in the long run, is probably controlled by growth processes accounting for different-sized capillaries. The width of a given capillary, in an acute situation being exposed to changes in blood pressure, appears to be stabilized by the GBM which is a strong elastic structure224 and, together with the mesangial cell bridges (see above), is capable of developing wall tension.147,225 In addition, the tensile strength of the GBM is reinforced by podocytes. Podocytes are a kind of pericyte; their foot processes represent a unique type of pericyte process which, like elsewhere in the body, counteract the dilation of the vessel. Podocyte processes are firmly attached to the underlying GBM (see above); their cytoskeletal tonus counteracts the elastic extension of the GBM. Podocytes cannot be replaced by any other cell; failure in this function will lead to capillary dilation.

Parietal Epithelium of Bowman’s Capsule The parietal layer of Bowman’s capsule consists of squamous epithelial cells resting on a basement FIGURE 20.39 Schematic to show the mechanisms that stabilize the glomerular tuft against expansion (relevant structures are highlighted in dark gray). The folding pattern of the GBM is thus stained by mesangial cells from inside, and by specific podocyte processes located in the depth between two capillaries from outside. The width of capillaries against transmural pressure gradients is maintained by wall tension, which is generated by the rigidity of the GBM, by the mesangial cell processes that interconnect opposing turning points of the GBM, and by the tonus of podocyte foot processes.

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membrane (Figure 20.29). The cells are of polygonal shape and contain prominent bundles of actin filaments running in all directions. Microfilament bundles are especially prominent in the parietal cells surrounding the vascular pole, where they are located within cytoplasmic ridges that run in a circular fashion around the glomerular entrance.20 The basement membrane of the parietal epithelium (PBM) is, at variance with the GBM, composed of several dense layers which are separated by translucent layers and contain bundles of fibrils.226 Recent studies suggest a role of type XIV collagen in the organization of the multilayered PBM.227 In contrast to the GBM, the predominant proteoglycan of the PBM is a chondroitin sulfate proteoglycan.149 The transition from the GBM to the PBM borders the glomerular entrance. This transitional region is mechanically connected to the smooth muscle cells of the afferent and efferent arterioles as well as to extraglomerular mesangial cells. At the urinary pole, the flat parietal cells transform into proximal tubule cells. In some cases the flat cells may continue for a certain distance as a so-called neck segment of the tubule (rabbit)228 or the typical proximal tubule epithelium generally starts within the glomerular capsule. This is the case in the mouse,824 most pronounced in males. In rare cases, parietal epithelial cells may be replaced by podocytes (“parietal podocytes”) which display a process pattern identical to that of podocyte proper of the tuft.229 At such sites, the PBM is similar to the GBM and capillaries may attach from outside. As shown recently, parietal podocytes are regularly found when ß-catenin is deleted in renal epithelial cells during development at the S-shaped body stage.230 Recent observations suggest that a niche of glomerular epithelial stem cells resides within the parietal epithelium at the transition to the proximal tubule.231,232 It is an intriguing hypothesis that proliferating stem cells from this locus may transform into podocytes and may reach the tuft via the transitions of the epithelia at the glomerular vascular pole. Migration of parietal cells via the vascular pole and subsequent transition into podocytes has been shown to occur in the new-born mouse.233 However, evidence that such a process may be of any relevance in the adult has so far not been presented.233

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compartment and the interstitial compartment that communicates with the blood compartment. The epithelium consists of a single layer of cells, resting on a basement membrane composed of extracellular matrix. The cells are interconnected by junctional complexes that encircle each individual cell like a belt. The tight junction (zonula occludens) separates the luminal compartment from the lateral intercellular space and is the boundary between the apical plasma membrane domain, facing the tubular fluid, and the basolateral membrane domain, which lines the intercellular compartments and is in contact with the basement membrane. The intermediate junctions (zonula adherens), and the patches of desmosomes (maculae adherentes) provide mechanical adherence. Gap junctions that provide intercellular communication exist exclusively in the proximal tubule. This basic organization of the epithelium (Figure 20.40) implies two transepithelial transport pathways for solutes and macromolecules: (1) the paracellular pathway across the tight junctions and the lateral intercellular spaces (the passage of solutes through the paracellular pathway is driven by the transepithelial electrochemical and oncotic gradients); (2) the transcellular pathway across the luminal membrane domain, the cellular cytoplasm and the basolateral membrane domain, and vice versa (the passage of solutes via the transcellular pathway occurs mostly against electrochemical gradients and is energydependent).

Luminal plasma membrane

1

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Basolateral plasma membrane Basal lamina

STRUCTURAL ORGANIZATION OF RENAL ELECTROLYTE TRANSPORTING EPITHELIA General Overview of Renal Epithelial Organization The renal tubular epithelia function as selective barriers between the tubular fluid in the luminal

FIGURE 20.40 Schematic drawing, demonstrating the essential structural features of renal transporting epithelia. (1) Paracellular route through the tight junction and the lateral intercellular spaces; (2) Transcellular route, across the apical plasma membrane, which may be augmented by short microvilli, microfolds (not shown) or long microvilli of uniform length, called “ brush border,” across the cytoplasm, and across the basolateral plasma membrane; the latter may be augmented by infoldings of the basal plasma membrane or by basolateral processes of the cells, which narrowly interdigitate with each other. The lateral interdigitating processes contain large mitochondria.

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FIGURE 20.41 Tight junction and intercellular space. (a) Freeze-fracture electron micrograph of thick ascending limb cells. (b) The tight junction (TJ) consists of several densely arranged parallel strands (BL: basolateral membrane; L: luminal membrane; Rabbit: 3B45,000). (Cooperation with A.Schiller and R.Taugner).

Paracellular Pathway As seen in freeze-fracture replicas, tight junctions are composed of globular particles, arranged in one or several roughly parallel strands or in a net-like pattern234 (Figure 20.41a). The more-or-less densely packed particles in the strands presumably represent the transmembrane proteins that participate in the junction’s formation. The tight junctions function as barriers between the luminal compartment and the lateral intercellular spaces. At the same time they allow a selective, regulated paracellular flow of small inorganic cations,235,236 and the passage of some large organic cations and of some uncharged molecules.234,237 The selectivity of tight junctions for different solutes varies among the different tubular epithelia.238,239 Claudins, a large family of integral membrane proteins, make up the bulk of tight junctional strands,240
243 and play a key role in determining and regulating the paracellular permeability for small inorganic cations.244 They act as size-, charge-, ion concentration- and pH-dependent channels or pores in the intercellular space,245
247 and seem to be targets of the serine-threonine kinases WNK1 and WNK4.248
251 The dynamic regulation of paracellular flux does not seem to involve structural changes of the tight junctional complexes. Mutations in claudin members252,253 or defects in the WNK-signaling cascades may have major implications on volume homeostasis.250,251,254
257 Occludin, another integral membrane protein, is interspersed with claudins in the tight junctional strands. The cytoplasmic domain of occludin is associated with ZO1, thereby providing a linkage for the membrane to its scaffolding actin

cytoskeleton.234,244 The interaction of occludins with the actin skeleton may be important in regulating the paracellular passage for larger molecules, and for “macropermeation” across the epithelium, as well as in the transduction of signals from apoptotic cells.258 Further, ZO1 seems to be associated with the “fence” function of tight junctions,258 i.e., the ability of the tight junction to prevent diffusion of lipids from the apical to the basolateral membrane domain. Via transcription factor zonula occludens 1 (ZO-1)-associated nucleic acid binding protein (ZONAB), it can also be involved in controlling cell proliferation.259 Cell adhesion proteins at the extracellular face of the basolateral surface of renal tubular cells maintain a basal level of cell
cell adhesion, in addition to strong cellular adhesion provided by the junctional complexes and/or desmosomes. The cell adhesion proteins in the intercellular spaces can be made visible by electronmicroscopy with specific fixation procedures.260 The “classical” cell adhesion proteins N- and Ecadherin,261
264 as well as the “atypical” kidney-specific (ksp) cadherin 16265,266 have been located by immunostainings on the basolateral membranes all along the tubular system. The classical cell adhesion molecules are linked to the scaffolding actin skeleton, as well as to β-catenin, at the cytoplasmic face of the basolateral cell membranes.267 This connection provides a pathway for coupling extracellular signals (among others, binding of a hormone to its receptor, mechanical stresses) to intracellular signaling cascades that control various cellular responses, such as endocytosis, ubiquitination of proteins, transcription, proliferation or apotosis.267 Transcellular Pathway The prerequisite for transcellular vectorial transport of solutes across epithelia is the asymmetric or polarized allocation of co-transporters, exchangers, channels, and enzymes, to the luminal and basolateral plasma membrane LUMINAL MEMBRANE DOMAIN

The uptake of most solutes into the cell is coupled to passage of sodium via solute-specific co-transporters, via channels or via exchange against protons (H1) in the luminal plasma membrane. The given assembly of transport proteins in the luminal membrane of the cells of a segment determines the segment-specific solute transport pattern. Enzymes (e.g., phosphatases, peptidases) in the luminal membrane hydrolyze poorly permeable organic compounds to readily permeable ones, and various receptor proteins (e.g., megalin, cubilin etc.268) mediate the uptake of their ligands into the cell. Many of the apical transport proteins are linked to the actin-based cytoskeleton under the plasma

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FIGURE 20.42

Augmentation of apical and basolateral plasma membrane surfaces by microvilli and interdigitating lateral cell processes. (a) Three-dimensional model of a rabbit proximal tubule cell. (From Welling, L. W., and Welling, D. J. (1975). Surface areas of brush border and lateral cell walls in the rabbit proximal nephron. Kidney Int. 8(6), 343
348 ,with permission.) The dark line indicates the position of the tight junctional belt between the apical and basolateral membrane domains; the apical membrane domain is amplified by microvilli, which form a brush border; the basolateral membrane domain is augmented by interdigitating lateral cell processes that split in an apico
basal direction to primary and secondary processes and basal plicae; the latter are anchored in the basement membrane. (b) and (c): Sections through S1 proximal tubule (Psammomys obesus); the dark contrast of the intercellular spaces (black lines), and the differential contrast of adjacent cells result from fixation with reduced osmium; (b) the section, cut approximately in parallel to the basement membrane through the center of the cell reveals the complex interdigitation of the lateral cell processes; (c) in the section in an apico
basal direction apical interdigitation by lateral processes is revealed by the different contrast in the brush border; the lateral interdigitating processes increasingly split up towards the cell base; the larger ones are filled out with mitochondria (TEM: 3B9000).

membrane via adaptor proteins containing PDZ interactive domains,269
273 namely NHERF1, 2, 3, and are thereby maintained in the specific cell membrane areas.274 The given assembly of transport proteins in the apical plasma membrane of a segment confers the specificity for transported solutes. The rate of solute permeation across the membrane critically depends on type (co-transporter, exchanger, channels) and quantity of active transport systems in the apical cell membrane domain. The latter is ultimately related to the available

surface area. Thus, in many epithelia with high transport rates the apical membrane area is much larger than the area of a virtual plane at the level of the tight junctional belt. Three modes of amplification of apical plasma membrane surface are distinguished (Figure 20.40): (1) densely arranged finger-like microvilli, all of similar dimensions, evenly distributed over the entire cell surface, forming the so-called “brush border” (Figures 20.42 and 20.44). The microvilli have an axial cytoskeleton of actin filaments, arranged in a 6 1 1

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pattern, associated with villin and fimbrin in the microvillar core.275 The actin filaments extend into the terminal web, located in the subapical cytoplasm immediately beneath the base of the microvilli. Brush border formation characterizes the proximal tubule: (2) short microvilli, found on all other tubular cells; their density and distribution on the cell surface varies considerably; (3) microfolds, found on cells in which regulation of the permeation rates for given solutes is associated with rapid transient modulation of the luminal cell surface area (subtypes of intercalated cells and occasionally also in collecting duct cells; see below).

S1 S2

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The entry of sodium-coupled solutes across the luminal membrane is driven by the enzymatic splitting of ATP by the Na-K-ATPase, the so-called sodium pump.276 In renal epithelia ATP is mainly made available by mitochondria. The Na-K-ATPase is inserted in the basolateral membrane domain of all tubular cells, and is firmly linked to the actin cytoskeleton by interacting proteins, such as ankyrin, spectrin/fodrin, and NHERF.277 Segment-specific differences in Na-K-ATPase activity/protein278,279 per unit tubular length rely on cell type-specific differences in the density of the enzyme molecules per area basolateral membrane, and on the available surface area of basolateral membrane per unit tubular length.280,281 Basically two modes of increases of the basolateral membrane surface per unit tubular length are distinguished in renal epithelia: 1. Lateral folding and interdigitation (basolateral interdigitations): this mode increases the lateral membrane area282 and implies an increase of the lateral intercellular space, the common compartment of para- and transcellular transport routes.40 The width of the lateral intercellular spaces (about 20
50 nm) varies little with function. In interdigitated epithelia the tight junctions are composed of one or several parallel strands with more-or-less high particle density. The tall lateral plasma membrane folds, equipped with Na-KATPase, narrowly enclose large mitochondria. The folds split into complex basal ridges with densely packed actin filaments (but no Na-K-ATPase283,284), arranged in a circular manner,285
287 which provide attachment to the underlying basememt membrane. This arrangement prevails in proximal (Figures 20.42, 20.43 and 20.44) and distal tubules (Figures 20.52, 20.53, 20.54, 20.56 and 20.57), and causes the characteristic basal striation of these segments in histological sections. 2. Infoldings of the basal plasma membrane into the cell body; the spaces between the infolded (Na-K-

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c S3

O S S3 d

FIGURE 20.43 Survey on location and ultrastructure of proximal tubule segments. (a) S1 segments start at the urinary pole of the renal corpuscle in the cortex, and transform gradually to S2 segments within the labyrinth, S2 segments give way to S3 at different levels (depending on the nephron generation) within the medullary rays; S3 terminates at the border (dashed line) of the outer stripe (OS) and the inner stripe. (b) Salient features of S1, S2, and S3 proximal tubule cells; neighbouring cells are shaded in order to reveal the interdigitation by lateral cell processes; the vacuolar apparatus in the subapical cytoplasm, mitochondria, ER, Golgi apparatus, lysosomes (black spots), and peroxisomes (cross-hatched) are indicated; in rat S3 segments (c) the brush border micovilli are the highest, in rabbit (d) and most other species they are the shortest. (Adapted from ref. [5], with permission).

ATPase carrying) membranes open via so-called basal slits directly towards the underlying basement membrane, and have no continuity with the lateral intercellular spaces. Consequently, trans- and paracellular solute transport pathways are largely separated. The tight junctional belt consists of networks of anastomosing strands with high particle density. The width of the intercellular spaces can be narrow or dilated, depending on the functional conditions. The lateral membranes carry small finger-like villi or folds, and are often interconnected by small desmosomes, These might help to maintain mechanical cohesion under

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FIGURE 20.44 Proximal tubule (rat). (a) Profiles of S1, S2, and S3 segments of juxtamedullary proximal tubules; note the differences in brush border length, in cell height, cytoplasmic density, and outer diameter (c: Peritubular capillaries; Rat: 1 mm Epon section; 3B1000). (b) Ultrastructure of S1, S2, and S3 proximal tubule cells (Rat). The mitochondria in S1 and S2 are located in lateral cell processes, in S3 they are mainly scattered throughout the cytoplasm; the endocytotic apparatus in the subapical cytoplasm (roughly delimited by broken lines) is most prominent in S1 and early S2; endosomes (stars) and lysosomes (L) are located deeper in the cytoplasm; in S3 the vacuolar apparatus and lysosomes are virtually absent, whereas peroxisomes (P) are more frequent than in S1 and S2; interdigitation by lateral folds is almost lacking (TEM: 3B5400).

functionally-induced dilation of the intercellular space. This epithelial organization characterizes the collecting duct system. Increases in membrane area by lateral interdigitating folds and basal infoldings may well be found in the same cell (e.g., connecting tubule cells; Figures 20.56 and 20.58).

Correlation Between Structure and Transport In defined nephron segments rather constant ratios have been found between Na-transport rates, Na-KATPase activity, basolateral plasma membrane surface area, and mitochondrial density.276,279
281,288
294 Acute changes in flow rates for a given solute across the luminal cell membrane may be sensed by local purinergic signaling and responded to by rapid adjustment in transport rates.295,296 They depend on the

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availability of active transport systems in the cell, and can be effected by several mechanisms, e.g., by gating of transport channels (e.g., ENaC in collecting duct cells), that are present in the luminal plasma membrane,277 by redistribution of the given protein between microdomains in the apical membrane (e.g., NHE3 in the brush border of proximal tubuli,297,298 by cycling of luminal and vesicular membrane domains containing the respective transport proteins (e.g., AQP2 in collecting duct cells; H1ATPase in intercalated cells) between the cell surface and intracellular vesicles299 or by exocytotic insertion and endocytotic retraction of specific transport proteins into and from, respectively, the luminal membrane (e.g., ENaC300; NaPiIIa301,302). Prolonged duration of increased transcellular flow rates stimulates, in addition to the acute responses, the transcription rates for the given transport proteins,303 and finally results in cellular and epithelial hypertrophy, including cell proliferation.304,305 Inversely, chronic decreases in Na-transport rates may result in epithelial hypotrophy, including a reduction of cell mass by apoptosis.293,305
307 On this background it is tempting to interpret the internephron heterogeneity as a reflection of their different filtration and transport rates. In rats the juxtamedullary nephrons with the largest glomeruli and highest filtration rates308 display the largest tubular diameter, the largest basolateral membrane area, Na-K-ATPase activity, and mitochondrial volume density.41 The superficial nephrons have the smallest glomeruli, filtration rates, and tubular dimensions.

Primary Single Cilia All renal cell types, except the intercalated cells, carry a central single primary cilium on their luminal surface. Primary cilia are regarded as mechanosensors that sense changes in luminal flow rate and circumferential stretch.309
312 The extracellular mechanical stimulus caused by the urinary flow is transduced via the transmembrane proteins Polycystin 1 (PC-1) and Polycystin 2 (PC-2),313 located in the membrane of the cilium.314,315 Both together form a complex required for flow-mediated calcium entry in response to the deflection of the axoneme.316 This subsequently results in release of calcium stores from the endoplasmic reticulum, possibly mediated by Polycystin 2.309,311 On the one hand, this might induce local purinergic signaling and modulate renal tubular transport317; on the other hand, the primary cilia may be involved in the functional differentiation of polarized cells,318 in the maintenance of normal tubular architecture,309 in regulation of tissue morphogenesis,319 and in gene transcription.320 Cilia seem also to help to regulate and control mTOR and temper the response of this pathway to growth factors.321 Loss-of-

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function mutations in the genes for PC-1 or PC-2 cause ciliary abnormalities322 and the autosomal dominant form of polycystic kidney disease (ADPKD).315,323

PROXIMAL TUBULE The proximal tubule takes up the glomerular filtrate and recovers the major fraction of water, sodium, and solutes by reabsorption from the tubular lumen back to the blood compartment, and it clears the blood of various organic compounds by uptake from the blood compartment and secretion into the tubular lumen.324,325 Furthermore, the epithelium removes almost completely filtered proteins from the tubular fluid by endocytosis. The proximal tubule begins at the urinary pole of the renal corpuscle, and ends at the transition to the descending thin limb of Henle’s loop which defines the border between the outer and the inner stripes. It has a convoluted part, situated in the cortical labyrinth, and a straight part (pars recta: the thick descending limb of Henle’s loop), located in the cortical medullary rays and in the outer stripe (Figure 20.43). The volume fraction of proximal tubules is about 48% in the rat cortex and about 54% in the outer stripe.41 From the collected tubular volume in the cortical labyrinth of an adult rat the convoluted proximal tubule takes a fraction of 80 to 85%.38

Morphology of Proximal Tubular Epithelium The proximal tubule is lined by cells with complex, interdigitating folding of the basolateral plasma membrane and characteristic formation of a brush border at the apical pole. The largely amplified apical and basolateral plama membrane surfaces correspond to the high transcellular solute transport rates (see “Organization of Electrolyte Transporting Epithelia”). The lateral foldings narrowly ensheath large mitochondria. At the base the foldings are split into numerous basal ridges, which are densely filled with circular running f-actin filaments, and provide the anchoring of the cell to the underlying extracellular basement membrane. The tight junctions are shallow, mostly consisting of a single strand with low particle density,275 in agreement with the low-resistance shunt pathway in parallel with a high-resistance pathway across the limiting cell membranes.326,327 The proximal tubule cells are electrically coupled by gap junctions. The subapical cytoplasm immediately under the base of the brush border microvilli is a membrane-rich region, called the “vacuolar apparatus”.268 It is the structural correlate of the early endocytotic apparatus

(Figure 20.45a) and contains intermicrovillar more or less deep infoldings (“clefts”) into the cytoplasm, small clathrin-coated vesicles, uncoated “dense apical tubules” (DAT) 70
90 nm in diameter, and large uncoated vesicles. The dimensions of the vacuolar apparatus are very variable, and depend on the rate of endocytosis (see below). The more or less abundant lysosomes, present in the center of the cells, are functionally related with the degradation of proteins. The nucleus is encircled in its equatorial plane by welldeveloped Golgi apparatus. Cisterns of rough ER are preferentially extended in parallel with the lateral cell membranes; ribosomes are abundant throughout the cytoplasm. Fenestrated cisterns of smooth ER are particularly abundant. In the terminal portions of the proximal tubule the cisterns of smooth ER contain xenobiotc-metabolizing enzymes328 which contribute to detoxification processes. The cisterns of the smooth ER often extend along the lateral membranes and narrowly enwrap mitochondria and peroxisomes.329 The latter are generally situated in the basal portions of the cells.330 The amount of lysosomes, peroxisomes, and lipid droplets in proximal tubule cells strongly varies with the functional stage of the animal, food intake, and sex hormones.331
333 The proximal tubule is subdivided into three segments, S1, S2 and S3.334,335 The subdivision is based on more-or-less gradually occurring quantitative changes along the proximal tubule. S1 cells line the initial half of the convoluted portion, and have the largest basolateral plasma membrane surface, Na-KATPase activity per unit membrane area, and mitochondrial density. They transform to S2 cells within the second half of the convoluted portion. All proximal tubule segments touching the renal capsule are S2 cella,336 and S2 cells also form the beginning of the straight part in the medullary rays. In rats and rabbits the microvilli in S2 are markedly shorter than in S1 (in rat S1: B4.5 to 4.0 μm; S2: B4.0 to 1.5 μm). S2 cells usually have very prominent lysosomes. The basolateral surface area, Na-K-ATPase activity per unit membrane area, and mitochondrial density decrease from S1 to S3. S3 cells supersede S2 cells at various levels (depending on the nephron generation) in the medullary rays34,113,268,336 and line the terminal portion of the proximal tubule. In rabbits, dog, and human the height of microvilli further decreases along S3.5,114,334,335 In mice differences in the length of the brushborder microvilli among the three segments are little apparent,113 whereas in rats the brush border microvilli of S3 are the longest from the three proximal tubule subsegments (Figure 20.44a). Lateral folding of the plasma membrane is lacking, and the S3 cells usually have a polygonal outline and a comparably small basolateral plasma membrane surface and

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FIGURE 20.45 (a) Schematic representation of receptor-mediated endocytosis in the proximal tubule, exemplified for the megalin- and cubilin-mediated uptake of three vitamin carrier protein complexes: DBP-vitamin D3, TC-vitamin B12, and RBP-retinol in renal proximal tubule. Likewise, the cubilin chaperone protein amnionless, AMN, is indicated. Following receptor-mediated endocytosis via apical coated pits, the complexes accumulate in lysosomes for degradation of the proteins, while the receptors recycle to the apical plasma membrane via dense apical tubules. Megalin mediates the uptake of cubilin and its ligands. The mechanisms for the cellular release of the vitamins remain to be clarified. (From ref. [346].) (b)
(d) Transmission electron micrographs of S1 cells of the proximal tubule of rats; (b) control; (c) 15 min and (d) 60 min after a single PTH-injection; PTH induces rapid downregulation of the sodium phosphate co-transporter NaPi-IIa in the brush border membrane by endocytotic mechanisms, associated with a transient expansion of the vacuolar apparatus (between arrows) in the subapical vesicular compartment (TEM B10,000). (Modified from ref. [356].)

Na-K-ATPase activity. Yet, the volume density of mitochondria in S3 of rat and mice is rather high. The mitochondria are scattered throughout the cytoplasm. Structural correlates for endocytosis and lysosomes are almost absent in S3, whereas amount and size of peroxisomes increases from S2 towards S3.

Functional Aspects Structural Correlate for Receptor-Mediated Endocytosis Receptor-mediated endocytosis (Figure 20.45a) is the most efficient mechanism for cellular uptake of filtered proteins268,337,338 and plays an important role in the acute downregulation of transport rates by selective retraction of transport proteins from the microvillous membrane rapid (e.g., NaPiIIa, Figures 20.45b,c,d; see below). By multiphoton microscopy the passage of

proteins across the different endocytotic compartments has been directly observed in vivo.339,340 The first requirement for cellular uptake of a protein by endocytosis is binding of a ligand to a receptor protein on the surface of the tubular cell. The multireceptors megalin, cubilin, and amnionless341,342 have all been located in the proximal tubule, mainly on the base of the microvillous plasma membrane, in the intermicrovillar membrane invaginations (“clefts”), and in subapical clathrin-coated pits. Megalin belongs to the LDL-receptor family, and is bound with its cytoplasmic tail to cytoplasmic adaptor proteins.343,344 It forms a tandem with the peripheral protein cubilin which is associated with the membrane by amnionless.345,346 Megalin is responsible for the internalization of its own ligands and of cubilin with its ligands.338 The receptor
ligand complexes are gathered in the clathrin-coated membrane pits, and are directed by clathrin-coated vesicles to larger, uncoated early and late

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endosomes, located slightly deeper in the cytoplasm. In the endosomes the receptors are cleaved from the ligands and travel back to the luminal membrane via uncoated “dense apical tubules” (DAT).347
350 The DAT form an elaborate, moving dynamic network of anastomosing tubules,348,351 which are transiently connected to the larger endosomes, and which display at their other end small clathrin-coated domains.352
354 From the endosomes the ligands are sorted either for degradation to lysosomes or for ubiquitination via the proteasome pathway. The trafficking of internalized material from the vacuolar apparatus to lysosomes critically depends on the microtubular system.355 Microtubules normally form a loose network across the proximal tubule cells, and become highly oriented in the apico
basal direction during vesicular transport of endocytosed cargo to lysosomes.356 The dimensions of the vacuolar apparatus and the abundance of megalin in proximal tubule cells are correlated with the rate of endocytosis. If endocytosis does not take place, either due to paucity of ligands in the tubular fluid (e.g., normally in S3) or due to lack or low levels of the endocytosis receptor,338,357,358 the vacuolar apparatus is barely developed. The processing of material in the vesicular compartments of the endocytotic pathway relies on acidification. NHE3, the proton-ATPase, and the chloride channel ClC-5 (for a review see 359), are all highly expressed and co-localized in the intermicrovillous clefts and in the vesicular membranes of the early endocytotic pathway.360
362 Dysfunction of one or several of these acidifying proteins may cause primary defects in endocytosis. Knockout of the ClC-5 channel, for instance, impairs the clearance of PTH from the tubular fluid, bringing about hyperphosphaturia and hypercalciuria.361,362 This mechanism can explain the high incidence of kidney stones in Dent’s disease, with functionally impaired ClC-5 channels.363
367 The role of basolateral endocytosis is interesting, since the basolateral cell membrane is the site of different hormone receptors,368 e.g., the insulin receptor. After binding to the receptors peptide hormones seem to be, at least in part, taken up by the cells and are transported to the lysosomes.369 Sodium Proton Exchange Apical Na1-H1 exchange in the proximal tubule and the reabsorption of the bulk of filtered sodium is mediated by the sodium/hydrogen exchanger NHE3 in the microvillous plasma membrane361,370
372 and in the plasma membrane of the intermicrovillous invaginations.297 The sodium/hydrogen exchanger is enriched in the intermicrovillar microdomain,370 where it interacts with the scavenger receptor megalin (see above, 298). Changes in Na/H exchange activity

correlate with changes in cell surface expression of NHE-3, mediated by sgk2.373 Rapid and reversible redistribution of NHE3 between the two microdomains in the microvillous plasma membrane domain and the intervillous plasma membrane invaginations (“clefts”) may also alter the surface expression of NHE3 and activity of Na/H exchange.298,374
377 Reabsorption of Water and Solutes The plasma membrane of the microvilli is covered by a glycocalyx containing hydrolases (phosphatases, peptidases, nucleotidases) which cleave their substrates in the tubular fluid (ecto-enzymes). The microvillous membrane holds a large variety of transport proteins for uptake of water and solutes from the tubular fluid. The density of a given transport protein in the microvillous membrane can be dissimilar along the segments of the proximal tubule and among nephron generations. Many of the transport proteins are anchored by adaptor proteins, such as PDZ-proteins and NHERF1/2, to the underlying apical scaffold.372,373,378,379 Transcellular water reabsorption in the proximal tubule is mediated by the constitutive water channel, aquaporin 1 (AQP1) located in the microvillous- and basolateral plasma membrane domains.380
384 Orthogonal arrays of intramembrane particles in the basolateral membranes of S3 of mice385 are associated with another water channel, AQP4, AQP7, which is probably involved in the reabsorption of glycerol (see review in 386) and is expressed in the brush border, especially of S3 in rats and mice, as shown by immunocytochemistry.387,388 Sodium-coupled solute uptake from the lumen into the cells is mediated by co-transport proteins located in the plasma membrane of the microvilli. The proximal tubule usually recovers all filtered glucose. The sodium
glucose co-transporter SGLT2 is found primarily in S1, and is responsible for 90% of glucose reabsorption. SLGT1 is located in S3, and is responsible for only 10% of reabsorption (reviewed by Hediger and Rhoads389,390). SGLT1 is more highly expressed in females than in males.391 The exit of glucose across the basolateral plasma membrane occurs by the glucose transporters GLUT2 (low affinity in S1) and GLUT1(high affinity in S3).392 Inorganic phosphate (Pi) transport is mediated by at least three different brush border Na1/P(i) co-transporter proteins, the electrogenic transporter NaPi IIa, Pit-2, and the electroneutral transporter NaPi Iic.393,394 Their expressions and activities appear to be tightly regulated. Low dietary intake of Pi increases mRNA and brush border expression of NaPi IIa.395 High dietary Pi intake, parathyroid hormone (PTH) and activation of dopamine receptors358 rapidly downregulate NaPiIIamRNA395 and NaPiIIa in the brush border,396
398 and induce phosphaturia. Downregulation of NaPiIIa in

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the brush border involves receptor-mediated endocytosis (see above) and subsequent lysosomal degradation.355,356,358,399,400 The passage of NaPi-IIa across the successive endocytotic compartments namely, the megalin-containing clefts, the clathrin-coated-vesicle compartment,401 through the early and late endosomal compartment, and finally its disposal in lysosomes, where NaPi-IIa is degraded, has been tracked by immunofluorescence.301 The shifting of the protein through the early endocytotic compartments goes along with a dramatic, rapidly transient expansion and remodeling of the vacuolar apparatus in the subapical compartment356 (Figure 20.45). PTH also reduces Pit-2 expression and activity, whereas NaPi-IIc is inhibited and internalized with a delay of several hours after PTH application.394 Recently Klotho has been recognized as a phosphatonin, and an important regulator of phosphate homeostasis. In partnership with the FGF-R, Klotho functions as an obligate co-receptor for FGF23.402 Secreted soluble Klotho inhibits Pi transport by altering the trafficking of the proximal tubule Na-coupled phosphate transporter.402 Neutral amino acids, which represent about 80% of circulating amino acids, are transported by the low affinity Na1-co-transporter B(0)AT1, located in the early proximal tubule. The high affinity transporter B (0)AT3 is located in the late proximal tubule, at least in mice. In addition, there are several other apical and basolateral amino acid transporters (for a recent review see 403). Similarly the short-chain peptide, di-, and tripeptide carriers PEPT1, high capacity, low affinity, and PEPT2 low capacity, high affinity are located in mainly S1 and S3, respectively.404,405 Secretion of organic amphiphilic electrolytes from the blood into the tubular fluid is a pathway for clearance and detoxification of xenobiotics and drugs, including diuretics.325,406
410 The uptake into the proximal tubule epithelium proceeds via multispecific organic anion transporters (OAT) and organic cation transporters (OCT) in the basolateral membrane domain. The majority of members of the OAT- and OCT-family have been immunolocalized to the basolateral cell membrane of S3 proximal tubule,411
413 yet OAT 1 has been detected mainly in S2,414 a few also in S1. The expression of the OATs and OCTs is strongly regulated by sex hormones.415
420 The export into the tubular lumen of both conjugated and unconjugated lipophilic anionic substrates involves various OATs and primarily active transporters with ATP-binding casette motifs, belonging to the MRP-family,421 and located in the brush border membrane of S1, S2, and S3 proximal tubule segments.421,422

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The role of basolateral endocytosis is interesting, since the basolateral cell membrane is the site of different hormone receptors,368 e.g., the insulin receptor. After binding to the receptors peptide hormones seem to be, at least in part, taken up by the cells and are transported to the lysosomes.369

THIN LIMBS OF HENLE’S LOOP (INTERMEDIATE TUBULE) The intermediate tubule comprises the thin tubular portions, interposed between the proximal and the distal tubules (Figure 20.46). Ultrastructurally, the intermediate tubule has four structurally different segments: (1) the descending thin limbs of short loops (SDTL); (2) the upper part; (3) the lower part of descending thin limbs of long loops (LDTLup and LDTL lp); and (4) the ascending thin limbs (ATL). This pattern has been observed in various species, including rat,423,424 mouse,34,425 golden hamster,426 rabbit,5,427 Perognathus,428 Octodon degus,429 Meriones shawii,116 and Psammomys obesus.430
432 An additional subsegment of thin limbs has been identified in Chinchilla.433 The thin limbs in human have so far not been studied in comparable completeness.434
436 Surprisingly, by lightmicroscopy these simple-looking epithelia are strikingly different from each other with respect to ultrastructure and function, not only the ascending from the descending limbs but, most remarkably, the descending limbs of short from those of long loops. Furthermore, within the descending segments (SDTL, LDTLup, LDTLlp) the proximal portion, although structurally no different from the distal portion, displays considerable functional differences. In the IM a high percentage of thin limbs was found that consisted of a patchwork of descending and ascending type epithelia.437,438 Beyond all these heterogeneities, there are prominent differences among species. This complex situation appears to account for the persistent discussion about the integrated function of thin limbs in the urine concentrating process. The type I epithelium (Figure 20.47), which is characteristic for descending thin limbs of short loops (SDTL), has a simple and uniform organization. It is composed of flat, non-interdigitating cells reposing on a thin basement membrane. The luminal cell membrane bears only a few short microvilli that are mainly found along the cell borders. The tight junction consists of several anastomosing junctional strands; desmosomes are frequently encountered. The SDTLs in the rat have, among all other thin limb segments, a particularly prominent cytoskeleton with a high content of cytokeratins and desmoplakins.439 Cell organelles, such as mitochondria, profiles of rough and smooth

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Short Loop

Long Loop

2

1

3

4

1

Complex 2

Simple

FIGURE 20.47 Thin descending limbs of short loops. (a) Crosssectional profile (rat; TEM: 3B4100). (b) The simplicity of the epithelium is demonstrated (J: Junctional complex; Rat: TEM: 3 B11,000). (c) Freeze-fracture electron micrograph. The tight junction consists of several anastomosing strands (L: luminal membrane; BL: basolateral membrane; D: desmosome; Rabbit: 3B71,000).

3

4

FIGURE 20.46

Survey of thin limb ultrastructure. Four thin limb segments are discernible: (1) Descending thin limb of short loops. (2) Descending thin limbs of long loops, upper part. This segment is differently developed among species: a complex type (upper panel) found e.g., in rat, mouse, and Psammomys is distinguished from a simple type (lower panel) found e.g., in rabbit and guinea pig. (3) Descending thin limb of long loops, lower part. The transition between upper and lower parts is gradual. (4) Ascending thin limb. (Adapted from Kriz, W., and Schiller, A. et al. (1980). In “Comparative and Functional Aspects of Thin Loop Limb Ultrastructure. Functional Ultrastructure of the Kidney,” 239
250, Maunsbach, A. B. Academic Press, London, and Kaissling, B., and Kriz, W. (1992). In “Morphology of the Loop of Henle, Distal Tubule and Collecting Duct. Handbook of Physiology: Section on Renal Physiology,” 109
167, Windhager, E. E. Oxford University Press, New York, with permission).

endoplasmic reticulum, etc., are exceedingly sparse in type I epithelium. Apart from an initial stretch of maximally 400 μm in about 10% of SDTLs in mouse, these nephron segments do not show any labeling for aquaporin 1 (AQP1), thus they are rather impermeable to water.440 The lower parts of SDTLs contain the urea transporter UT-A2 in its cell membranes.382,441,442 In species with complex vascular bundles (e.g., rat, mouse), the short descending limbs lie within the vascular bundles115; in these surroundings, the thin limbs are in an ideal position to recycle urea from the ascending vasa recta into the short loop nephrons (see below).

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This simple type I epithelium is also found in many descending loop profiles in the inner stripe of feline and canine kidneys which, by microanatomical definition, possess only “long” loops. Consequently, it may be assumed that these simple profiles belong to those long loops that descend into the inner medulla for only very short distances, frequently less than 500 μm.443 The epithelial characteristics of these loops may be more important than their short descent into the inner zone for determining their functional role. From this point of view, “short loops” are also present in the cat kidney. The short descending thin limbs of cortical loops
studied in the minipig444 and guinea pig (unpublished results from our laboratory)
are also established by the simple type 1 epithelium. The descending thin limbs of long loops are generally much larger in diameter, and have a thicker epithelium than those of short loops. Moreover, the LDTLs are heterogeneous; obviously, those of the “longest” long loops begin in the inner stripe as a much thicker tubule than those of “shorter” long loops. The character of the epithelium gradually changes as the limbs descend toward and into the inner medulla. The subdivision of these thin limbs into an upper part (type 2 epithelium) (Figure 20.48a) and a lower part (type 3 epithelium) (Figure 20.48b) is an approximation, and reflects the gradual change to a more and more structurally simplified epithelium. Moreover, this process of epithelial simplification appears to be individually related to the length of each loop. It occurs earlier and more quickly in “short” long loops and is delayed in the longest of the long loops.34,260,424,432,445 This explains the heterogeneity among descending thin

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limb profiles in a given cross-section through the medulla: profiles lined with the lower-part epithelium (type 3) may already be found at the end of the inner stripe. Even deep in the inner medulla, profiles with the upper part epithelium (type 2; in reduced elaboration) are still present. Furthermore, considerable interspecies differences, concerning in particular the upper parts of LDTLs, complicate understanding of the long descending thin limbs. Two patterns may be distinguished260; in one group of species (mouse, rat, golden hamster, Perognathus, Psammomys, O. degus, cat), the epithelium (type 2) of the LDTLup is characterized by an extremely high degree of cellular interdigitation. In a single cross-section, more than 100 cell processes may be encountered (Figures 20.48a and 20.49a,b). The tight junctions are extremely shallow, usually consisting of one junctional strand. Thus, the most characteristic features of this epithelium are the prominent paracellular pathways. The junctions are “leaky,” and the amount of junctional area available per unit area of epithelial surface is increased several-fold by the tortuosity of the junction due to cellular interdigitation. The lateral cellular spaces form an elaborate “labyrinth,” bordered by correspondingly amplified basolateral membranes. Additional structural characteristics of the epithelium are numerous apical microvilli, considerable numbers of mitochondria, and a strikingly high density of uniform intramembranous particles in the luminal and basolateral membrane. In addition, cytochemical and immunohistochemical studies have revealed that the LDTLup exhibits a sodium
potassium ATPase in both membranes,446,447 suggesting active salt secretion. In addition, salt transport may occur through the tight

FIGURE 20.48 Descending thin limbs of long loops, upper part. (a) Complex type; note the many tight junctions (arrows) (Psammomys: TEM: 3B3000). (b) Simple type; only three junctions are encountered (arrows) (rabbit: TEM: 3B3000).

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FIGURE 20.49 Descending thin limbs of long loops, upper part. (a) The complex epithelium is characterized by numerous tight junctions (arrows) indicating the extensive intercellular digitation. The interdigitation of the basolateral membrane forms a “labyrinth” of extracellular spaces within the cell body (*) (rat: TEM: 3B11,000). (b) Freeze-fracture electron micrograph exhibiting the luminal aspect of the complex epithelium demonstrating the extensive cellular interdigitation. The tight junction consists of one strand only (arrow) (L: lumen of the tubule; Rat: TEM: 3 B13,000). (From Kriz, W., and Schiller, A. et al. (1980). In “Comparative and Functional Aspects of Thin Loop Limb Ultrastructure. Functional Ultrastructure of the Kidney,” 239
250, Maunsbach, A. B. Academic Press, London, with permission.) (c) Freeze-fracture electron micrograph of the simple type epithelium. The tight junction (T) consists of several junctional strands. Note the dense pattern of intramembrane particles on the P face of luminal (L) and basolateral (BL) membranes (an equally dense particle pattern is also found in the complex type) (L: Tubular lumen; Rabbit: 3 B66,000). (From Schiller, A., and Taugner, R. et al. (1980). The thin limbs of Henle’s loop in the rabbit. A freeze fracture study. Cell Tissue Res. 207(2), 249
265, with permission.)

junctions, which contain claudin 2.261,448 LDTLups are highly permeable to water due to the abundancy of the constitutive water channel aquaporin 1 (AQD1) in both membranes,449 probably correlating with the high density of intramembrane particles. Carbonic anhydrase activity was found in both short and long descending thin limbs.450 In a second group of species that includes rabbit,427,451 minipig444 and guinea pig (unpublished data from our laboratory), the upper parts of LDTLs are more simply organized. The prominent paracellular pathway typical of the first group is lacking. The epithelial cells in this group do not interdigitate, and are joined by much deeper tight junctions consisting of several anastomosing junctional strands

(Figures 20.48b and 20.49c). In other respects, however, the epithelia are similar in the two groups. Numerous luminal microvilli, many mitochondria, and the dense assembly of intramembrane particles in luminal and basolateral membranes are present in type 2 epithelium also in this group. The high density of intramembrane particles may be partially due to the high density of aquaporin 1 (AQP1) channels in both membranes; corresponding to the decrease of particle density along its descending course the density of AQP1 channels decreases.452
454 The epithelium of the lower part of LDTL (type 3 epithelium) is comparably simple (Figure 20.50); interspecies differences are no longer prominent. The epithelium consists of relatively flat, noninterdigitating

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THIN LIMBS OF HENLE’S LOOP (INTERMEDIATE TUBULE)

FIGURE 20.50 Descending thin limbs of long loops, lower part. (a) Cross-sectional profile (rat: TEM: 3B3800). (b) The epithelium is simply organized; basal infoldings (arrow) are regularly encountered (J: Junctional complex; Rabbit: TEM: 3B10,200). (c) Freeze-fracture electron micrograph demonstrating the regular pattern of basal infoldings within the basal cell membrane (*) (Psammomys: 3B12,800). (In cooperation with A. Schiller and R. Taugner.)

cells bearing some sparse microvilli; in the rat it is covered by an unusually thick surface coat.424 The tight junctions are of an intermediate apicobasal depth, composed of several junctional strand (in rabbit: 138 6 37 nm and 3.5 6 0.7 strands; in Psammomys, 51 6 28 nm).260 The basolateral membrane regularly forms basal infoldings, similar to those found in the simple type of LDTLup.260 The fluid spaces between the infoldings are not continuous with the lateral intercellular spaces, and thus they are not part of a paracellular pathway route. The pattern and density of intramembrane particles in the luminal and basolateral membranes are inconspicuous; the dense packing,

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typical for the upper parts, has disappeared. This appears to correlate with the decrease in density of AQP1 channels that, in the terminal portions of this segment, may completely disappear. Thus, the water permeability probably decreases toward the loop bend452,453; the terminal segment may accordingly be thought to have a very low water permeability. Regarding the permeability to urea and the distribution of the urea transporter UT-A, conflicting data are published, especially when comparing data from different species.438,442,452,455
457 In the mouse in antidiuretic conditions, the UTA2 urea transporter is upregulated in LDTLep.458 Also, claudin 8 has been found in this segment.240 With respect to the descending thin limbs of the human kidney, the published data do not allow a final conclusion. In an older TEM investigation459 a thin limb profile is shown with a heavily interdigitated epithelium corresponding to the thin limb epithelium described above as the complex type in other species. However, in the text the descending thin limbs in the human kidney are described as being outlined by a simply structured epithelium. In 1967, when this paper was published, it was not yet known that there were four different thin limb epithelia. The axial, the internephron, as well as the interspecies differences in descending thin limb epithelia, is surprisingly prominent compared to all other nephron segments. Differences among thin limb segments were also found with respect to the cholesterol content of their cell membranes.460 Binding studies with various lectins have revealed distinct labeling patterns in the descending, as well as the ascending, thin limbs in rat and rabbit.461
463 The ascending thin limb is present only in long loop nephrons, and is uniformly organized among mammals (Figure 20.51). Generally, the transition from the type 3 epithelium of the descending limb to the type 4 epithelium of the ascending limb occurs a short, but fairly constant, distance before the bend (“pre-bend segment34,120,121,431). Therefore, functionally, the entire bend should be regarded as part of the ascending thin limb. The type 4 epithelium is characterized by very flat but heavily interdigitating cells joined by shallow tight junctions, consisting of only one prominent junctional strand. This leaky organization of the paracellular pathways corresponds with functional studies,464,465 which all demonstrate that the ascending thin limbs are highly permeable for ions. The change from the type 3 epithelium to the type 4 epithelium coincides with the disappearance of the urea transporter UT-A 2, and the abrupt beginning of the expression of the chloride channel ClC-K1437,452,466; aquaporins are completely lacking. Thus, the ascending thin limb is water- and urea-impermeable, but highly

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the canine467 and human kidney,459 a gradual transition between the thin and thick ascending parts of the limb has been observed.

THICK ASCENDING LIMB OF HENLE’S LOOP

FIGURE 20.51 Ascending thin limbs. (a) Cross-sectional profile; note the many junctions (arrows) (Psammomys: TEM: 3B3500). (b) Epithelium exhibiting extensive intercellular interdigitation; numerous tight junctions are encountered (arrows) (C: Capillary; Golden hamster: TEM: 3B13,500). (c) Freeze-fracture electron micrograph. Luminal aspect of the tubule demonstrating the mode of cellular interdigitation and the shallow tight junction (arrow) (L: luminal membrane; BL: basolateral membrane; Psammomys: 3B4800). (From Kriz, W., and Schiller, A. et al. (1981). Freeze-fracture studies on the thin limbs of Henle’s loop in Psammomys obesus. Am. J. Anat. 162(1), 23
33, with permission.)

permeable for Cl2 and also Na1. The relevance of the expression of claudin 4261 is poorly-understood. Surprisingly, in mouse, rat, and rabbit a high fraction of “mixed” thin limbs was found consisting of alternating stretches of descending and ascending type epithelia. In most species the transition from the ascending thin limb to the thick ascending limb (distal straight tubule) is abrupt over the length of one cell. The level of this transition defines the border between the inner medulla and the inner stripe of the outer medulla. In

The Thick Ascending Limb (TAL 5 distal straight tubule
DST) absorbs NaCl in excess of water.468 The subtraction of salt from the tubular fluid contributes to rendering the surrounding interstitium hypertonic, a crucial prerequisite for the urinary concentration process. The tubular fluid delivered by the segment into the cortex becomes progressively diluted. In addition, the TAL plays a prominent role in acid
base homeostasis, and recovers important fractions of filtered Mg21 and Ca21 via the paracellular transport route. The beginning and end of the TAL epithelium are sharply demarcated from the preceding thin limb epithelium, and the successive DCT epithelium. The TAL of nephrons with long loops begins at the border between inner and outer medulla, and that of nephrons with short loops at various levels within the inner stripe of the outer medulla (in cortical loops even in the medullary rays in the cortex). It ascends through the outer medulla and the cortical medullary rays, enters the cortical labyrinth for a short distance, and contacts with the “macula densa,” the vascular pole of its parent glomerulus (Figure 20.52). After a short “post-macula” segment the TAL transforms to the distal convoluted tubule (DCT). The length of the post-macula segment varies not only among species (, 500 μm in rabbits), but also among nephrons within the same kidney.5,37,260 An association of its length with nephron types has not been established. Long-looped nephrons have a thinner epithelium than nephrons with short loops.5,308,469 The thickness of the epithelium decreases gradually, although considerably in the flow direction along the segment (Figures 20.52 and 20.53).308 The organization of the TAL-epithelium is exemplary for electrolyte transporting epithelia (see above: “Organization of Electrolyte Transporting Epithelia”) (Figure 20.52). The cells display prominent lateral membrane foldings (Figure 20.22a)5,470 which narrowly interdigitate with adjacent cells and enclose plate-like large mitochondria, and occasionally a few cisterns of rough endoplasmic reticulum (rER)470 (Figure 20.53). Except in the deep inner stripe the lateral folding extends over the entire cell height. Hence, the luminal outline and tight junctional belt are much longer in upstream portions than in the deep inner stripe,471 evident by the very frequent hits of the tortuous tight junctional belt in sections of cortical TAL

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THICK ASCENDING LIMB OF HENLE’S LOOP

FIGURE 20.52 Survey on location and ultrastructure of the thick ascending limb of Henle’s loop. (TAL: distal straight tubule, including macula densa; C: cortex; IS: inner stripe; OS: outer stripe; IZ: inner zone.) The direction of the urinary flow is indicated by white arrows, interdigitated cells with large mitochondria, enclosed in the lateral processes; (a) medullary part; (b) cortical part; (c) macula densa; note the difference in the organization of the lateral intercellular spaces between macula densa cells and other TAL cells. (Adapted from Kaissling, B., and Kriz, W. (1979). Structural analysis of the rabbit kidney. Adv. Anat. Embryol. Cell Biol. 56, 1
123, and Kaissling, B., and Kriz, W. (1992). In “Morphology of the Loop of Henle, Distal Tubule and Collecting Duct. Handbook of Physiology: Section on Renal Physiology,” 109
167, Windhager, E. E. Oxford University Press, New York, with permission).

C

C

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epithelium (Figure 20.53b). The tight junction is organized by a few strands, arranged in parallel, and with a high particle density (Figure 20.41a). The large nucleus usually (except in the deep inner stripe) spans the entire cell height. The cytoplasm in the nuclear region displays small, round mitochondrial profiles, a particularly extensive Golgi apparatus,308 polyribosomes, and some short cisterns of rER. The varying amounts of narrow tubular profiles and smooth vesicles in the subapical cytoplasm might be related with trafficking of apical transport proteins (see below). The apical membrane of the cells carries short stubby microvilli,114,472 which usually border the tight junctional belt471 and are less abundant in the center of the cell and in the vicinity of the single cilium. Scanning electron microscopy revealed that “rough” cells with numerous microvilli may be present side-by-side with rather smooth cells with only a few microvilli.469 The latter cells display strong immunoreactivity for EGF, which seems to play a role in the regulation of growth and differentiation of cells in the loop of Henle.473

Role of the TAL in NaCl Reabsorption The major fraction of salt reabsorption by the TAL (including the macula densa cells) proceeds via the

Na1,K1,2Cl2 (NKCC2) symporter468 in the luminal membrane474
476 which is specifically inhibited by loop diuretics, such as bumetanide and furosemide.477 The apical entry of Na1,K1 and 2Cl2 is driven by the Na-K-ATPase in the basolateral plasma membrane. The density of Na-K-ATPase in the TAL exceeds by far that of more proximal tubular sites.278,279 The inwardly rectifying renal outer medullary K channel, ROMK, which recycles the K1 ions entering the cell via NKCC2 over the apical membrane, is particularly abundant in the apical plasma membrane of TAL cells.365,478
483 The basolateral extrusion of Cl2 occurs passively through ClC-K and ClC-Kb channels.479,484

Role in Bicarbonate Reabsorption In addition to its role in salt reabsorption, the TAL has also an important function in maintaining acid
base homeostasis. It reabsorbs about 15
20% of the filtered bicarbonate485
488 via the sodium/hydrogen exchangers NHE3 and NHE2 in the luminal plasma membrane of the TAL cells370,489
491 and the NEMsensitive vacuolar H1-ATPase.492 The apical Na/H exchange is tightly coupled with the basolateral Cl2/ HCO32 exchange that proceeds by the Cl2/HCO32

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FIGURE 20.53 Thick ascending limb cells. (a) Deep level of the inner stripe; the lateral interdigitated foldings contain large mitochondria and do not reach up to the lumen; (b) Cortical part; in the much lower cells the lateral interdigitated foldings reach up to the lumen, causing a folded course of the tight junctions (arrows) (rat: TEM: 3 B14,500). (Adapted from and Kaissling, B., and Kriz, W. (1992). In “Morphology of the Loop of Henle, Distal Tubule and Collecting Duct. Handbook of Physiology: Section on Renal Physiology,” 109
167, Windhager, E. E. Oxford University Press, New York, with permission).

exchanger AE2.493,494 The abundance of the NHE3 protein in the apical plasma membrane of the TAL has been shown to increase with functional adaptation to reduced renal mass,487 under metabolic acidosis,495 and with high levels of glucocorticoids.496

Role in Mg21 and Ca21 Recovery Although virtually impermeable to water, the junctions in the TAL display a selective permeability for Mg21 and Ca21. 40
70% of the filtered Mg21 is recovered by the TAL in a passive paracellular manner facilitated by tight junction proteins claudin-16 and claudin-19 protein.252,497The much greater length of the tight junctional belt in the CTAL than in the MTAL might explain the higher paracellular movement of Mg21 and Ca21 in the CTAL than in the MTAL.497,498 Impaired function of paracellin 1 (claudin 16) leads to urinary losses, specifically of magnesium and calcium.499
506 Regulation of salt transport rates in the TAL involves peptide hormones, among them vasopressin and glucagons, which bind to receptors on the

basolateral plasma membranes. Via the cAMP messenger system they increase the abundance of the NKCC2 co-transporter and the K-channel in the luminal membrane.106,117,474,468,507
512 The acute response to decreases or increases of cAMP seem to involve endocytotic and exocytotic, respectively, membrane translocation.513 The various amounts of NKCC2-displaying vesicular and tubular structures in the apical cytoplasm of TAL cells270,510,514 might well be part of the membrane pool, available for translocation. Similar to the proximal tubule, endocytosis requires NHE3-mediated acidification, and needs the chloride channel CLC5. NHE3 and ClC5 are both located in the apical cytoplasm of TAL cells.480,515 The basolateral extrusion of Cl occurs passively through ClC-K and ClC-Kb channels.479 The trafficking of ClC-K to the basolateral membrane depends on the protein barttin.479 Cyclooxygenase 2 (COX2) has been located in the TAL cells, including the macula densa cells, and contributes through local production of prostaglandins479,516
518 to the handling of ions by the TAL. All maneuvers that chronically affect the salt transport rates by the TAL finally result in structural hyperor hypotrophy of the TAL epithelium. The plasticity of the TAL epithelium in response to variations in plasma levels of vasopressin (ADH) or cAMP had been revealed by studies on Brattleboro rats, which genetically lack ADH and suffer from diabetes insipidus (DI).519 In these rats the medullary and cortical portion of the TAL are equally thin.520 In healthy rats with chronic low plasma levels of ADH due to chronic high water intake the structural appearance of the TAL resembles that seen in DI-rats.521 Several weeks of substitution of ADH in DI rats or of endogeneously increased ADH-levels, associated with chronic water restriction, restore the normal axial heterogeneity.522,523 The transport rates by the TAL epithelium are correlated with the DNA synthesis rate of TAL cells. Specific inhibition of NaCl reabsorption in the TAL of rats by furosemide transiently reduces the incidence of TAL cells showing DNA synthesis (assessed by nuclear detection of the proliferating cell nuclear antigen, PCNA, and incorporation of the thymidine analog bromodesoxyuridine) from about 1%, the basal rate in the rat TAL epithelium, to zero.305 With the given background it is likely that the structural heterogeneity regarding cell height, mitochondrial density, and basolateral plasma membrane surface along the TAL represents the physiologically lower tubular salt load, and ensuing lower transport rates in the cortical than in the medullary portions.468 In rabbits the overall reduction of the cell height, membrane area and mitochondrial volume (per unit tubular

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length) along the TAL is much more pronounced than in rats and mice. Disruption of genes coding for NKCC2 (SLC12A1) or for one or several proteins and/or channels associated with the NaCl-transport via NKCC2 (e.g., chloride channels ClCKA, ClCkB or the Barttin subunit, CLC5, NHE3), involved in regulation of its surface expression, or of the respective signaling cascades causes more or less severe renal salt-wasting, characteristic of “Bartter” syndrome. Symptoms of the Bartter syndrome are, e.g., lowered blood pressure, hypokalemic metabolic alkalosis, and hypercalciuria, with variable risk of kidney stones.479,483
484,524
527 The Tamm-Horsfall glycoprotein (THP), the most abundant urinary protein in mammals, is synthesized exclusively by the renal TAL epithelium. It is located in high density on the apical plasma membrane, in low density also on the basolateral plasma membrane.528,529 Uromodulin has been linked to water
electrolyte balance and to kidney innate immunity.530 THP is thought to be relevant in the pathogenesis of cast nephropathy and urolithiasis. By its property to compete efficiently with urothelial cell receptors, such as uroplakins, in adhering to type I fimbriated Escherichia coli, it may play a role in defense against urinary tract infection.531 Mutations in the gene encoding uromodulin lead to rare autosomal dominant diseases, collectively referred to as uromodulin-associated kidney diseases.532 Recently, it has been shown that THP-deficient (THP2/2) mice showed moderately impaired urinary concentrating abilityuromodulin plays a permissive role in TAL reabsorptive fu/uromodulin plays a permissive role in TAL reabsorptive function.533

SEGMENTS DOWNSTREAM OF THE TAL: DISTAL CONVOLUTED TUBULE, CONNECTING TUBULE, AND COLLECTING DUCT Electrolyte transports by the tubular epithelia distal of the loop of Henle provide the fine tuning of urinary electrolyte- and water-excretion. Located downstream of the macula densa, their transepithelial solute transport rates are no more directly submitted to tubuloglomerular feedback control but, rather, they are regulated by systemic hormones and a multitude of local factors.296,317 The structural subdivision of these portions in the cortex into the distal convoluted tubule (DCT), the connecting tubule (CNT), and the cortical collecting duct (CCD) goes back to light microscopic observations made in sections and microdissected tubules from kidneys of rabbits, human, mouse, sheep, cat, pig, beef,

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and dolphin by Karl Peter early in the 20th century.37 More than half-a-century later, microdissection studies of nephrons from rabbits and mice by the group of Morel534 revealed that the distribution of sensitivities for several peptide hormones was bound to the morphological segmentation. Detailed electron microscopic investigations of the distal tubular portions in rabbits,5 rats,291,535,536 Psammomys obesus,35 and mice537,538 further confirmed and extended the earlier findings. The few studies on the human nephron459,539
541 agree with the data obtained from experimental animals. In contrast to the preceding tubular portions, the epithelial lining of each of the segments following the TAL display at least two distinct cell types: one cell type is segment-specific and called accordingly DCT cell, CNT cell, and CD cell; the other one, the intercalated cell (IC cell), is interspersed in differing amounts among the specific cells of each segment.542 The transition from one segment to the next may be sharp and unequivocally definable, e.g., as in rabbits5,268 or they may develop gradually, involving more-or-less long transitional portions with a mixture of cells from the successive segments or with cells showing features intermediate between the segment-specific cell types of the given segments.535 The presence of a rather long transitional portion between the definite DCT and CNT in rats,543,544 in mice,537,538 and in humans539 claimed a subdivision of the DCT in these species into the “early” DCT and the “transitional portion”538,545 or the DCT 1 and the DCT 2,537 respectively. Taken the morphological data of the various species (except rabbits!) together it is obvious that the segmentation of the cortical tubular portion distal of Henle’s loop is a matter of definition.544 The nowadays conventionally-used segment definitions for the DCT, the CNT and the CCD (Figure 20.55) take into account the structural data and the distribution of the major apical salt and water transport proteins (Figure 20.55). The uptake by the cell type-specific transport proteins in the luminal plasma membrane into the cell and the movement across and out of the cell is facilitated by a bunch of “auxiliary” cytoplasmic and/or basolateral membrane proteins (e.g., Na-K-ATPase, Ca-binding proteins, K-channels, hormone receptors, etc.). These “auxiliary” proteins are not restricted to the given cell type and thus, are not segment-specific. The inventory of gene expressions (in situ hybridization of mouse kidney sections with annotations) in given segments can be looked up in the “Euregene Expression Database.” “The Kidney Atlas” (http://www.euregene. org/portal/pages/index.html). The segmentation of the CD into cortical (CCD), outer medullary (OMCD), and inner medullary CD (IMCD) is based mainly on the location in the given zones. The structural differences between the CCD and

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b a C b a c

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FIGURE 20.54 Survey on organization of the cortical distal segments and collecting ducts (left panel) and on ultrastructure of the segment-specific cells (right panel). (C: cortex; OS: outer stripe; IS: inner stripe of the outer medulla; IM: inner medulla; Dashed line: delimits the medullary ray; (a): distal convoluted tubule (DCT) and DCT-cell; (b): connecting tubule (CNT) and CNT-cell; (c): CCD and CCD cell; (d): inner medulla (IM) and IMCD cell; Black semicircles indicate the occurrence of intercalated (IC) cells.) Each DCT opens into one CNT. In superficial nephrons the CNT opens directly into a CCD; connecting tubules of deeper nephrons join to form an arcades which ascend in the cortical labyrinth, before they open into a CCD. The collecting ducts descend in the medullary rays and through the outer and inner stripes of the outer medulla; the lower two-thirds of the collecting duct are lined by IMCD cells exclusively; the IMCD open as papillary ducts on the renal papilla. (Adapted from and Kaissling, B., and Kriz, W. (1992). In “Morphology of the Loop of Henle, Distal Tubule and Collecting Duct. Handbook of Physiology: Section on Renal Physiology,” 109
167, Windhager, E. E. Oxford University Press, New York, with permission).

d

d

OMCD cells are quantitative rather than qualitative. Usually, the IMCD does not display any more IC cells in its two lower thirds and the lining cells (IMCD cells) are regarded as a separate cell type546 (Figure 20.54).

Distal Convoluted Tubule (DCT) The DCT reabsorbs 5
10% of the filtered Na-load547 and determines the final urinary Mg21 concentration through active transcellular transport.548 In addition, the transitional portion (DCT2) participates (together with the subsequent CNT; see below) in regulation of calcium excretion by transcellular calcium reabsorption.538,549 The DCT epithelium is water-impermeable, similar as the preceding TAL.

The abrupt increase in epithelial height (Figure 20.56)550,544 marks the beginning of the DCT. This prominent feature in the tubular epithelium has been observed in all mammalian species investigated so far, and it coincides exactly with the replacement in the luminal membrane of the NKCC2, characterizing the TAL cells, by the thiazide-inhibitable sodium chloride co-transporter, NCC, characterizing the DCT cells.547 The NCC characterizes all DCT cells (DCT1 and DCT2). The breaking off of NCC expression defines the end of the DCT.537 It is sharp in rabbits,551 but it drops off over a more or less long distance in mice and rat.538 The DCT epithelium is organized by laterally interdigitating cells (Figures 20.57 and 20.58a) similar to the TAL, yet the lateral folding excludes the apical cell portion in DCT cells. The amount of Na-K-ATPase,279 the

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FIGURE 20.55

Schematic distribution of the major apical transport proteins (NKCC2, NCC, TRPM6, TRPV5, ENaC, and AQP2) along the cortical distal segments. (1) In rabbit; and (2) in rat, mouse, and human (MR: medullary ray; TAL: thick ascending limb; G: renal corpuscle; DCT: distal convoluted tubule; CNT: connecting tubule; CCD: cortical collecting duct). Sharp beginning and stop of a transporter along the cortical nephron is indicated by vertical bars, the continuation along the CD by arrows.

surface area of basolateral membranes and the volume density of mitochondria280 are the highest of all tubular cells41 (Figure 20.26). The large lamella-like mitochondria are narrowly enveloped by the lateral interdigitating plasma membrane foldings, all other cell organelles are situated in the apical cytoplasm: the nucleus; the distinct Golgi apparatus; numerous small mitochondrial profiles; short cisterns of rough endoplasmic reticulum; and abundant smooth small, invaginated vesicles closely beneath the apical plasma membrane.260,552 Lysosomes are less frequently observed in DCT cells. The microtubular system in DCT cells is much more prominent than in proximal tubule cells. The tight junctional belt has a similar organization as in the TAL, but is shorter since the apical portions of the DCT cells have a polygonal outline.The intercellular space has a regular width of about 50 nm and is bridged by an intercellular skeleton.553 The apical plasma membrane carries numerous stubby microvilli. Single cilia are present on the center of all DCT cells. In the transitional portion (DCT-2) the lateral folding is progressively superseded by infoldings of the basal plasmalemma which may extend into the apical cell pole. The infolded membranes carry a few caveolae on their cytoplasmic face.260 This structural observation is confirmed by the finding of caveolin in late DCT cells.554,555 Size and volume density of mitochondria slightly decrease along the DCT2. In rats, mice, and humans the appearance of basal plasmalemma infoldings of the DCT cells coincides with the most upstream appearance of intercalated cells. In rabbits, a species that lacks a transitional segment (DCT2), infoldings of the basal plasmalemma and

FIGURE 20.56 Distal tubular segments beyond the macula densa. Small arrowheads delimit the macula densa, large arrowheads point to the transition from the epithelium of the thick ascending limb to the distal convoluted tubule (DCT) (CNT: connecting tubule; CD: cortical collecting duct). The tubule profile in the upper portion of the micrograph has a mixed cell population, composed of CNT cells (arrow), CD cells (double arrow), and IC cells (asterisk) and represents the transition from a CNT to a cortical CD (rat: TEM: 3B500).

the most upstream appearance of IC cells mark the beginning of the CNT. Functional Data Sodium chloride reabsorption by the DCT proceeds via the electro-neutral Na1-Cl2 co-transporter (NCC) in the apical plasma membrane of DCT cells. NCC is specifically inhibitable by thiazide diuretics,547,556 which are frequently used in the treatment of hypertension.547 The driving force for influx of NaCl via NCC is generated by the Na-K-ATPase activity in the basolateral membrane of DCT cells. The basolateral chloride channel, subunit b (ClC-Kb),479,556,557 extrudes Cl2 ions at the basolateral side of DCT cells. Potassium handling, associated with NCC-mediated transport,

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FIGURE 20.57 Organization of distal and collecting duct cells in the renal cortex; (a) and (b) Psammomys obesus; (c) rat; fixation by reduced osmium. (a) DCT cell, interdigitating, lateral cell processes (arrows) narrowly enclose large mitochondria; (b) CNT cell, displaying a few interdigitating lateral cell processes and abundant infoldings of the basal plasma membrane (arrows), extending up into the apical cell half; most mitochondria are aligned between the infolded membranes; (c) non-interdigitating CCD cell; all infoldings of the basal plasma membrane are restricted to the basal cell portion; the location of mitochondria above the basal rim of infolded membranes is characteristic for CD cells (TEM: 3B10 000).

involves ROMK, detectable by immunomethods in the cytoplasm of DCT1,481,558 and in the apical membrane of DCT2 cells, and BK channels.558,559 The NaCl transport rates by the DCT epithelium are linked with NCC surface expression. It is regulated (among others) by the luminal NaCl-load,560 dietary salt,561 by angiotensin II,562
564 and by sex hormones.565 Although mineralocorticoid receptors have been detected in the DCT,547 aldosterone has no effect on NCC-mediated transport since beta hydroxysteroid-dehydrogenase, which confers mineralocorticoid specificity to the receptor, is lacking in DCT1.562 Changes in NCC surface expression are effected by trafficking of the co-transporter from the subapical vesicular compartment into564 and removal by endocytosis566 from, respectively, the apical plasmalemma, and by altering the NCC degradation rate through the lysosomal pathway.567,568 Kinases, such as the serum- and glucocorticoidinducible kinase, SGK1, with-no-lysine kinases WNK1 and WNK4, both themselves controlled by NaCl intake, play an important role in this regulation.559,561,569
571 WNKs promote NCC-targeting to the lysosome for degradation.572 WNK signaling is implicated in the coordination of transcellular and paracellular flux to achieve NaCl and K1 homeostasis.250 Recent data obtained in genetically vasopressin-deficient Brattleboro rats suggest that vasopressin and the vasopressin-V2 receptor-NCC signaling cascade might play a role in the short-term regulation of NCC in the apical plasmalemma of DCT cells.573,574Vasopressindependent increases in cAMP had not been recorded in the DCT of rats and other species.534,575

Chronic increases in the NaCl-transport rates in the DCT, induced in rabbits by high dietary Na-intake combined with low K-intake280,281 or in rats by rises in NaCl-delivery due to impaired NaCl-reabsorption in the preceding TAL, provoke extensive structural compensatory hypertrophy in the DCT,290,292,304,576,577 including substantial increases in the DNA synthesis rate in DCT cells.305,578 These changes occur in the presence, but also in the absence, of increased plasma levels of mineralocorticoids,578 and are mediated most probably by angiotensin II.562 In line with these earlier structural observations are recent studies in a ROMK-deficient mice model for Bartter’s syndrome with loss of TAL function. The ROMK-deficient mice reveal hypertrophy of the DCT epithelium, with compensatory upregulation of NaCl reabsorption via the thiazide-sensitive NCC cotransporter.579,580 The renal abundance and the NCC-labeling in DCT were found to be profoundly and selectively decreased in aldosterone-escape rats, suggesting that the thiazidesensitive NaCl co-transporter may be the chief molecular target for regulatory processes responsible for mineralocorticoid escape via a post-transcriptional mechanism.581 The DCT determines the final urinary Mg21 concentration through active transcellular transport.504,548,582,583 The transient receptor potential channel melastatin subtype 6 (TRPM6),548,584 co-localizes with NCC, at least in the early DCT (DCT1), and is regarded as a likely candidate for influx of Mg21 across the luminal membrane. This influx apparently requires the presence of the gamma subunit of the renal Na-K-APTase in the basolateral membrane of the

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calbindinD28k, and is prominent in the early part of the DCT of mice.537,538 PV seems also to play a role in the endogeneous NCC expression in DCT cells by modulating intracellular Ca21 signaling in response to ATP.590 The key players for paracellular Mg21-transport, Claudin 16 (paracellin1) and Claudin 19, are both detected in the DCT tight junction,497 and may enable paracellular Mg21-movement across the DCT epithelium in addition to the transcellular Mg21-transport. The tight junction protein Claudin 7 has been found to be highly expressed in the distal convoluted tubules (and collecting ducts) of the mature kidney, suggesting that it may play a role in paracellular NaCl and K handling.240,591,592 DCT 2

FIGURE 20.58 Ultrastructure of distal convoluted tubule cells (rat kidney). (a) Cell in the early and (b) late portion of the DCT; in (a) characteristic apical position of the nucleus and location of the mitochondria in basolateral interdigitating cell processes; the volume density of mitochondria is high; in (b) the amount of basal plasma membrane infoldings is higher than in (a), the amount of mitochondria is lower; the most upstream appearance of intercalated cells (IC) is in the late DCT (TEM: 3B5400).

This transitional segment expresses, in addition to NCC, the amiloride-sensitive epithelial sodium channel, ENaC. The onset of ENaC in the apical plasma membrane coincides with the most upstream appearance of intercalated cells (see below), apical immunoexpression of ROMK. The eyecatching beginning of prominent cytoplasmic immunostaining for Vitamin Ddependent calbindin-D28k, and the marked increase in immunostaining for PMCA and NCX in the basolateral plasma membrane go along with the onset in the luminal membrane of the epithelial calcium channel, TRPV5,538 the gatekeeper for renal epithelial Ca21 transport.593 ENaC and TRPV5 are coexpressed in the CNT and will be discussed there. Rabbits have no DCT2; in this species the transition from the DCT to the CNT is sharp and marked by an abrupt change in cell structure,280 coinciding with the abrupt onset of ENaC538 in the apical membrane, as well as the onset of the TRPV5594 (Figure 20.55), and also the appearance of IC cells.

DCT cells.276,585 Transcription factor HNF1B (hepatocyte nuclear factor 1 homeobox B) is proposed to regulate the expression of the g-subunit of the Na1/ K1-ATPase.586 In addition to the Na-K-ATPase, the DCT cells weakly display the plasma membrane Ca21(Mg21)-ATPase (PMCA)307,587 and the sodium
calcium exchanger (NCX).544 The epidermal growth factor (EGF) expressed by the DCT epithelium473,588,589 seems to be involved in TRPM6-mediated regulation of active Mg21 reabsorption. Transcellular magnesium reabsorption via TRPM6 seems to critically depend on low levels of free intracellular magnesium, putatively kept low by the cytoplasmic calcium-binding protein parvalbumin (PV). PV has a several-fold higher binding capacity for magnesium than the calcium-binding protein

Dysfunctions of NaCl Reabsorption in the DCT Inhibition of NCC in rats treated for three to four days with thiazide diuretics induces massive rates of apoptotic cell death of DCT cells in the early part of the DCT, while the late part of the segment with the additional sodium entry pathway ENaC remains intact.307 If the transport activity of the early DCT cells is inhibited for only a few days, the epithelium rapidly and fully recovers within a few days after removal of the drug. In loss-of-function mutations of the NCC gene in mice,595 permanent, dramatic atrophy of the early DCT portion is seen.545 These data highlight the eminent importance of the transport activity in maintaining and modeling the tubular epithelium. Loss-of-function mutations in the NCC-gene in humans cause “Gitelman’s syndrome.” This syndrome is characterized by mild

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renal sodium-wasting, hypocalciuria, hypomagnesaemia, hypokaliemic alkalosis, and reduced blood pressure in humans.596
599 Mutations in the NCC-regulating WNK1 and WNK4 increase NCC activity, and cause Gordon’s Syndrome (Pseudohypoaldosteronism type II - PAH II). The symptoms of this disease comprise arterial hypertension, hyperkaliaemia, hypercalciuria and hypermagnesaemia, and mirror Gitelmans disease.251,567,600 Lossof-function mutations of one or several of the genes involved in Mg21 reabsorption are associated with hypomagnesemia,601 characteristic for Gordon’s syndrome.602

Connecting Tubule (CNT) In all species the epithelium of the CNT is lined by two distinct cell types (Figure 20.59): the segment-specific CNT cells; and the intercalated cells (IC cells) (see below). The segment-specific CNT cells display the calcium channel TRPV5 and the amiloride-sensitive epithelial sodium channel (ENaC) in their apical plasma membrane. In rats, mice, and humans they display, in addition, the vasopressin-regulated water channel, aquaporin-2 (AQP2) (Figure 20.55). In these species the emergence of AQP2 in the apical plasmalemma in the epithelial lining defines the beginning of the CNT, since TRPV5 and ENaC already appear in the transitional region (DCT2) (see Figure 20.55). Contrastingly, in rabbits vasopressin-regulated water channels are lacking in the CNT. In rabbits the beginning of the CNT is defined by a distinct change in epithelial structure, expression of TRPV5 and ENaC, and the first incidence of intercalated cells544 (Figure 20.55). Evidently, the CNT shares cytological and functional features ascribed to both the nephron (derived from the metanephrogenic blastema) and the collecting duct (derived from the ureteric bud). The assignment of the CNT to either the nephron or the collecting ducts is disputed.36,37,603,604 CNT Cell Organization The organization of CNT cells (Figures 20.54b, 20.57b, and 20.59b) is similar to that of DCT2 cells, i.e., intermediate between the DCT cells with basolateral membrane surface augmentation by interdigitating lateral folds and the non-interdigitating epithelia (CD cells) with basal plasma membrane infoldings. The apical and the basal outlines of CNT cells approach a polygonal shape, and the cells are smoothly apposed to each other. The basolateral plasma membrane area is increased predominantly by folding of the basal plasma membrane into the cell. The infolded plasma membranes may extend into the most apical

FIGURE 20.59 Connecting tubule (rat kidney). (a) The epithelium is composed of CNT cells and IC cells, (asterisks). (b) Characteristic CNT cell with abundant infoldings of the basal cell membrane; the arrows point to the tight junction. Insert: The infolded plasma membranes reveal numerous caveolae (TEM (a): 3B1400; (b): 3B6100).

cell portion, and are endowed with abundant caveolae on their cytoplasmic face260,554 (Figure 20.59). The extracellular spaces between the basal plasma membrane foldings and the lateral intercellular spaces have no direct continuity and are usually narrow.260 The apical plasma membrane with short slender microvilli is delimited from the lateral plasma membrane by rather deep tight junctions, composed of several anastomosing strands.427 The nucleus, the Golgi apparatus, polyribosomes, very short profiles of rER, and elongated and small round mitochondrial profiles are located in the cytoplasm between or above the infolded membranes. Smooth vesicles are particularly abundant in the apical half of CNT cells. In contrast to DCT cells, small lysosomes are frequent in CNT cells.

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From the beginning to the end of the segment, the height of the CNT cells, the extent of basal plasma membrane, and their volume density of mitochondria decrease. The steepness of the axial changes is more pronounced in rabbits280 and mice than in rats, and varies with the functional conditions.605,606

Functional Aspects Calcium Reabsorption Microperfusion607 and micropuncture studies had located active Ca21 reabsorption to the distal convolution, including the DCT and CNT (for review see 608) a long time before the specific Ca21-channel in the distal segments, TRPV5, was known.. The localization of TRPV5609 in the apical plasmalemma of the late distal tubule (DCT2 cells and CNT cells538) (Figure 20.55) unequivocally identified these segments as sites for active transcellular Ca21 reabsorption in the kidney. The paracellular pathway in these segments is impermeable for Ca21. The cytoplasmic calcium-binding protein, calbindin D28k, the sodium calcium exchanger, NCX, and the plasma membrane calcium-Mg ATPase, PMCA, located in the basolateral plasma membrane, are auxiliary proteins necessary for TRPV5-mediated transcellular calcium movement. All three reveal very heavy immunostaining in DCT2 and CNT cells.537,538 Upon its entry into the cell via TRPV5, calbindinD28k buffers Ca21 and the basolateral Ca21 transporters NCX and PMCA extrude Ca21 into the interstitial compartment.593 Interestingly, in tubular flow direction immunostaining for TRPV5 progressively shifts from the apical plasma membrane into the cytoplasm,538 associated with parallel decreases of immuno-traceability for cytoplasmic calbindinD28k, for basolateral PMCA and for NCX. These changes most probably indicate respective changes of transcellular calcium transport rates. Regulation of transcellular Ca21 transport rates involves changes in the apical channel abundance and direct TRPV5 channel activation.610 Via binding to its receptor (PTH/PTHrP) in the basolateral plasma membrane of CNT cells,611
613 parathyroid hormone (PTH) increases the protein expression of TRPV5593,614 and via a cAMP-PKA signaling pathway PTH increases the channel opening probability.615 Transcription of the Ca21 channel is regulated by the active form of vitamin D3, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3).594,616 The male and female sex hormones, estrogens and androgens, also play a role in renal Ca21 handling.617 Urinary klotho stimulates TRPV5 channel activity at the apical membrane, whereas intracellular klotho enhances basolateral Na-K-ATPase surface expression

649

that activates NCX-mediated Ca21 efflux.593 Urinary tissue kallikrein (TK) activates a bradykinin receptor (BK2) in the apical membrane of segment-specific CNT cells,618 and thereby stimulates TRPV5-mediated Ca21 influx.615,619
621 The sites of TK synthesis in the kidney are approximately congruent with the sites of calcium reabsorption, i.e, the late DCT and CNT.618 These findings suggest that TK may be a physiologic regulator of renal tubular calcium transport.622,623 Kallikrein synthesis in the CNT and its subsequent release into the urine are stimulated by aldosterone,624 dietary Na1 restriction,618 and in particular by dietary K1 loading.625,626 TK knockout mice display a somewhat delayed kaliuretic response to potassium loading.623,627 Sodium Reabsorption The amiloride-sensitive sodium channel, ENaC, is the key player in the final sodium recovery by the kidney.628,629 ENaC is a heteromultimeric channel composed of three homologous subunits (α, β, γ).630 Full activity of ENaC requires the co-expression of all three subunits in the luminal membrane. The activity of amiloride-sensitive transport is under the tight control of aldosterone.631 Therefore, all segments with ENaC-mediated sodium reabsorption
DCT2, CNT, and CD
are collectively designated as “aldosterone-sensitive distal nephron” (ASDN632). While the mineralocorticoid receptor (MR) is expressed in all distal segments,633 only the renal ENaC-expressing portions display the enzyme 11-β-hydroxysteroid dehydrogenase type 2 (11βHSD2),605,606,634
639 which confers mineralocorticoid specificity to the MR. The rate-limiting factor for transepithelial Na1 transport in the ASDN is the activity and abundance of ENaC in the luminal membrane of the ENaC-expressing cells. Under control conditions605,606 all three EnaC subunits are well detectable by immunostainings in the apical plasma membrane of the DCT2- and CNTcells.605,606 Along the course of the CNT and CD-segments all three subunits become undetectable in the apical plasmalemma, but heavily accumulate in the cytoplasm.605,606,639 The decline in available channels in the luminal membrane is paralleled by reduction of basal infoldings and of mitochondria, most evident along the CNT epithelium and by a respective progressive decline of Na1 transport activity along the axis of the ASDN (CNT . CCD).604,640 The physiological relevance of ENaC-mediated Na1 transport in the CNT is highlighted by the observation that the collecting duct-specific deletion of the alpha ENaC gene in mice604 is fully compensated by the residual activity of ENaC in the upstream located CNT (and in the DCT2).604

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In fact, recent data on the three-dimensional reconstruction of the mouse nephron show that five to seven nephrons are connected via a CNT to a single CCD.34 Thus, the collected luminal surface for ENaC-mediated sodium reabsorption in the late DCTs and the CNT,37,641 is several-fold greater than that available in the CCD itself. All factors involved in regulation of sodium transport rates in the ASDN (hormones, proteases, intra-, and extracellular ion concentrations, tubular flow rate, as well as kinases and interacting proteins (for review see 562,642
644), ultimately target the ENaC channel activity or abundance in the apical plasma membrane. Changes in ENaC abundance in the luminal plasma membrane involve channel synthesis, exocytotic delivery of subunits to the cell surface, and endocytotic retrieval of channels from the luminal membrane and their degradation.300,562,628,644,645 Endogeneous increases in plasma aldosterone levels rapidly induce (within hours) activation and redistribution of ENaC subunits from intracellular compartments to the apical plasma membrane605,631,639,646 and a decrease of internalization of ENaC through the synthesis of SGK1.647
649 Prolonged changes in EnaCmediated sodium transport promote respective changes in cell height, abundance of basolateral plasma membrane infoldings, and the density of mitochondria,280,290,292,293 which all together reflect the changes in Na1 transport rates. ENaC channel activity and abundance in the apical plasma membrane of the ASDN is also target of other hormones. The co-expression of ENaC with vasopressin receptors (V1 and V2) and vasopressin-sensitive water channels AQP2 in rat-, mice-, and human-segment-specific CNT (not in rabbit CNT cells) suggests the mutual interaction of sodium and water transport.650,651 Indeed, vasopressin facilitates the translocation of ENaC to the apical membrane652 and on removal of a V2R agonist ENaC is endocytosed from the membrane surface and reorganized into recycling vesicles, with a mechanism similar to that described for AQP2 regulation.653 The delivery of somewhat water-depleted tubular fluid from the CNTs to the cortical collecting duct might enhance the urinary concentration process in the CD. Interestingly, in rabbits, in which the CNT lacks vasopressin-sensitive water channels, the arcades open at a much higher cortical level into the cortical collecting duct than in rats, mice or humans.37 Insulin and insulin-like growth factor,610 angiotensin 654
656 II kinases, interacting proteins, intra-, and extracellular ion concentrations, osmolarity (for review see 645), locally released nucleotides, and tubular flow rate (for review see 657) have also been shown to modulate ENaC-mediated Na-transport activity.

Potassium Transport In all ENaC-displaying cells renal outer medulla potassium channel ROMK is strongly expressed in the luminal membrane, where it co-localizes with PDZ proteins (NHERF2).272 ENaC-mediated sodium reabsorption is coupled in a fixed ratio with K secretion via the ROMK. K1 enters the cell by the activity of the NaK-ATPase in the basolateral membrane, and exits into the tubular fluid via ROMK. The ratio of sodium-reabsorption and K-secretion by the segment-specific (CNT; CD cells)658 can be modulated by intercalated cells which are bound to ENaC-displaying epithelia. The proton secretion by IC cells via a H-K-ATPase can apparently be coupled with K reabsorption.659 Therefore, the ASDN is also the tubular site for net renal potassium (K1) excretion.482,660 The main factors regulating K1 secretion are dietary K1 intake and aldosterone (for review see 562). Mutations in the genes coding for ENaC subunits,628,629 and for proteins involved in ENaC-associated K-secretion (ROMK),661 as well as the correct targeting into or removal from the membrane (e.g., SGK1, Nedd4-2; for review see 645) are associated with severe disturbances of blood pressure regulation.662 Transition From CNT tO CCD In rodents and humans no marked structural change indicates the transition from the CNT to the CCD. Morphologically, the CCD can be defined by its location in the medullary ray. In difference, in rabbits the clear-cut onset of vasopressin-regulated water permeability marks the beginning of the CCD. It is associated with the appearance of dilated intercellular spaces in the epithelium5,280 and the change of segment-specific cells, i.e., from CNT- to CD-cells. By immunostaining, the beginning of the CCD is defined in rodents and rabbits by the break-off of TRPV5 (Figure 20.55) and related proteins (NCX, calbindin D28k538,544). In humans, NCX and calbindin D28k have also been detected in the CCD.539

Collecting Ducts The CCD, the OMCD, and the upper part of the IMCD are composed of segment-specific cells (CD cells) and intercalated cells (IC cells; see subsequent sections) (Figure 20.54). The CD cells (Figures 20.54, 20.57, 20.60 and 20.61) have simple polygonal basal and apical outlines. Their most characteristic feature is the narrowly arranged basal plasmalemma infoldings of uniform height (Figures 20.54, 20.57, and 20.60) at the base of the cells, easily recognizable in light- and electron-microscopy as a basal light rim. All major cell organelles
the nucleus, small

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mitochondria, numerous small Golgi-fields, abundant profiles of smooth ER, and a few of rough ER, lysosomes, multivesicular bodies, and occasional glycogen accumulations
are located in the zone above the infolded membranes. The subapical zone often reveals small round or elongated vesicles, oriented either perpendicularly or at an oblique angle to the luminal membrane (Figure 20.61c). These vesicles contain aggregates of AQP2, and are called aggrephores. Many of the aggrephores carry spherical clathrin-coated heads. The tight junctional belt is deep and consists of anastomosing strands with high particle density.663 The apical plasma membrane generally bears only a few short slender microvilli or microfolds. The prominent central single cilia on the collecting duct-specific cells (Figure 20.62a) have been proposed as the key structural element in the Ca21 response to fluid shear stress.312,664 Short microvilli or folds of the lateral plasma membrane project into

FIGURE 20.60 Cortical collecting duct (rat kidney). (a) The epithelium is composed of CD cells and IC cells. (b) CCD cell
infoldings of the basal plasma membrane are restricted to the basal cell portion; all mitochondria and cell organelles are located above the infolded membranes (Arrows: Tight junctions; TEM (a): 3B2700; (b): 3B8500). (Adapted from and Kaissling, B., and Kriz, W. (1992). In “Morphology of the Loop of Henle, Distal Tubule and Collecting Duct. Handbook of Physiology: Section on Renal Physiology,” 109
167, Windhager, E. E. Oxford University Press, New York, with permission).

651

the intercellular space, and are connected by small desmosomes with those of adjacent cells (Figure 20.62b). In marked contrast to the waterimpermeable epithelia of the TAL and the DCT, the width of the intercellular spaces between CD cells as well as the space between the infolded basal plasma membranes may be largely dilated or narrow, correlating with bulk water flow across the epithelium (see below). The cytoskeleton is particularly prominent in CD cells. Actin filaments and microtubules form a dense meshwork along the apical and lateral plasma membrane. The cytoskeleton is essential for the shuttling of AQP2 to and from the plasmalemma (see below). Furthermore, the prominent cytoskeleton may be one mean, among others, to withstand the varying osmotic pressure in the collecting duct. The CD cell undergoes gradual, although considerable, changes from the deep cortex (CCD) downstream to the upper third of the inner zone (IMCD) (Figure 20.61). The extent of basal plasma membrane foldings and the volume density of mitochondria decrease from the cortex towards the inner zone, whereas the volume density of lysosomes and the density of cytoskeletal proteins increase. The degree of changes along the CD differs among species.5,260,540 The CD cells in the lower two-thirds of the inner medulla are distinguished as inner medullary collecting duct cells (IMCD cells).552 In rabbit5 and guinea pig, IMCD cells increase in height toward the papilla up to 20-fold. A substantial, albeit less dramatic, increase occurs in rhesus monkey665 and in human kidney.540 In other species (e.g., rat, mouse, Psammomys, and dog260) the epithelium near the tip of the papilla is cuboidal or low columnar. The luminal membrane of IMCD cells is covered by numerous stubby microvilli, and generally lacks the central cilium.546 The lateral intercellular spaces are conspicuous by their dense assembly of microvilli and microfolds, projecting from the lateral cell membranes. In the beginning of the inner medullary collecting duct (IMCD) the tight junctions are complex and consist of several anastomosing strands.260 Toward the papillary tip in rat and rabbit, there is a considerable decrease in the number of strands, and in the apico
basal depth of the junction.260 Sodium Reabsorption in the Collecting Duct Together with the vasopressin-regulated waterchannel AQP2, the CD cells consistently express the amiloride-sensitive Na-channel ENaC (see CNT)666 (Figure 20.55) and ROMK. The coexistence of the differentially regulated pathways for water- and Na-reabsorption in the same cell suggest the possibility for mutual interactions.650,666,667

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a

b

c

d

FIGURE 20.61 Inner medullary collecting duct. (a) Tubular profile showing the homogenous epithelium (rat: TEM: 3B3400). (b) Epithelium of the middle portion of an IMCD. Within the epithelium three zones are seen: a basal zone with basal infoldings, a middle zone containing Golgi fields, mitochondria and lysosomal elements, and a thin apical zone with tubular and vesicular profiles. Note the deep tight junction (rat: TEM: 3B17,000). (c) Apical zone of a CD cell with many elongated tubular profiles (arrows) which are believed to represent agrophores (rat: TEM: 3B38,000). (d) Freeze-fracture electron micrograph to show the multistranded tight junction of the collecting duct epithelium (L: luminal membrane; BL: basolateral membrane; Rabbit: 3B34,000).

Recently, a second pathway for electroneutral NaCl absorption has been revealed in the collecting duct epithelium, This pathway is located in the intercalated cells (see below) and is insensitive to amiloride but inhibited by thiazides, and couples 2 anion exchangers, pendrin and Na-driven chloride/bicarbonate exchanger (NDCBE)668,669 (see “Intercalated Cells”). Paracrine and autocrine regulation by the purinergic system296,657,670,671 mediates flow-and metabolic ratedependent changes of Na1 and water transport in the collecting duct. Vasopressin-Regulated Water and Urea Reabsorption in the Collecting Duct Collecting ducts are the canonical targets for vasopressin-sensitive water and urea reabsorption.672 They display receptors for vasopressin (V1 and V2) in the basolateral plasma membrane of the segment-specific cells, the CD cells,519 and vasopressin-sensitive water channels AQP2. Water permeability of the luminal membrane is achieved by exocytotic insertion of the vasopressin-regulated water-channel AQP2 from subapical vesicles (aggrephores) into the apical cell

membrane. The exocytosis is triggered by binding of vasopressin (ADH) to the V2-receptor at the basolateral membrane of CD cells and the subsequent signal transduction cascade. The aggregates of AQP2 in the aggrephores are colocalized with dynein and dynactin.673 Many of the aggrephores carry spherical clathrincoated heads. With low levels of vasopressin, the AQP2-containing membrane portions recycle back into the subapical cytoplasm.299,674
676 The movement of aggrephores critically depends on microtubules and actin filaments in the apical cytoplasm. High levels of vasopressin-independent AQP2 surface expression have been observed under long-term677 and acute678 exposure of rats and mice to statins. Applied chronically, statins decrease membrane cholesterol679 and clathrin-mediated endocytosis of AQP2. Acutely, the statins seem to decrease endocytosis of AQP2 and vesicle trafficking by modulating RhoGTPase,678 which is involved in regulation of the cytoskeleton, endocytosis, and vesicle trafficking.680,681 The water channels AQP3 and AQP4 are both located in the basolateral membrane of CD cells.682 AQP3 is permeable to glycerol, urea, and water; AQP4 is associated with orthogonal arrays of intramembrane particles, as

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653

heat shock protein 70,688 are target genes of the tonicityresponse enhancer binding protein (TonEBP), a transcriptional activator of the REL-family. During kidney development, expression of TonEBP precedes that of the urea transporter. It is first detected in the renal medulla of mice at the fetal age of 16 days and increases up to postnatal day 21, when the medulla is fully developed and the urinary concentrating ability is achieved.689

Intercalated Cells (IC Cells) Intercalated cells (IC cells) are interspersed as single cells among the epithelium of the ASDN (i.e., among ENaC-displaying epithelia, the late DCT (DCT2), the CNT (Figure 20.59a), and the CD (Figure 20.60a). IC cells play a decisive role in the final regulation by the collecting system of acid
base excretion, in potassium reabsorption and secretion, in ammonia excretion and, as discovered recently, intercalated cells participate together with the segment-specific cells in electroneutral sodium reabsorption.

FIGURE 20.62

Intercalated cells from the rabbit. (a) Scanning electron micrograph of a cortical collecting duct with collecting ductspecific CD cells (CD) and intercalated cells (IC). The CD cells carry single cilia (C) and short microvilli (arrowhead). The straight ridges (open arrow) represent the cell borders between CD cells. From the IC cells one (IC 1) has a narrow, constricted apical cell pole, the other one (IC 2) a large apical cell pole, both adorned with numerous long microvilli (TEM: 3B13,000). (b) Section across corresponding cells. Note the position and accumulation of flat vesicles (asterisk) in the apical cell pole of the IC cells (TEM: 3B7500).

revealed by freeze-fracture studies in the outer medullary collecting duct.683,684 The IMCD cells co-express, in addition to vasopressin-regulated AQP2, the vasopressin-regulated urea transporters UT-A1/3 and possibly UT-A4 (for review see 148). The abundance of UT-A1/3 in the apical membrane is rate-limiting for transepithelial, vasopressindependent urea reabsorption.571,685 The basolateral membranes of the IMCD cells display the water channel AQP3. The abundance of UT-A1/3 in the apical membrane is rate-limiting for transepithelial, vasopressin-dependent urea reabsorption.571,685 In the basolateral membranes of the IMCD cells the water channels AQP3,682 permeability to glycerol, urea and water,686 and AQP4683,687 with an apparently low urea permeability have been demonstrated. The genes coding for proteins, which are involved in cellular accumulation of organic osmolytes, such as the vasopressin-regulated urea transporter UT-A and

Consistent Structural Features of Intercalated Cells Intercalated cells reveal conspicuous structural heterogeneity (Figure 20.61). IC cells usually do not form a continous epithelial layer, but at least their luminal poles are entirely surrounded by the segment-specific cells. The luminal outline of IC cells is in most cases rather circular (Figure 20.62a),291,540,690
692 IC cells generally reveal a specific surface pattern of microprojections (Figure 20.62a) and lack, at least in the cortex, the central cilium which is apparent on other cells (Figure 20.62b). Among the most consistent distinguishing intracellular features is the distribution pattern of mitochondria: the often rounded mitochondrial profiles lack the systematic association to basolateral cell membranes35,693,694 evident in other tubular cells. The generally small, more or less round vesicles often reveal an invagination bordered by a thin smooth membrane (“invaginated vesicles”5); they participate in endocytosis695
697; the elongated slender profiles
“tubules”
probably represent sections through flat saccules or collapsed large spherical vesicles (“flat vesicles”).5 Occasionally they are found to be continuous with the luminal membrane, and are often in close juxtaposition with mitochondria.5 Transitional forms between the two vesicle types can be seen; the presence of specific particles in one or several membrane domains (luminal, basolateral, and/or tubulo-vesicular) have been observed. In TEM preparations the membranes reveal a coat of densely arranged, approximately rectangular large particles, so-called “studs,” on their cytoplasmic face. The “studs” are 10 nm spherical

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structures (Figure 20.65a,c,d), which represent the H1ATPase.698,699 In freeze-fracture preparations dense arrays of intramembrane particles, so-called “rodshaped” particles appear on the P-face of the membranes (Figure 20.65b).543,700 The presence of “studs” and of “rod-shaped” particles on the cytoplasmic membrane faces often coincides. Clathrin-coated pits on either the luminal or the basal cell membrane and clathrin heads on the “studded” vesicles are regularly found. The nucleus generally reveals more heterochromatin condensations and looks darker than that of the adjacent CNT or CD cells.

Proteins Related to Intercalated Cell Functions All intercalated cells display high levels of cabonic anhydrase II in the cytoplasm.450,472,701
704 All IC cells express the electrogenic V-type proton-ATPase, the proton pump698 in at least one of their membrane domains, and all express an anion exchanger, either anion exchanger 1 (AE1, gene Slc4A1; band 3705) or Pendrin (Slc 26A4706) in one membrane domain. Pendrin is an aldosterone-sensitive Na1-independent Cl2/HCO32 exchanger that mediates Cl2 absorption and HCO32 secretion in the cortical collecting duct (CCD).669 Furthermore, studies on isolated CCDs suggested that the parallel action of the Na1-driven Cl2/HCO32 exchanger (NDCBE/SLC4A8) and the Na1-independent Cl2/HCO32 exchanger (pendrin/ SLC26A4) account for the electroneutral thiazide-sensitive sodium transport in the CCD (where the thiazide sensitive electroneutral NaCl co-transporter NCC is not expressed), a finding that challenges the current concept of a functional separation between principal cells for the regulation of sodium and potassium balance, and intercalated cells for acid
base regulation.668

All IC cells express the Rhesus glycoproteins, Rh B Glycoprotein (Rhbg707) and Rh C Glycoprotein (Rhcg708). These proteins are recently recognized ammonia transporters in the distal tubule and collecting duct. Rhcg is present in both the apical and basolateral plasma membrane, is expressed in parallel with renal ammonia excretion, and mediates a critical role in renal ammonia excretion and collecting duct ammonia transport. Rhbg is expressed specifically in the basolateral plasma membrane.709 Subtypes of Intercalated Cells By morphological criteria and distribution patterns of specific transport proteins (Table 20.1) three different manifestation of IC cells have been described, type A cells, type B cells,694 and type nonA-nonB cells.710 TYPE A IC CELLS

Morphologically, the type A IC cells usually have a broad protruding apical cell pole, which is adorned with numerous slender microfolds and/or finger-like microvilli giving rise to a very complex surface pattern5,691,694(Figures 20.63a and 20.64a). The cytoplasmic membrane face of the microprojections and of the esicles in the apical cell pole are “studded”594 (Figure 20.65a). “Rod-shaped” particles (Figure 20.64b) have also been demonstrated.700 The mitochondria are particularly numerous, and are accumulated in the apical cell pole above the large round nucleus (Figure 20.64a). They are often found very closely adjacent to the luminal cell membrane. They possess narrower cristae, and their matrix appears more electron-dense than in other cell types. The Golgi apparatus and other cell organelles are only slightly apparent among the numerous mitochondria. Polyribosomes may be exceedingly frequent. Some profiles of RER are

TABLE 20.1 Proteins with Defined Functional Relevance (see text) in Renal Intercalated Cell (IC) Subtypes Intercalated Cells Protein

Type A

Type B

Type nonA-nonB

References

Carbonic anhydrase II

yes; a

yes; c

yes; c

450,812
819

H -ATPase

yes; a

yes; b

yes; diff

698,699,714
719

AE1 (Slc4A1; Band 3)

yes; a

absent

absent

705,709,721,740,820

Pendrin(Slc 26A4)

absent

yes; a

yes; a

740,820

RhBG

yes; b

absent

yes; b

494,709,730

1

RhCG

yes; a

absent

yes; a

709,730,732,821,822

H 1 -K-ATPase (gastric and non-gastric)

yes; a

?

?

823
830

50 NT

yes; a

yes; a (b)

?

57,753

Localization in: a: apical membrane domain; b: basolateral membrane domain; (b): occasionally; diff: diffuse vesicular; c: cytoplasmic; ?: not determined. bold: marker combination for IC subtype diagnosis.

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655

FIGURE 20.63 Survey of ultrastructure of intercalated cells. (a) A-type IC cell; (b) B-type IC cell. . (Adapted from and Kaissling, B., and Kriz, W. (1992). In “Morphology of the Loop of Henle, Distal Tubule and Collecting Duct. Handbook of Physiology: Section on Renal Physiology,” 109
167, Windhager, E. E. Oxford University Press, New York, with permission)

FIGURE 20.64

generally found in basal cell portions. Basal infoldings can be extensive in the rat,694,711 yet in the rabbit they are virtually absent.712 Some structural variation within type A cells exist among and even within individuals. They concern essentially the extent of studded membrane projections on the luminal cell surface, and the abundance of studded tubulo-vesicular profiles in the apical cell pole. The membrane surface area of the microfolds seems to be inversely related that of the tubular-vesicular profiles; both vary with functional conditions. For instance, under acute metabolic and respiratory acidosis711 and/or potassium depletion the vesicular pool decreases and the apical membrane increases. Mitochondria with rather short profiles and narrowly arranged cristae are amassed in the apical cell pole, in particularly close vicinity to the apical cell membrane. Many microtubules and clathrin-coated vesicles are apparent between the tubulo-vesicular profiles and

the mitochondria. The nucleus is shifted to the basal cell portion (Figures 20.63a and 20.64a) (for review see 492,698,713). IC cells in the outer medulla and the initial part of the inner medullary CD (rats) appear slightly different from the cortical type A cells. In rats, the apical cell pole is often narrower than the basal cell pole.268 The mitochondrial profiles are fewer and smaller than in cortical type A, the Golgi apparatus and the SER are less apparent than in cortical type A cells, the basal infoldings are less extensive compared to cortical type A cells and the nucleus has a characteristic elongated flattened profile. Among the “studded” vesicles in the apical cell pole very large round profiles and

Intercalated cells (rat). (a) Type A cell with many luminal microfolds and abundant mitochondria in the apical cell pole. (b) Type B cell with a rather narrow apical cell pole, a narrow rim of dense cytoplasm with no vesicles under the apical plasma membrane, abundant smooth-surfaced vesicles in the apical cytoplasm, and a huge Golgi complex (G), with abundant mitochondria along the basolateral plasma membrane (Arrows: Tight junction; TEM: 3B7000).

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20. STRUCTURAL ORGANIZATION OF THE MAMMALIAN KIDNEY

particularly long flat vesicles often predominate over other cell organelles. Type A IC cells are considered as the proton-secreting cells. Their apical membrane domain possesses various subunits of H1-ATPase, among them the B1- and d-subunits which possess a high selectivity for intercalated cells.698,699,715
719 The proton-ATPase functions in series with a bicarbonate/Cl (HCO32 /Cl2) exchanger located in the basolateral membrane domain (for review see 492). In type A IC cells the HCO32 /Cl2 exchanger is the anion exchanger AE1 (SLC4A1, band 3), a splice variant product of the erythrocyte band 3 gene.705,720 The presence of AE1 in the basolateral membrane is decisive for diagnosis of type A IC cells.705,721 In addition to the V-type proton-ATPase type A IC cells
at least in the outer stripe
also display a P-type (gastric-type) K-H-ATPase, shown so far in rat and rabbit.578,659,722
726 This K-reabsorbing ATPase seems to be associated with clusters of rod-shaped particles, revealed by freeze-fracture studies on the P-face of cell membranes in rabbit IC cells.700 Thus, the type A IC cells could be involved in recovering potassium, secreted via ROMK, in association with EnaC-mediated Na-reabsorption by the segment-specific cells. Type A cells also express the chloride channel ClC 5.727 The secretory isoform of the Na-K-Cl-co-transporter, NKCC 1, has been detected in the basolateral membrane of type A cells in the outer stripe.728 The type A IC cells express apically and basolaterally non-erythroid Rh-associated glycoproteins Rhcg and basolaterally Rhbg,492,494,709,729
731 which mediate transport of ammonia/ammonium (NH41/NH3)494,732 when expressed in Xenopus laevis oocytes.733 In chronic metabolic acidosis and prolonged high proton secretion the type A IC cells hypertrophy (for review see 492), and IC cells in the OMCD and IMCD show increased RhCG expression.734 However, genetic ablation of the RhBG gene is not a critical determinant of NH41 excretion by the kidney under acidic or under control conditions.729 Under chronic acidosis the type A IC cells proliferate, as evidenced by upregulation of cell cycle proteins, by incorporation of the thymidine analog bromo-deoxyuridine (BrdU) and mitotic figures.735

a

b

c

d

TYPE B IC CELLS

FIGURE 20.65 Intercalated cells (rat). (a) Luminal membrane; its cytoplasmic face is coated with studs (arrows) (TEM: 3B61,000). (b) Freeze-fracture electron micrograph showing the rod-shaped intramembrane particles (stars) of a luminal membrane (TEM: 3B32,000). (c) Apical cytoplasm of a type A cell. The specific vesicles are coated with studs (TEM: 3B64,000). (d) Membrane of basal infoldings of type B cell; its cytoplasmic face is covered with studs (arrows) (TEM: 3B61,000).

Morphologically, Type B IC cells reveal a relatively small, occasionally slightly polygonal luminal outline and protrude only slightly into the lumen (Figures 20.60a, 20.63b, and 20.64b711). The cells seem to be partly covered by the adjacent CD cells, and their sectional profiles often appear almost elliptical (Figures 20.63b and 20.64b). The luminal membrane, with only a few short microprojections, lacks “studs”.736 In contrast, “studs” may be apparent on

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fragments or even along the entire lateral and often extensive formation of infoldings of the basal plasma membrane (Figure 20.65d). The small short mitochondria are accumulated in the basal cell portion and along the lateral cell faces (Figures 20.63b and 20.64b), but they are never found immediately beneath the luminal membrane. The cytoplasm above the plane of the tight junctional belt may be completely devoid of any cell organelles. The nucleus often reveals some basal indentations,711 and is often situated eccentrically. The center of the cell is occupied by a conspicously developed Golgi apparatus, a few short profiles of RER, polyribosomes, and microtubules, as well as lysosomes of varying dimensions, and autophagosomes containing frequently recognizable remnants of mitochondria or membranes. A striking feature of these cells is the high abundance of narrowmeshed profiles of smooth ER, often with clathrincoated heads.329 The SER is intermingled with a great amount of small, generally “unstudded” invaginated vesicles, which are preferentially found in the apical cell portion, but may be accumulated also in the direct vicinity of the basal cell membrane. “Studded” vesicles are sparse or lacking. The type B cells also display different manifestations. Between cells which are densely stuffed with SER and display very few mitochondria and very few “studded” membrane domains, and cells which contain large amounts of mitochondria, of “studded” vesicles, and which may be even densely covered with short microvilli, all intermediates can be found. Another configuration of possibly type B cells is found in the cortex of rabbits. These cells appear “constricted” at the level of the tight junctional belt where a prominent web of microfilaments is evident. Some elongated profiles of “studded” vesicles are found within and beneath this web. The narrow, apical cell pole is adorned with a tuft of long microvilli.260 Type B IC cells mediate secretion of HCO32 through apical Cl2/ HCO32 exchange, which functions in series with H1-ATPase-mediated H1 efflux across the basolateral plasma membrane.737 They are characterized by apical pendrin (Slc4A1)738
740 and basolateral H1ATPase. Type B cells reveal less carbonic anhydric activity than type A cells,702 and they express the chloride channel ClC 3727 in the apical cell pole. Metabolic alkalosis is compensated in the kidney by reducing bicarbonate reabsorption and increased bicarbonate secretion by type B IC cells. Type B IC cells adapt under chronic metabolic alkalosis with cellular hypertrophy.741 DNA synthesis and mitoses of type B cells have been recorded under this situation.742 Adaptive downregulation of pendrin in metabolic acidosis indicates the important role of this exchanger in acid
base regulation in the CCD.741 The type B

657

intercalated cell does not express either Rhbg or Rhcg detectable by immunohistochemistry. NON A-NON B CELLS

A third type of IC cells without evident polarity with respect to proton-APTase, and so far with undefined function, are called “non A-non B” cells.738 These cells are often much larger and protrude much more into the lumen than type A or B.709 In contrast to type A and B IC cells, neither the luminal nor the basolatreal plasma membranes reveal “studs,” at best “studs” are found on membranes of vesicular profiles in the cytoplasm. In mice this latter population of intercalated cells is more frequent than in rats.743 It decreases in either pronounced chronic metabolic acidosis or pronounced chronic metabolic alkalosis.492,711,744 On their apical plasma membrane they display, similar to type B IC cells, the anion exchanger pendrin (Slc4A1) and diffusely distributed H1-ATPase, occasionally also in the apical membrane. They express apical, but not basolateral, Rhcg and basolateral Rhbg.709 The observations on non A-non B cells, the striking structural diversity713,745 and the apparent plasticity of IC cells raised the question whether one, two or more distinct cell types are subsumed in the IC cell population or whether the different appearances are manifestations of different functional stages of the same cell type. Based on studies in collecting ducts in vitro from adult rabbits and IC cells cultured in vitro,746
749 (IC cells with the morphology of type B were identified by apical binding to peanut lectin),750 it was speculated that the IC cells might reverse their polarity in response to specific functional environmental conditions. Type A would present the terminal differentiation of IC cells. This hypothesis received support from studies in vitro showing that the matrix protein hensin could reverse the functional phenotype of cultured intercalated cells (for review see 492), and induce the type A IC cells. The non A-non B cells might represent intermediate stages. The diminution of this latter population under chronic acidotic or alkalotic conditions would agree with this hypothesis. Another view was that the non A-non B cells could be precursors for either A or B cells or only of B cells (for review see 492). This hypothesis would also be supported by the finding of a diminution of non A-non-B cells under chronic acidosis or alkalosis. The observations on mitosis in fully-differentiated type A,735,751 as well as in type B IC cells742 do not agree with the hypothesis on reversal of polarity, nor with the hypothesis claiming a common precursor cell of type A and B. Most IC cells also display on at least one membrane domain (more often on the luminal and vesicular than on the basolateral) the AMP-degrading phosphatidyl-inositol-anchored ecto-enzyme 50 nucleotidase

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20. STRUCTURAL ORGANIZATION OF THE MAMMALIAN KIDNEY

(50 NT)57,60,740,752,753 and the protein Connexin 30, that might function as plasma membrane ATP channel.754 It has been shown that increased flow in the distal nephron induces K secretion through the large-conductance, calcium-activated K channel (BK), which is primarily expressed in intercalated cells (IC). High distal flows and shear stress induce BK-dependent K efflux and ATP release from IC cells.295 These mechanisms might play a role in the purinergic autocrine and/or paracrine regulation of salt and water reabsorption.317 The intergral membrane proteins syntaxin 3755 and synaptotagmin VIII demonstrated in the basolateral membrane of IC cells,756 are possibly involved in the targeting of acid
base transporters and may participate in the basolateral membrane remodeling of IC cells in response to systemic acid
base perturbations. Interestingly, it had been reported that IC cells lack significant levels of Na-K-ATPase in the basolateral membranes.283 However, more recently, weak to moderate Na-K-ATPase was revealed in cortical and outer medullary IC cells, whereas IC cells in the upper part of the inner medullary collecting duct showed a staining intensity that was similar or even stronger to that in adjacent IMCD cells.757 Distribution of IC Cells The ratio of intercalated cells to other tubular cell types varies among and within species, and along the distal segments (DCT, CNT, upper and lower half of cortical collecting duct, outer and inner stripe collecting duct). Possibly, it may be altered by some functional conditions.744 Discrepancies in reported data for a given species may be rooted in poor definition of the investigated kidney region, and also in the criteria used for the recognition of cells. The relative number of intercalated cells (all forms) in the various segments is roughly B25
30% in the CNT, B40% in the CCD and OMCD, and B10% in the initial IMCD.699,718,743,758 IC cells are absent in deeper levels of the inner medulla in most species. In the rat, taking the morphological, cyto-, and immunochemical data together, it can be deduced that among the IC cells in the CNT the type A cells prevail. Type B cells are in the minority among the IC cells in the CNT in rat, but not in mouse. Type B IC cells constitute the majority of IC cells in the CCD, of which a varying proportion may present proton-ATPase in the basolateral cell membrane. Based on the different distribution of the proton-ATPase in non-type A intercalated cells, it has been suggested type B IC cells with only basolateral proton-ATPase staining and non-A/ non-B IC cells with bipolar or luminal proton-ATPase staining should be distinguished.743 However, both subpopulations carry pendrin staining, and may represent different states of activity.

In the CCD type A cells are in the minority, and often appear in their apparently functionally less active form, with less microprojections, but more intracellular “studded” vesicles than type A cells in the CNT.759 Accordingly, they display a slightly weaker luminal, but often a diffuse cytoplasmic, staining for the protonATPase. Apparently only one type of IC cells exists in the OMCD of rats, mice, and humans. It resembles the type A cells in the cortex. Disruption of IC Cell Characteristic Genes Inheritable forms of distal renal tubular acidosis (dRTA) most often affect the physiology of type A IC cells.760 Disruption of one of the IC cell characteristic genes leads to profound structural alterations of IC cell types. In mice with functional deletion of carbonic anhydrase II, the frequency of IC cells is drastically reduced.701 The genetic disruption of pendrin (Slc26a4) leads to marked reduction of type B cell size, with reduced H1/OH2 transporter expressions.506,738 In mice with disruption of the Foxi1 gene, upstream of several anion transporters, proton pumps, and anion exchange proteins expressed by intercalated cells, and of the collecting ducts cells, the normal collecting duct epithelium with its two major cell populations
collecting ducts cells (principal) and intercalated cells
has been replaced by a single cell type positive for both principal and intercalated cell markers.761

ARCHITECTURAL
FUNCTIONAL RELATIONSHIPS So far we have always emphasized the relationships between structure and function. However, we have neglected the important relationships between architecture and function
i.e., arrangements through which the close relationships between certain nephron and vascular portions permit the carrying out and coordination of complex regulatory functions. The two most obvious examples in this respect are the juxtaglomerular apparatus (JGA), regulating glomerular perfusion and renin secretion, and the renal medulla permitting the production of urine, varying in dilution and concentration.

Juxtaglomerular Apparatus The juxtaglomerular apparatus (JGA) is a composite assembly of specialized structures at the vascular pole of the glomerulus (Figures 20.28 and 20.29). The thick ascending limb of Henle’s loop (TAL) returns to its parent glomerulus and extends through the angle

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FUNCTIONAL RELATIONSHIPS

659

the terminal portion of the afferent arteriole housing the renin producing granular cells; the initial portion of the efferent arteriole; and the extraglomerular mesangium (EGM). The latter is in continuity with the intraglomerular mesangium, and has intimate relationships with the parietal epithelium of Bowman’s capsule.20 The JGA, more precisely the granular cells and the smooth muscle cells of afferent and efferent arterioles, are richly innervated by sympathetic nerves.

Macula Densa

FIGURE 20.66 Juxtaglomerular apparatus. (a) Meridional section through a glomerulus which runs through both glomerular arterioles (rat). The macula densa (MD) is attached to the extraglomerular mesangium (EGM), which fills the angle between the afferent (AA) and efferent (EA) arteriole. Within the wall of the afferent arteriole granular cells (G) are seen. Note the intraglomerular segment of the efferent arteriole (TEM: 3B1850). (b) Meridional section through a glomerulus running in between both arterioles (rabbit). The macula densa (MD) is a prominent cell plaque within the thick ascending limb. It covers the extraglomerular mesangium (EGM). Within the glomerular stalk the EGM continues into the mesangium (M). The EGM interconnects opposing parts of the GBM (one arrow) to the basement membrane of Bowman’s capsule (BCBM) (two arrows), as well as the first parts of the BCBM (three arrows). Note the dilated intercellular spaces between macula densa cells (TEM: 3B8100).

between afferent and efferent arterioles, where it is firmly attached to the extraglomerular mesangium (Figure 20.66a). At the attachment point, the TAL changes its character: a plaque of specialized cells, known as the macula densa (MD), represents the contact site of the tubule. Around this attachment, other specialized structures are developed which, together with the macula densa, comprise the JGA. These are:

Shortly before its end, the TAL passes between the afferent and efferent arterioles of its original glomerulus. At this site the basal face of the tubule is affixed to the extracellular matrix, enveloping the cells of the extraglomerular mesangium (EGM), tying together both arterioles. The TAL cells in contact with the EGM are transformed into the “macula densa” (MD). The MD is a cell plaque comprising some 20 to 30 specialized epithelial cells (in juxtamedullary nephrons more than in superficial nephrons). The MD completely and consistently overlaps the EGM, and may extend over variable portions of the afferent and efferent arterioles.429,762 The specific histo-topographical relationship of the epithelial tubular cells and the other components of the JGA at the glomerular vascular pole are established already during nephron formation. The prospective MD cells are affixed to the mesenchymal cells accompanying the capillary loops that invade the distal cleft of the S-shaped body of the nephron-anlage to form the glomerular tuft.763 This occurs before the epithelial cells of the prospective loop of Henle have elongated into a tubule. The cells of the MD (Figure 20.66) differ from the surrounding cells of the thick ascending limb in several aspects. The most eye-catching feature of the MD are the closely packed nuclei, usually located in the apical cell pole.5,764 This feature, well recognizable even in light microscopic preparations at low magnification, conferred the name “macula densa” to the cell plaque. Most importantly, and in marked contrast to the cells of the thick ascending limb, MD cells do not interdigitate with each other by large lateral folding; rather, the lateral cell membranes of MD cells run in a fairly straight fashion from the tight junction toward the base of the epithelium.5,764 They possess slender microplicae or microvilli that protrude into the lateral intercellular spaces, and contact (frequently by desmosomes) corresponding protrusions from opposite cells. At the very base the cells ramify into slender processes. They are fixed to the basement membrane of the MD cells which is fused with the basement membrane-like material

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surrounding the extraglomerular mesangial cells. The tight junctions are morphologically similar as in the TAL, but they may be slightly deeper (e.g., in rabbit). Like all other TAL cells, MD cells do not display gap junctions. The cytoplasm of MD cells is relatively sparse and displays the usual organelles comprising some small mitochondria. The Golgi apparatus is large, smooth endoplasmic reticulum and free ribosomes are abundant, but rough endoplasmic reticulum is infrequent. The luminal cell membrane is densely studded by short stubby microvilli and displays, like the other tubular cells, single cilia. In some species (e.g., rabbit5) the MD cells are distinctly taller than the surrounding TAL cells, so that the entire plaque of the MD protrudes into the tubule lumen. The inventory of transport proteins in MD cells is essentially the same as in the other TAL cells, i.e., they display the bumetanide-sensitive NKCC2 co-transporter,537,765,766 ROMK,767,768 and NHE3769 in the apical plasma membrane, and express cyclooxygenase-1.516 They specifically express cyclooxygenase-2516 and nitric oxide synthetase 1.770,771 In contrast to the TAL, MD cells lack the Tamm-Horsfall protein.528 Recent findings772 detected by RT-PCR and by immunohistochemistry demonstrated olfactory-related adenylate cyclase 3 (AC3) and the olfactory G-protein limited to the distal convoluted tubule and especially the MD.772 These findings suggest a role of the olfactory machinery in the regulation of renin secretion and glomerular filtration rate.772

In contrast to all other TAL cells, the lateral intercellular spaces in the MD epithelium have been found to be dilated under most physiological conditions, usually regarded as “normal” conditions.754,764,773
776 In agreement with the suggestion that water flow through the MD-epithelium is secondary to active sodium reabsorption, compounds that block sodium transport by MD cells (e.g., furosemide), as well as high osmolalities of impermeable solutes in the tubular fluid (e.g., mannitol), are associated with narrow intercellular spaces.764,773 These observations suggested that the MD epithelium might be a water-permeable cell plaque within the water-impermeable TAL epithelium,764 but so far direct evidence for this suggestion is missing. The lack of immunoreactivity for TRPV4, a nonselective cation channel of the transient receptor potential (TRP) family, gated by hypotonicity, had been interpreted as indirect support for this assumption.777 The granular cells (often termed juxtaglomerular cells) (Figure 20.67)778 are assembled in clusters (up to 15 cells, but generally not more than 4 or 5) within the wall of the terminal portion of the afferent arteriole, replacing ordinary smooth muscle cells. Occasionally, they are also found within the wall of the efferent arteriole, again occupying the space where one would otherwise expect to find an ordinary smooth muscle cell. In rare cases, extraglomerular mesangial cells may also be replaced by granular cells. The name “granular” cell points to the specific cytoplasmic granules which may densely fill the cell body cytoplasm. They are electrondense, membrane-bound, and irregular in size and

FIGURE 20.67 (a) Juxtaglomerular portion of an afferent arteriole. Smooth muscle cells are replaced by two granular cells (rabbit: TEM: 3B2700). (b) Granular cell. Renin granules are membrane-bound. Granules with a crystalline substructure are considered as “protogranules” which will develop into mature amorphous granules (rat: TEM: 3B48,000).

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ARCHITECTURAL
FUNCTIONAL RELATIONSHIPS

shape. Small granules with crystalline substructure represent protogranules, which are developed within the prominent Golgi apparatus and are then transformed into the major amorphous granules. Immunocytochemical studies with two antibodies against the renin prosegment and against mature renin have shown that only protogranules are prosegmentpositive, whereas a signal of mature renin was found in mature as well as protogranules. These findings show that the cleavage of the prosegment, i.e., the maturation of renin, takes place in the juvenile granules; mature renin is then stored in the electron-dense granules. However, it is suggested that a major fraction of prorenin never matures to renin, but is constitutively secreted as pro-renin
together with an unknown fraction of renin. Mature renin is segregated into storage granules for regulated release. The release mode of renin is not fully-understood. In addition to classic exocytosis, other mechanisms may also be involved.18 It is important to know that renin release occurs into the surrounding interstitium, not into the lumen of the afferent arteriole, as has been frequently suggested. Granular cells are modified smooth muscle cells. Within the peripheral parts of the cytoplasm, especially within the many cell processes, granular cells contain myofibrils. In situations that require enhanced renin synthesis (e.g., volume depletion or stenosis of the renal artery) additional smooth muscle cells located upstream in the wall of the afferent arteriole transform into granular cells.19 Granular cells have processes of manifold shapes.99 Because of them, granular cells have extensive

661

membrane contacts to all surrounding cells, e.g., other granular cells, smooth muscle cells, and extraglomerular mesangial cells. At these contacts, gap junctions are frequently encountered.19 Like ordinary smooth muscle cells, granular cells also have membrane contacts to endothelial cells, in the manner that foot-like processes of endothelial cells penetrate the basement membrane and come into contact with granular cells; gap junctions are found at these contact sites.99 Peripolar cells have first been described in sheep, where they are regularly found779; in most other species, including man, they are rare.780 Peripolar cells are parietal cells of Bowman’s capsule which are located around the glomerular hilum (i.e., at the vascular pole, therefore: peripolar), and which contain numerous cytoplasmic membrane-bound granules filled homogeneously with electron-dense fibrillogranular material.779 Subsequent studies have shown that these granules contain a neuron-specific enolase-like protein781 and transthyretin782; their function is unknown. The number of cells and the number of granules per cell vary greatly among species and, furthermore, are dependent on age.780 In the rat kidney, granulated peripolar cells have only rarely been found.780 Extraglomerular mesangial cells (EGM-cells, Goormaghtigh cells, lacis cells) together with the surrounding matrix establish the extraglomerular mesangium (polar cushion). The EGM represents a solid cell complex that is not penetrated by blood vessels or lymphatic capillaries. Nerves pass on both sides of it from the afferent to the efferent arteriole, but do not enter the cell complex.99

FIGURE 20.68 (a) Schematic of the extraglomerular mesangium (EGM). The glomerulus is shown as a globe. Its outer aspect is represented by the parietal basement membrane of Bowman’s capsule (PBM). The EGM lies between the two arterioles above the opening of Bowman’s capsule (broken line). It is attached to the PBM and has extensive contacts with the two arterioles. The macula densa and the smooth muscle layers of the arterioles are not shown. (b) Schematic cross-section through the vascular pole just above Bowman’s capsule. Afferent and efferent arterioles (AA, EA) are cut transversely. Extraglomerular mesangial cells (EGM) are shown in moderate gray, smooth muscle cells in dark gray, and endothelial cells in light gray. Note differences in the walls of AA and EA; the AA already displays endothelial fenestration on the side facing the EGM. Conversely, the EA has a continuous endothelium with many cell bodies. In both AA and EA the smooth muscle layer (SM) is not complete; towards the center of the EGM the SM cells are replaced by EGM cells. (Elger, M., and Sakai, T. et al. (1998). The vascular pole of the renal glomerulus of rat. Adv. Anat. Embryol. Cell Biol.139, 1
98, with permission).

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The EGM is located within the triangular space bordered by the two glomerular arterioles and the macula densa (Figures 20.66 and 20.68).778 Reconstruction studies have shown that EGM-cells are flat and elongated, separating into two bunches of long cell processes at their poles.783 They are arranged in several layers parallel to the base of the macula densa. The cells nearest to the glomerular stalk, thus filling the deepest portion of the triangle, lose this parallel grouping, but extend into the stalk of the glomerular tuft mixing with mesangial cells proper. The cells are separated by a conspicuous matrix which appears to be different from

a

b

FIGURE 20.69

c

Flat section through the extraglomerular mesangium (rat). (a) The section crosses the afferent (AA) and the efferent (EA) arteriole; it grazes the top of Bowman’s capsule, showing the basement membrane of Bowman’s capsule (BCBM), and the parietal epithelium (PE), as well as the urinary space (US). The extraglomerular mesangium forms a complicated texture by which the structures of the vascular pole are interconnected. Note that toward their insertion in the BCBM the extraglomerular mesangial cells fall apart into many processes (stars) (G: Granular cells; TEM: 3B2650). (b) Higher magnification of extraglomerular mesangial cells. Note the microfilament bundles within the periphery of cell bodies, as well as within cell processes (arrows). Note the irregular extracellular spaces filled with a matrix of varying appearance (star) (TEM: 3B6000). (c) Gap junction between two mesangial cells (rat: TEM: 3B147,000).

the intraglomerular mesangial matrix by the fact that microfibrils are rarely found in the EGM20; details are largely unknown. EGM cells are characterized by the scantness of their cytoplasm and their extensive ramifications (Figure 20.69).778,784 A Golgi apparatus and some profiles of granulated endoplasmic reticulum are regularly encountered. Although direct evidence is lacking, EGM-cells can be expected to be contractile for several reasons. First, they contain a good amount of microfilaments, mainly in their processes and peripherally within cell bodies. Second, intimate structural similarities are found among arteriolar smooth muscle cells, granular cells, and intra- and EGM cells, suggesting that they have the same origin. Third, they are extensively coupled by gap junctions. Gap junctions not only bridge different cells, but also regularly bridge individual processes of the same cell.785 Moreover, gap junction contacts consistently occur to all other cells of the JGA (except the macula densa!), i.e., to granular cells, to ordinary smooth muscle cells of both arterioles, and to the mesangial cells proper.19 From a biomechanical point of view, the contractile apparatus of EGM cells is conspicuous. Microfilament bundles are contained within the periphery of cell bodies and within the cell processes, which are connected to the walls of both glomerular arterioles and to the basement membrane of the parietal layer of Bowman’s capsule (PBM) surrounding the glomerular hilum (Figures 20.68 and 20.69). As a whole, the EGM can be considered as a spiderlike contractile clamp sitting above the glomerular entrance interconnecting all structures at this site.20 The EGM probably represents some sort of closure device of the glomerular entrance, maintaining the structural integrity of the entrance against the distending forces exerted on it by the high intraglomerular pressure. Moreover, from the viewpoint that the glomerular mesangium represents a high pressure compartment (mesangial interstitial pressures are expected to range in the same magnitude as glomerular capillary pressures147), the EGM would seem to be the structure which mediates a gradual pressure drop toward the cortical interstitium and toward the base of the macula densa.20 The function of the EGM cells is obscure. Because of their central position within the JGA, their constant relationships to the macula densa and their gap junction coupling to all smooth muscle-derived cells of the JGA, the EGM cells have repeatedly been considered as the necessary functional link between the macula densa and any possible effector cell within the regulatory mechanisms of the JGA.20,99 Thus, they are widely considered as an integrating system of signals derived

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THE RENAL MEDULLA

from the reabsorptive function of the MD and the function of the EGM as a pressure sentinel mirroring the blood pressure in the afferent
efferent arteriolar system, but details are unknown. The intimate and systematic juxtaposition of tubular and vascular cells within the JGA has given rise to early speculations about a feedback system between tubular and glomerular function.786 It has now become clear that the JGA serves two different functions: it regulates the flow resistance of afferent arterioles in the so-called tubuloglomerular feedback mechanism; and it participates in the control of renin synthesis and release from granular cells in the afferent arteriole.787 Researchers originally assumed that the two responses might be related to each other, in that renin released from the granular cells not only has systematic relevance, but locally triggers the formation of angiotensin II, and thus is responsible for afferent vasoconstriction as well; however, it now appears that the final activation of smooth muscle and granular effector cells occurs through largely independent pathways. Renin release from granular cells is the major source of systemic angiotensin II, and thus plays an essential role in controlling extracellular volume and blood pressure, whereas the vasoconstriction of the afferent arteriole locally serves to modulate the filtration of this nephron. For both mechanisms, it is well-established that a change in NaCl concentration in the tubular fluid at the MD initiates the appropriate signal. Thus, the MD, situated at the very end of the TAL, controls the work of the TAL; the short postmacula segment of the TAL may be interpreted to guarantee that the composition of the tubular fluid at the MD might not be influenced by the function of the subsequent DCT. Expressed in general terms, the MD translates changes in the tubular fluid Na-Cl concentration into a graded release of mediators that reach their target by diffusion, thus acting in a paracrine fashion. Note that the extraglomerular mesangium that mediates the contact between the MD and the effector cells is not vascularized, so that the build-up of any paracrine agent would not be perturbed by blood flow. With respect to renin release, the most likely paracrine mediators of this process are prostaglandin E 2 and nitric oxide.18,788
790 With respect to the vasoconstrictor response purinergic mediators, either ATP or adenosine, as first suggested by Oswald and colleagues791 appear to play the major role.62,787,792 For an up to-date discussion of the function of the JGA see the reviews by Schnermann and Levine,787,793 Persson and colleagues,794 and Komlosi and colleagues.795,796

THE RENAL MEDULLA During phylogeny the renal medulla has developed in response to the necessity to conserve water by excreting concentrated urine.29 Loops of Henle, collecting ducts, and a specific blood supply through vascular bundles have developed into a complex structural system that accounts for this function. However, the details are insufficiently understood. The overall mechanism (Figure 20.70) is clear: reabsorption of NaCl from the MTALs in the outer medulla represents the driving force to produce an interstitial cortico-medullary osmotic gradient that provokes osmotic water withdrawal from the collecting duct when the latter descend toward the papillary tip. The reabsorbed water is brought back into the systemic circulation by venous vasa.2,797 The unresolved problem is the generation of a cortico-papillary solute gradient, notably in the inner medulla. In discussions concerning the formation of a medullary solute gradient “countercurrent multiplication” has occupied a center-stage as the decisive mechanism. This mechanism has been experimentally established in artificial tubes,798 and has been imposed on the renal medulla, conceding immense deviations from the original conditions. From a structural point of view, the preconditions for countercurrent multiplication in the renal medulla would appear to be quite incompletely developed: at no site are the limbs of Henle’s loop juxtaposed to each other. Even when allowing a mediating interstitial space between both loop limbs, the DTLs do not case behave homogenously in an adequate way, but they change their function gradually on their descent, and even change their transport characteristics to the ascending limb type a considerable distance before the bend; most relevant in the present context is that the terminal third of the SDTLs in the mouse kidney is equipped with TAL epithelium.16,34 Without going into more details, everyone who has been engaged in this problem knows that the principle of countercurrent multiplication has been extensively bent to make it fit with the structural organization of the renal medulla
in our view, with little benefit in facilitating the understanding of the function of the renal medulla. If at all, this principle can only be applied to describe the mechanism in the outer medulla. In the inner medulla, a process that could be regarded as a “single concentrating effect” is not apparent. Several “passive models”452,799
802 attempting to explain the concentrating mechanism in the inner medulla have greatly refined our understanding of the problem, but have never reached the level of a convincing theory.

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FIGURE 20.70 Schematic to show the functional interactions in the medulla as they are derived from the histotopography of the structures, the distribution of channels and transporters and direct measurements of transport characteristics in the various tubular and vessel segments. A long looped nephron, a short looped nephron, and a collecting duct are shown in light gray. Descending vasa recta (DVRs) derived from the efferent arteriole of a juxtamedullary glomerulus are shown in white (including the capillaries), ascending vasa recta (AVRs) in dark gray: both together establish a vascular bundle. The osmolar concentration in the medulla rises from the cortico-medullary border to the papillary tip from 300 to 12,000 mosmol/l, mainly established by the increase in the concentration of salt (indicated by dark dots) and urea (indicated by open circles). The driving force of the concentrating mechanism is the dumping of salt into the medullary interstitium from TALs (thick black arrows), leaving behind a diluted fluid (indicated by an osmolar concentration of 100 mosmol/l at the re-entry into the cortex). Osmotic water withdrawal from CCDs (slim arrows) into the cortical circulation again elevates the tubular urine to 300 mosmol/l upon re-entry into the medulla. Continuous water reabsorption along the MCDs (slim arrows) will produce a final urine concentration of about 12,000 mosmol/l (in humans). The source for the inner medullary solute gradient is shown to consist of: (1) dragging of salt from the IS into the IM by LDTLs (arrow heads); and (2) re-entry of urea from the CDs into the terminal portion of the IM (hatched arrows). The gain in osmotic energy by urea re-entry originates from urea recycling, which starts with a shift of urea from the AVRs into the SDTLs in the IS (follow the hatched arrows), and concentration of this urea by water reabsorption in the CCDs, OMCDs, and starting portions of IMCDs (see text for further explanation). The removal of the water from the medulla regained from the CDs (thus the final step in urine concentration) is effected by AVRs (follow the slim arrows). These vessels are the core structures in the complex countercurrent exchange system of the medulla equilibrating at any level with the local concentrations of salt and urea. Open thick arrows show passive movements of salt (see text).

In this situation it might be worth an attempt, opposite to the usual, to start with the available functional data
including the recent data on the distribution of transporters
confronting them with the structure, i.e., the architecture of the renal medulla, as well as the cellular organization of the individual

components, in order to arrive at a novel view of “function
structure
correlation.” Let us first regard the three regions of the medulla with such an approach. The most constant region is the IS; there is no renal medulla known without an IS. In contrast, an OS is frequently quite incompletely developed, and an IM may be fully absent.

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The Inner Stripe of the Outer Medulla The IS (Figures 20.20 and 20.24) is made up of two portions: the vascular bundles (VBs) and the interbundle region (IBR). The IBR contains the tubules (DTLs, TALs, CDs) supplied by a dense capillary plexus which is drained upwards by the gradual transition of capillaries into AVRs that directly ascend into the OS. Since all the salt reabsorbed by MTALs accumulates in this area, the interstitium of the IBR is rich in salt.803 The VBs, structurally, are part of the IS but, functionally, they belong to the IM. They represent a quite perfectly developed countercurrent exchange system primarily handling the blood descending to and ascending from the IM by respective DVRs and AVRs. However, since the DVRs also supplying the capillary plexus of the IBR of the IS are contained within the VBs (not the respective AVRs), the VBs provide the possibility of shifting solutes coming up from the IM into the IBR of the IS. Since the dominating solute of the IM is urea, the VBs are rich in urea. The handling of urea as a main function of the VBs becomes most obvious in the complex bundles (see below).

The Inner Medulla The IM (Figures 20.20 and 20.27) including the papilla, at the transverse level, is homogenously organized; a separation into VBs and an IBR is no longer possible. Even if there may be a certain prevalence that the ATLs are more frequently gathered around CDs than DTLs,34,804 it appears quite doubtful that this is of any functional relevance. The AVRs (including the capillaries) are homogenously distributed among all other components and, most importantly, a wide homogenous interstitial space permits the interaction of every descending tube with every ascending tube. The IM provides strict countercurrent arrangements of all involved structures, but without giving prevalence to any specific lateral interaction. Thus, the IM as a whole may be considered as a countercurrent system that allows countercurrent exchange
mediated by the interstitium
between all descending (DVRs, DTLs, CDs) and all ascending tubes (AVRs, ATLs), according to the transport characteristics of the individual tubes. A most important feature of the IM is its particular shape reflecting its longitudinal organization. The inner medulla tapers from a broad basis to a tiny papilla2 (see also above). This shape perfectly reflects what happens with the structures within the inner medulla: loops of Henle, vasa recta, and collecting ducts (by fusing together) all decrease rapidly in number from the base to the tip of the papilla.119,805 For the rat, it has been calculated that, of an estimated 10,000 long loops entering

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the inner medulla at its base, only about 1500 reach the papillary half of the inner medulla, and only a few of these the papillary tip.2 The majority of long loops, the “short” long loops, turn back shortly after entering the inner medulla, a smaller but still substantial number of long loops reach the middle part of the inner medulla, and only a small population of “long” long loops really reach the papilla.

The Outer Stripe of the Outer Medulla The OS (Figures 20.20 and 20.23) is a transitional region which separates the medulla from the cortex, mediating the transition between an hyperosmotic and an isoosmotic environment. The OS does not seem to make any particular contribution to the creation of the cortico-medullary solute gradient, but it greatly helps to maintain it. The OS contains the nascent (or dissolving) VBs (performing the same function as in the IS but quantitatively of minor importance) and the AVRs which are directly coming up form the IBR of the IS (Figure 20.71). Together with the AVRs spreading out from the dissolving VBs, the AVRs as a whole traverse the OS as individual vessels intimately associated with the tubules of this region; actually they represent the major “capillary” supply of the OS (see above). In addition, among all regions of the kidney, the OS exhibits the smallest fraction of interstitial space, thus the vessels are most closely juxtaposed to the tubules (note: lymphatics are absent from the entire medulla25). Since the PSTs of juxtamedullary nephrons, in contrast to their name, take a tortuous course when descending through the OS, the majority of tubular profiles in the OS consists of PSTs (S3 segments). This arrangement
AVRs closely associated with descending PTs (and, to some extent, also CDs) represent an ultimate countercurrent trap to prevent the loss of osmotic energy into the systemic circulation (Figure 20.71 right panel). Since reabsorption from PTs is isoosmotic, the hypertonic environment created by AVRs will allow water withdrawal not only from CDs (starting the concentrating process), but also from the PTs, increasing their osmolarity already at the level of the OS
with a clear prevalence of the PTs of juxtamedullary nephrons (tortuous course!) that give rise to the “long long loops.” The TALs of the OS are already of the cortical type (equipped with a comparably flat epithelium; see above) capable of maintaining and even increasing a large salt gradient, but incapable of transporting large quantities.308 So far, we have a summary of the essential architectural features of the three medullary regions; let us now talk about the functional connections between them. This needs, first, to talk about the overall mechanism

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(a)

(b)

(c)

C

OS

IS

IM

FIGURE 20.71 Schematics to demonstrate the possible recycling routes within the medulla. A short loop and a long loop of Henle and a collecting duct are shown. The straight proximal tubules are hatched; the thin limbs, collecting ducts, and capillaries are white; the thick ascending limbs are gray. Ascending vasa recta are drawn black en bloc (C: cortex; OS: outer stripe; IS: inner stripe; IM: inner medulla). (a) Simple type of medulla: recycling route from the ascending vasa recta in the inner stripe, via descending vasa recta, to inner stripe tubules. (b) Complex type of medulla: recycling route from ascending vasa recta in the inner stripe to descending thin limbs of short loops. (c) Recycling route from the ascending vasa recta in the outer stripe to descending tubules (proximal tubules and collecting ducts); valid for both the simple type and the complex type of medulla. (Adapted from Kaissling, B., and Kriz, W. (1979). Structural analysis of the rabbit kidney. Adv. Anat. Embryol. Cell Biol. 56, 1
123, and Kriz, W., and Barrett, J. M. et al. (1976). The renal vasculature. In “Anatomical
Functional Aspects. Kidney and Urinary Tract Physiology II, 1
21, Thruau, K. Tokyo University Park Press, Baltimore, London.)

underlying urine concentration in some more detail, and afterwards to talk about the individual mechanisms. The Basic Mechanism in Some Detail The renal medulla contains the phylogenetically ancient “diluting segments” of the nephron, i.e., the TALs which separate salt from water (Figure 20.70). The salt is dumped into the medullary interstitium, the water is carried up into the cortex and
in the case that ADH is available
is recovered by the systemic circulation through osmotic withdrawl from the CCDs. Thus, the tubular urine that re-enters the renal medulla in the collecting duct is isoosmotic with respect to plasma concentration, and considerably reduced in quantity compared to the amount that originally entered the renal medulla in descending limbs after filtration.

Thus, the salt that is available to drive the concentrating mechanism has emerged from a much larger quantity of isoosmotic fluid than the quantity of isoosmotic fluid that is subject to concentration. Moreover, along with the increasing concentration of the CD urine, less and less water has to be reclaimed to achieve the same increment in concentration; the work that is necessary to account for a progressively increasing urine concentration decreases steeply toward the tip of the papilla. In the IM, in addition to salt, urea is a major solute accounting for the solute gradient toward the tip of the papilla. Since in the IM neither any up-hill transport of salt nor of urea is known, the crucial problem consists of explaining the increasing concentration of salt (flat increase) and of urea (steep increase) in the IM toward

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the papillary tip. If both depend on the work of the TALs in the OM, how can part of the osmotic energy be carried down from the OM into the IM and piled up there to a steep solute gradient toward the tip of the papilla? In our view, three major mechanisms have become apparent that are responsible for the distribution of salt and urea into a cortico-papillary gradient in the IM (Figure 20.70). They all depend on salt reabsorption by TALs in the OM; they all fit with the morphology, even more: specific structural elaborations in highly concentrating species support their relevance. These are: (1) salt dragging by flow to deeper medullary levels; (2) countercurrent exchange of solutes and water to maintain the cortico-papillary gradient; and (3) urea recycling by short loops of Henle as a major mechanism to create the solute gradient toward the papilla. Dragging of Solutes by Flow to Deeper Medullary Levels In all descending tubes of the renal medulla (DVRs, DTLs, CDs) solutes are dragged by flow to deeper medullary levels. This appears to be relevant for salt in the DTLs and for urea in the CDs (see urea recycling). Continuous uptake and dragging of salt by SDTLs down to their bends at the end of the IS has been considered as an essential mechanism in the original countercurrent multiplication concept. In the light of current knowledge such a mechanism in short DTLs may not be of crucial importance. In contrast to previous reports, SDTLs (apart from some of them; see above) do not express aquaporin, and thus must be considered as fairly water-impermeable. Also, the Na1 and Cl2 permeability was found to be low.806 Thus, the increasing concentration towards the bend of SDTLs seems to be predominantly accounted for by urea entry into the lower part (see below: urea recycling). However, in any case the salt left from filtration at the end of the proximal tubule that enters the SDTLs in the OS will be dragged down by flow until the loop bends and will return in the TALs to be reabsorbed there. In case that, despite low salt permeability, a small part of the reabsorbed salt may
via the interstitium
re-enter the descending limb, thus being trapped within the SDTLs, this salt transfer from the TALs to the SDTLs represents the only esssential step that would be left from the countercurrent multiplication principle. In contrast, continuous uptake of salt by LDTLs in the OM and dragging it by flow down into the IM appears to be the essential mechanism for salt accumulation in the IM; no other source of salt (apart from a small quantity reabsorbed by IMCDs) for the IM is

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obvious. Available models of the inner medullary concentrating process do not put so much emphasis on this mechanism, but the structural data strongly do suggest it. Layton and colleagues452 were the first to include this idea into a model. The structural arguments are the following: 1. The upper portions of the LDTLs, most obvious in the IS, have an epithelial organization that suggests ion transport through extensively developed (Figure 20.48a) claudin 2 (cation pore) and claudin 10 (anion and cation pore) containing tight junctions.261,807 Since the salt concentration is certainly higher outside the LDTLs (see below), salt can readily be expected to diffuse into the lumen. This process may become reinforced by active saltsecretion into the lumen, based on the abundant occurrence of Na1-K1-ATPase in the upper portions of LDTLs.446,447 Moreover, the LDTLups are the only thin limb segments with abundant expression of aquaporin 1 channels (see below). No question, the salt concentrations within LDTLs at the transition from the OM to the IM may be expected to be quite high. Measured data are not available, but it seems reasonable to suggest considerably higher salt concentrations inside than outside. 2. The LDTLs on their descent through the inner stripe always pass through the sodium richest area, i.e., distant from the vascular bundles among the TALs of short loops803 (Figure 20.22). In the mouse kidney, two specific modifications in this system appear to reinforce the ability of LDTLs for salt uptake. First, the LDTLs take a tortuous course when descending through the IS (increasing their length by 27%34) and, second, at the end of the IS before entering the IM, they traverse the so-called “innermost stripe,” which has a thickness of about half of the IS proper and in which the SDTLs are already equipped with the TAL epithelium.16,34 Thus, the density of TALs dumping salt into the interstitium at this level is dramatically higher (5 versus 3 per unit area) than at any other level of the OM. Consequently, the interstitial salt concentration should be very high. The LDTLs traverse this region and may readily be expected to take up some of the accumulated salt. 3. After challenging urine concentration in rats by water deprivation (or treatment with ADH) the TALs hypertrophy, with the most prominent increase in epithelial salt transport capacity of the initial portions in the deep IS.520,808 Thus again, a prominent “surplus” in salt accumulation occurs just at the border to the inner medulla. 4. As described above, the LDTLs are quite heterogeneous with respect to their actual length,

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and with respect to the epithelial differentiation of their upper parts.435,803 Most important, the actual length of an LDTL strictly correlates with the degree of epithelial elaboration of its upper part in the IS. The small fraction of longest long loops extending down into the papilla clearly have the most prominent upper parts in the IS: they are thicker, with larger lumina and with more elaborate epithelia composed of heavily interdigitating cells; also their abundance in Na-K-ATPase is spectacular.446 Comparing these longest long loops with those extending down into the IM for only short or intermediate distances, there is a continuous spectrum also with respect to the epithelial elaboration in the IS. Thus, the idea is plausible that salt is taken up by the upper portions of LDTLs, and subsequently carried down through the salt impermeable lower portions to the bend region. Beginning abruptly with a prebend segment, the loop becomes ion-permeable, allowing the salt to be dumped into the interstitium. In the mouse, the prebend segments are quite prominent comprising a length up to 700 μm.34 Thus, the entire loop bend appears to represent a loop segment that delivers salt into the interstitium. Since the lower portions of LDTLS are also waterpermeable (to what extent is not clear452,453), water withdrawal may further increase the salt concentration in these segments. The problem consists of the driving force for such a process. A possibility offered from a structural view is that the heterogeneous longitudinal distribution of loops within the inner medulla (as describe above) might account for a cascade-like transport of salt toward the papilla. The large fraction of “short” long loops carries salt into the first third of the IM. Together with some urea (emerging from reabsorption by papillary CDs and subsequent recycling; see later) the total interstitial osmolality will be elevated above DTL fluid osmolality at the respective level allowing reclaimation of water from DTLs, elevating the total osmolality and salt concentration in all the DTLs underway to deeper medullary levels. A major fraction of those will reach an intermediate level of the IM before bending, dumping a major fraction of its salt at this level into the interstitium. Again, together with some urea a driving force will be established accounting for some water withdrawal from the small fraction of “long” long loops that will carry their salt into the papillary tip. No question, this is quite a hypothetical idea, but from a structural point of view would be worth modeling. Ideas in this direction have been published previously,435 and also mathematical models for some features of such a mechanism have been presented.452

Countercurrent Exchange of Solutes and Water Countercurrent exchange in a U-type countercurrent exchanger may have two functions: (1) trapping of solutes within the system by transfer of solutes from the ascending to the descending limb; and (2) preventing water from entering the system by short circuiting from the descending to the ascending limb. Both mechanisms per se do not build up any solute gradient; they are only capable of maintaining a gradient (with little loss). However, incorporated into the complex countercurrent arrangement of the renal medulla they decisively participate in the creation of the cortico-papillary gradient. In agreement with others,797 in our view, the relevance of countercurrent exchange has always been underestimated compared to countercurrent multiplication in the urine concentrating mechanism. Actually, from a structural point of view, the renal medulla should best be considered as an extremely complex countercurrent exchange system that is fuelled by two mechanisms at two sites: by salt reabsorption through TALs in the OS; and by urea addition through terminal CDs in the IM. These solutes are distributed by countercurrent exchange into a cortico-medullary gradient that
due to continuous fueling
allows water withdrawal from CDs and the transport of this water into the systemic circulation in the cortex. As already discussed above, a more-or-less direct countercurrent exchange between juxtaposed countercurrent tubes may only occur within the VBs of the IS. In contrast, in the IBR of the IS, and within the entire IM, the countercurrent tubes are consistently separated from each other by comparably wide interstitial spaces, thus any exchange at a transverse level is mediated by the interstitium. No doubt, this decreases the effectiveness of the exchanges, but allows countercurrent exchanges between more than two structures. Any solute dumped into the inner medullary interstitium, i.e., predominantly salt from LDTLs (at their bends) and urea from CDs (at their terminal portion) or left in ATL and AVRs at their beginning in the IM, will be subject to countercurrent trapping (or used to drive countercurrent short-circuiting of water; see below), between AVRs and ATLs on the one site and DVRs and DTLs on the other. In addition to solute trapping, countercurrent exchange of water preventing the flow of water to deeper levels by short circuiting seems of major relevance. The recent elucidation of the distribution of aquaporins in medullary structures has shed considerable light on the handling of water in the urine concentrating process. Among the thin limbs only the LDTLups have regularly been found to be equipped with aquaporin 1 channels; in rat, also, the LDTLlps have been reported to express AQP1453; DVRs are abundantly equipped

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with AQP1 channels453; the water permeability of CDs is ADH-dependent (see above). The only ascending structures that are water-permeable are the AVRs (based on the hydrophilic fenestrae providing a direct route of water flow through the endothelium). Thus, any water withdrawn from any descending structure finally enters an AVR, by which it is brought back to the cortex. This cardinal function of AVRs has recently been analyzed in detail by Pallone and colleagues.797 The ability of AVRs to take up water appears to be based on an elevated oncotic pressure (resulting from short circuiting of water between DVRs and AVRs within the VBs), and on the reasonable assumption that the blood in capillaries at any level of the medulla is in osmotic equilibrium with the interstitial fluid (due to the hydrophilic fenestrae in capillary and AVR endothelia). Thus, when capillary blood at any level of the medulla starts to assemble in AVRs and to ascend, it will
after any small ascent
become hyperosmotic compared to the surrounding interstitium. Due to the abundant hydrophilic fenestrae of the AVR endothelium osmotic equilibrium will most easily be achieved by water uptake. The water to be taken up originates mainly from the CDs, but water may also originate from DTLs. In LTDLs, the upper parts have abundant aquaporins (type 1) in both membranes surprisingly contained in an epithelium that exhibits the features of an ion-permeable epithelium, i.e., an extremely lengthened leaky tight junction due to extensive cellular interdigitation. Thus, this epithelium, in addition to being water-permeable, obviously allows the transport of large amounts of ions. From what we have argued above, that salt dragging by flow in LDTLs appears to be the only mechanism to bring down the salt into the IM, large volumes of salt-enriched urine would be the best precondition for this goal. It appears to us that the actual transport capabilities of the upper portions of LDTLs have not been completely elucidated. Along the lower portions of LDTLs in the IM, the water permeability progressively decreases and fully ceases at the transition to the prebend segment.437,438 This offers the possibility (described already above) that water withdrawal from the LDTLs by an osmotic driving force established at each medullary level by salt and urea might lead to a “cascade-like” transport of salt toward the tip of the papilla.

Urea Recycling Solute recycling is a different mechanism compared to solute trapping by countercurrent exchange; this difference is rarely appreciated. Urea appears to be the only solute, recycling of which plays an essential role

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in the urine concentrating process. Recycling of urea defines a process that starts with a molecule of urea in the inner medullary interstitium and brings this molecule back into the IM via the normal tubular route, finally re-entering the inner medullary interstitium via exit from the terminal IMCD (Figure 20.70). As described above, the terminal portion of the IMCDs contains the urea transporters UTA1 and UTA3 which allow the facilitated diffusion of urea from the CD into the surrounding interstitium.809 A precondition for this reuptake of urea is that the concentration of urea in the tubular urine of IMCDs has reached a higher level than in the medullary interstitium at the same level. This is achieved by water removal through urea-impermeable tubular segments, i.e., CNTs and CCDs in the cortex, OMCDs and upper IMCDs in the medulla. The reabsorption of urea through IMCDs compromises the reclaiming of water through IMCDs, decreasing urine concentration instead of increasing it. Thus, urea does both: on the one hand, reabsorption of urea increases the inner medullary solute gradient, thereby decreasing urine osmolality; on the other hand, the increased solute gradient increases the driving force for water reabsorption, thereby increasing urine osmolality. This sounds like a story from Baron Mu¨nchhausen, who was able to help himself getting out of a swamp by pulling on his own hairs. However, the two opposing functions of urea have a solid base: a given molecule of urea may become active a second (or a third) time, this is what urea recycling means. After entering the inner medullary interstitium from an IMCD, a given molecule of urea will travel towards the cortex within an AVR, thereby balancing (together with other molecules and other solutes) a certain amount of water that enters the AVR along its ascent, and that finally has to be delivered into the systemic circulation at the cortico-medullary border. However, this specific molecule of urea participates in water balancing on its way from a hyperosmotic to an isoosmotic environment only up to an intermediate level, i.e., up to the IS. Here it reenters the nephron; its relevance in balancing water that continuously enters the AVR and needs to be transported further up into the cortex is taken over by salt which, due to the active Na1 reabsorption by TALs, is available in abundance at this level. Thus, the urea molecule has spent only part of its osmotic energy for the transport of water before it re-enters the nephron, precisely the SDTL, and becomes again subject of a concentrating process in subsequent ureaimpermeable, but water-permeable, tubular segments. This urea molecule started the intratubular concentrating process at a higher energy level than a urea molecule that entered the tubule by filtration. In our view, this difference in osmotic energy between a recycling

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and a freshly filtered urea molecule, entering the nephron in the IS versus in the cortex, and becoming subject to a concentrating process by water removal along the DCT, CNT, CCD, OMCD, and upper IMCD represents a real gain in osmotic energy that is finally available for water reabsorption in the inner medulla. Entry of urea to the LTDLs somewhere in the IM (i.e., recycling of urea via long loops) would not have a similar effect, simply because at this site there is no surplus salt available to replace urea in its role of water balancing. At the present stage of knowledge, urea recycling via short loops appears as an essential process in urine concentration, and represents the most obvious evidence for the numerical dominance of short loops of Henle in every highly concentrating species. Note that also species which by definition are commonly reported to have 100% long loops (e.g., Psammomys obesus) in reality have a majority of short loops. Moreover, as described above, the SDTLs in such highly concentrating rodent species are directly incorporated into the vascular bundles, and thus provide a more direct route for urea recycling than the simple bundles (Figures 20.22, 20.25, 20.26, and 20.71).16,32,115,435 Within the VBs of such species, AVRs coming up from the IM are arranged in a countercurrent fashion not only with DVRs but, most prominently, also with the SDTLs. Thus urea, by countercurrent exchange, may directly enter the SDTLs through the urea transporter UT-A2441,809 (the hydrophilic fenestrae of the endothelium of the AVRs may readily be expected to be highly permeable for urea). In a renal medulla with “simple” vascular bundles (as they are found in most species) countercurrent exchange of urea first occurs from AVRs to DVRs (which contain the urea transporter UT-B1810,811), which afterwards will deliver their blood to the capillary plexus of the IBR in the IS, which perfuses the SDTLs on their descending way. Thus, even if probably much less effective, urea from the inner medulla has access to the SDTLs435,803,809 and may start its recycling route to the terminal CD in the papilla.435,809,811 The locus of urea redelivery, i.e., in the papilla, thus at the “ultimate bend” of the complex countercurrent exchange system in the papilla, is optimal, since by countercurrent exchange in vasa recta and thin limbs, urea is largely trapped and distributed in a longitudinal gradient within the inner medulla. The fraction of urea that escapes this process in the inner medulla is subject to recycling via short loops (starting in the IS) back into the inner medulla, and is thus available another time within the papillary interstitium ready to withdraw and/or to balance water reabsorption from the collecting duct.

CONCLUSION The urine concentrating mechanism certainly does not belong to the most urgent of problems in medicine, but it represents a highly challenging biological enigma. We are aware that several aspects of our view are hypothetical, but they are based on particular structural features (the ultrastructural organization and particular histotopographical relationships of LDTLs, the incorporation of SDTLs into the VBs in highly concentrating rodent species) for which no better functional relevance is available. Whether the proposed mechanisms are sufficient to build up an effective solute gradient in the IM (in rodents up to several osmoles) or whether there are additional sources complementing the solute gradients (continuous production of osmotic active substances) is presently unknown.

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