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The glomerular capillary wall
© 1970 by Academic Press, Inc.
526
J. ULTRASTRUCTtJRER~SZARC~I32, 526--544 (1970)
The Glomerular
Capillary Wall s
HARRISON LATTA
Department of Pathology, School of Medicine, University of California, Los Angeles, California 90024 Received February 13, 1969 Observations of the glomerular capillary wall of rats and rabbits in normal and experimental conditions, including hemoglobin excretion, clarify the fine structure of the different components involved in the process of glomerular filtration. During excretion of large amounts of hemoglobin, the protein fills the inner and outer layers of the basement membrane, indicating a fairly loose gel structure. The lesser density of the central layer of the basement membrane and of the surface coat on the foot processes indicate rather compact structures without much space for hemoglobin to accumulate, although it must pass through both layers. The absence of hemoglobin between most foot processes when other parts of Bowman's space are filled with it, suggest that normally in vivo the surface coats between adjacent foot processes are usually in contact for the entire height of the processes. The glomerular filtrate must pass through the two surface coats filling the slit approximately 240 It wide between the foot processes. Several lines of evidence support the following conclusions. Glomerular endothelial fenestrations offer little or no barrier to filtration. The central layer of the basement membrane is a dense structure normally stopping molecules larger than about 100 It. The slit membrane and the polysaccharide surface coat beyond it appear to act as a finer filter, but are permeable to hemoglobin (64 in largest diameter) and smaller molecules. Earlier electron microscopic studies with Thorotrast (15) and ferritin (4) showed that the central layer of the basement m e m b r a n e acted as a coarse filter. M o r e recent studies with peroxidases and catalase have suggested the epithelial slits at the level of the slit m e m b r a n e as a finer filtration barrier for relatively small proteins (6, 8). In the course of experiments designed for various purposes, observations have been made which clarify the structure and function of the peripheral capillary wall. They give a new concept of foot processes in vivo with sides in contact. They emphasize the role of the slit m e m b r a n e and surface coats in filtration, and indicate that the basement m e m b r a n e in vivo really has three layers. 1 This work was supported by Research Grant No. AM-06074 from the National Institute of Arthritis and Metabolic Diseases, US Public Health Service.
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MATERIALS AND METHODS The animals used in the present report have been selected from a larger number of experimental and control animals studied over the past two years. Four animals showed material in Bowman's space thought to be hemoglobin. Two rabbits were injected in the left renal artery with a blood clot fragmented in a Waring Blendor. Kidney tissue was removed and fixed after 1 hour (rabbit 35) and 24 hours. The left renal artery of rabbit 88 was clamped for 1 hour before tissue was removed for fixation. Rat 731 was given intravenously 1 ml of centrifuged Clostridium welchii type A toxin (Lederle, No. 379). When the animal became moribund after 15 minutes, renal blocks were removed for fixation. After the above animals were studied, 7 rats were injected with hemolyzed blood or crystallized hemoglobin. Two adult rats (including rat 750) were injected intravenously with hemolyzed rat blood (0.75 ml/100 g body weight) in normal saline. The kidneys were fixed within 30 minutes. One rat (749) was injected intraperitoneally with the same dose, and the left kidney was fixed after 1 hour by dripping buffered 1.5 % glutaraldehyde on the kidney in the living animal for 20 minutes. Two rats were injected intravenously with crystallized bovine hemoglobin (Mann Research Laboratories), 100 and 150 mg/100 g body weight, and after 5 minutes buffered glutaraldehyde was dripped on the kidney. No hemoglobin was found in Bowman's space in the 5 rats above, and the glomeruli seemed essentially normal. Crystalline bovine hemoglobin was then injected into two rats at the level of the renal arteries with the lower aorta clamped. One rat was given about 25 mg/100 g body weight and the other, rat 785, was given about 100 mg/100 g body weight, the aorta being clamped in addition above the celiac artery. The kidney of the latter turned dark during injection and the renal vein was clamped immediately afterward. After 10 minutes, blocks were removed and fixed in glutaraldehyde. Only rat 785 showed considerable hemoglobin in Bowman's space. Additional animals were utilized as follows. Two rabbits anesthetized intravenously or subcutaneously served as controls. Two rats (717 and 629) were immunized with small amounts of ferritin over a 3-month period, but the glomerular capillary walls appeared in the normal range. The kidneys of a Macaca mulatta adult female monkey were fixed by perfusion with glutaraldehyde, and appeared normal. (The author is indebted to Dr. Lydia Osvaldo-Decima for this material.) Rat 678 was injected intravenously with 100 mg of uranium sulfide (US~) suspended in normal saline. Five days later when the animal was moribund, it was sacrificed and renal tissue was removed. The small blocks of kidney fixed in glutaraldehyde buffered with phosphates were postfixed in osmium tetroxide, embedded in Epon, cut with diamond knives on a Porter-Blum MT 1 microtome and examined with a Siemens Elmiskop 1 at 80 k ¥ .
OBSERVATIONS
Capillary wail in control animals The o b s e r v a t i o n s o n n o r m a l a n d c o n t r o l animals are described here for c o m p a r i s o n with the animals showing h e m o g l o b i n in the filtration p a t h a n d to enable a m o r e precise d e s c r i p t i o n of structures in this path. The descriptions will a p p l y to the r a t unless otherwise stated.
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LATTA
The endothelium lining the peripheral capillary wall is perforated by fenestrations (Figs. 1-3) roughly about 1000 A in diameter in the rat. Only rarely is a diaphragm seen across a fenestration, although they are frequently seen in the rat medulla (17). Occasionally junctional complexes are seen where two layers of cytoplasm come in contact (Fig. 4, arrow). The endothelial layer is often loosened from the basement membrane, suggesting that it is not firmly attached (Fig. 2). The basement membrane has three layers in the rat (Figs. 1 and 2) totaling about 1500 A (1100-1600 A) in thickness when measured from the endothelial cell to the bottom of the epithelial foot processes. The inner layer is about 200-300 A and the central and outer layers about 500-600 A thick. The inner layer is sometimes quite variable in thickness, especially in poorly fixed or abnormal states. In the rabbit the basement membrane is also usually about 1500 A thick (1200-1900 A) but the inner layer seems wider than in the rat. The central layer varies somewhat in thickness and the edges are not clearly marked so that measurements are difficult, but often each layer forms about a third of the distance from the endothelium to the base of the epithelial cell. In the monkey (Fig. 3), the thickness is much greater, about 25003000 A. The inner layer is about 200-400 ~ thick, the central layer about 2000-2400 A, and the outer layer about 400 A. The inner layer is of light staining density and shows faint fine fibrils sometimes running from the endothelial cell to the central layer. Large fairly straight fibrils about 100 A wide are seen occasionally in the inner layer in rats and rabbits, sometimes appearing to run out of the central layer. Their surface stains more heavily so that they are sometimes seen longitudinally as pairs of dense lines and in cross section as circles (Fig. 2). They are not periodic, yet they are similar to fibers forming part of large bundles of collagen fibers outside Bowman's capsule, most of which are periodic. The central layer is moderately dense after exposure to osmium, uranium, and lead stains (Figs. 1-3). At high magnifications a faint fine fibrillar pattern is seen, something like a layer of felt. The fine poorly stained fibrils about 20-50 A wide are somewhat irregular in thickness and linearity but tend to run in the plane of the layer. Infrequently a large fibril about 100 A wide m a y be found. After uranium poisoning FIO. 1. Peripheral glomerular capillary wall. The endothelial fenestrations appear open. The inner and outer layers of the basement membrane are much lighter than the central layer. The foot processes are embedded in the outer layer of the basement membrane, the outer portion of which is marked by the slit membrane. The free portions of the foot processes appear to have shrunk away from each other and from the overlying epithelial cytoplasm. See Fig. 12 for labeling of these structures. Rat 749. x 102,000. FIO. 2. Glomerular capillary wall. Poorly staining fine fibrils may be seen in the three layers of the basement membrane. Some large fibrils are seen in longitudinal and cross section (arrows). Bundles of myofilaments (Mr) cut in cross section lie in foot processes and epithelial cytoplasm. Rat 749. × 102,000. Fie. 3. Monkey glomerular capillary wall. Thecentral layer (CL) is much wider than that of the rat. Fine fibrils are seen in all three layers, x 100,000.
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LATTA
a number of large fibrils were revealed in the central layer of one basement membrane (Fig. 5), similar to those sometimes seen in the inner layer. They run in the plane of the membrane and in a few places a rough periodicity is suggested. The spaces between the fibers are lighter than the usual density of the basement membrane. In the monkey the fine fibrils of the central layer are similar to those of the rat, but may be more apparent (Fig. 3). The outer layer is almost as light in staining density as the inner layer. Fine fibrils are a little more apparent here than in the inner layer and usually run from the central layer to the epithelial foot processes (Figs. 1-3). The foot processes of the epithelial cells are embedded in the outer layer for a distance of 400-500 A. In good preparations the surface of the outer layer in the slit between foot processes is usually covered with a darkly staining membrane about 40-60/~ thick, and is called the outer limiting membrane or slit membrane. Its width between the foot processes runs about 240 •. Where the foot processes of epithelial cells are embedded in the outer layer of the basement membrane they are usually wider than the free portion, which is sometimes only half the width of the base. Most foot processes are between about 3000 and 5000 ~ in height. The space between the free portions is often about 800 A wide, and a similar space often separates them from the overlying epithelial cytoplasm. The matching contours of adjacent foot processes and overlying epithelium suggest that the cytoplasm has shrunk during fixation and embedding, except for the base in the basement membrane (Figs. 1, 2, and 4). The plasma membrane of the foot processes has a triple-layered structure about 90/~ thick like that of other cells (Figs. 1-3, and 6). The surface coat is not usually revealed very well after conventional staining techniques; however, at times a layer of material 100-150 A thick may be seen on the plasma membrane and occasionally on the slit membrane (Figs. 3 and 6). Three types of fibrillar systems are clearly seen in the visceral epithelial cytoplasm in addition to the generally recognized structures. Microtubules about 200 A wide (Figs. 2 and 7) and small randomly oriented fibrils about 50-100 N wide seem more frequent in the cell body and primary processes. Bundles of parallel filaments about 50 N wide resembling myofilaments in smooth muscle are frequently seen as large masses in the larger portions of the cytoplasm (Fig. 7) and as smaller bundles in foot processes in longitudinal or cross section (Figs. 1-3, and 6). They often fill the outer portion of the foot processes so that the latter are club-shaped in cross section (Figs. 1 and 2).
Capillary wall during hemoglobin excretion In the animals injected intravenously or intraperitoneally with hemolyzed blood or hemoglobin, no definite masses of hemoglobin were seen in the blood plasma, the
FIG. 4. Tangential section of glomerular capillary. The endothelium shows attachment bodies (arrow). The foot processes fit together like pieces of a jig-saw puzzle that has shrunk. Most of them contain bundles of myofibrils. Rat 750. x 22,000. FIG. 5. Large fibrils ( ~ 100 ~ ) are found in the central layer of the glomerular capillary basement membrane of a rat poisoned with uranium. In cross section the profiles are ring-like (arrow). The density of the matrix between these fibrils is less than usual. Rat 678. × 100,000.
534
LATTA
basement m e m b r a n e or B o w m a n ' s space. Clamping the aorta before and the renal vein after intra-aortic injection of hemoglobin did enable moderate to large amounts of this material to be f o u n d in B o w m a n ' s space (rat 785). The greatest concentrations of hemoglobin in the filtration path f r o m plasma to B o w m a n ' s space were f o u n d in 3 animals. One, rabbit 35, was injected with a fragmented clot which was hemolyzing because loss of hemoglobin f r o m red cells was obvious in the supernatant of the injected suspension as well as in electron micrographs. The glomeruli of another, rabbit 88, showed focal necrosis an h o u r after clamping the renal artery. A third animal, rat 731, was injected with Clostridium welchii toxin. H e m o g l o b i n was the only apparent available substance in these animals which could increase the density of material in the capillary wall to the extent observed. All three animals showed high concentrations of h e m o g l o b i n in the capillary blood plasma, in the inner and outer layers of the basement membrane, and in B o w m a n ' s space (Figs. 8-11). The inner and outer layers were usually nearly uniformly filled, but sometimes they were not. The hemoglobin was occasionally more concentrated in the inner layer in the region of the filtration slit (Figs. 8 and 9). In the slits between adjacent foot processes hemoglobin was usually either absent or present in low concentration in small irregular masses of light density. Sometimes thin dense masses were seen midway between the foot processes and some of these were seen to connect with the large masses filling B o w m a n ' s space. The relatively clear space between the f o o t processes was a b o u t 600-800 • wide, which was several times width of the surface coat (about 120 fli). Over the free portions at the top of foot processes covered by dense concentrations of hemoglobin there was a narrower relatively clear space which was one to two times as thick as the surface coat (Figs. 8 and 9). The bundles of myofilaments at the top of the foot processes seemed to be associated with the lesser shrinkage there. These observations suggested that, except for the surface coat, the clear space was artifactual and that there was little space in the surface coat for FIG. 6. Surface coat on triple-layered plasma membrane of foot processes. It is somewhat wider than the plasma membrane. Rat 629. x 118,000. FIG. 7. Large bundles of myofilaments (Mr) in visceral epithelium. Microtubules (T) and randomly oriented fibrils (F) are also seen. Rat 717. x 32,000. FIG. 8. Hemoglobin masses in peripheral capillary, inner (/) and outer (O) layers of basement membrane and in Bowman's space. The relatively small amounts between adjacent foot processes indicate that most of them were in contact in vivo. Rat 731. x 88,000. FIG. 9. Hemoglobin masses in mesangial matrix (M) and outer (O) layer of basement membrane. The hemoglobin in Bowman's space is separated from the foot processes by a relatively clear space which is one to two times the width of the surface coat. Rat 731. x 86,000. FIG. 10. Hemoglobin masses in inner and outer layers of a rabbit glomerular basement membrane. Rabbit 35. x 87,000. FIG, 11. Hemoglobin extends from capillary lumen (C) through basement membrane to Bowman's space (BS). A pyknotic epithelial cell has contracted dark foot processes which alternate with the foot processes of a more normal cell. The relatively clear space over the contracted processes is about the thickness of the surface coat. Rabbit 88. x 58,000.
1
I
538
LATTA
hemoglobin. The looser portions of mesangial matrix and the outer layer of the basement membrane over the mesangial region were also penetrated by hemoglobin (Fig. 9). In the glomerulus of rabbit 88 some epithelial cells had died after clamping of the renal artery and the foot processes were quite shrunken and dense (Fig. 11). These foot processes alternated with less shrunken foot processes, probably from a different epithelial cell. Hemoglobin filled most of the space between these foot processes which apparently shrank before fixation and embedding. These processes were not separated from the mass of hemoglobin in Bowman's space by a large clear space, but they were surrounded by a thin relatively clear space which was just about the thickness of the surface coat. This also suggested that the surface coat was compact enough to keep hemoglobin molecules from increasing its density appreciably. DISCUSSION The observations on normal and control animals confirm and emphasize certain findings in earlier studies (3, 4, 10, 15, 18). After intravenous injection of hemoglobin, it does not ordinarily appear in concentrated form in the glomernlus (2). What concentrated it in the present studies? The three animals with the highest concentrations probably had hemolyzing red cells in their glomerular capillaries. In addition local glomerular filtration was probably greatly reduced or absent. The main features of the reaction to Clostridium welchii type A toxin (primarily lecithinase) are hemolysis and a shock-like state (5). Emboli of clot fragments and clamping of the renal artery or aorta would reduce glomerular filtration to negligible levels. The layers of the glomerular capillary wall will be discussed in order.
Endothelium A variety of large molecules and particles pass through endothelial fenestrations in the rat and mouse without apparent difficulty, and have been observed by electron microscopy in the basement membrane. These include Thorotrast particles about 200 /k in diameter (15), ferritin [about 100 fit (4) or 110 ]i (1) in diameter], catalase [mol. wt. about 232,400 (24); dimensions approximately 95 × 80 × 70 ]l (25)], myeloperoxidase [mol. wt. about 160,000 (8)], hemoglobin [tool. wt. 64,500; dimensions 64 × 55 × 50 A (2•)], and horseradish peroxidase [mol. wt. about 40,000 (6); tool. dia. about 50 it (1)]. Diaphragms with central knobs have been described in the mouse glomerulus (23), but they do not seem to present a significant barrier to the peroxidase molecules (6). In a detailed study of their permeability, diaphragms in the capillaries of mouse
GLOMERULAR CAPILLARY WALL
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intestiDe presented little barrier to horseradish peroxidase although they did slow the passage of ferritin (1). Diaphragms are rarely seen in the rat glomerulus under the same conditions where they are well preserved in the medulla (17). Even if they should prove to be more frequent in the rat glomerulus than has hitherto been described, they apparently offer little barrier to the passage of Thorotrast (15), ferritin (4), or hemoglobin molecules.
Basement membrane Earlier work showing Thorotrast particles of about 140 ~ diameter (15) and ferritin (4) in the inner less-dense layer of the basement membrane indicated that this layer was functionally significant and probably had the structure of a loose gel. The filling of this layer with hemoglobin in the present studies and with horseradish peroxidase (HRPO) and myeloperoxidase (MPO) (6) reinforces this conclusion. The central dense layer of the normal basement membrane has been shown to stop the passage of Thorotrast (15) and ferritin (4) particles of about 100 A diameter. The observations that the central dense layer does not increase much in density at the time when the inner and outer light layers are filled with hemoglobin in these studies or with horseradish peroxidase (6) indicates that this layer is molecularly fairly dense and penetrated by relatively few channels narrower than about 100 A and wider than 64 A. (The channels or pores do not need to be cylindrical: they could be elliptical or slit-like in cross section.) Biochemical (9) and immunologic studies (22) indicate that a large portion of the glomerular basement membrane is collagen and that the remainder is composed of glycoproteins. Both of these proteins occur as elongated molecules. The observation of faintly stained fine fibrils in the plane of the central layer in the present studies with Epon embedding confirms earlier evidence with methacrylate embedding (3, 4, 10). Epon is generally considered superior to methacrylate in maintaining fine structure. Some caution regarding the presence and size of fibrils in the earlier observations was rightly advised (3, 4); but other considerations based on birefringence, artifactual layering, and a structural need to resist the force of the blood pressure suggest that elongated molecules run in the plane of the central layer (12). The large fibers in the inner layer have not been noted frequently in these studies or by others (4). The failure to observe a definite periodicity in them or any other structure in the basement membrane, even when a collagen periodicity was revealed in the mesangial region (13) indicates that, if collagen is present, it is not in a stainable form or the staining is blocked by some other substances, possibly mucoproteins. This idea is strengthened by the observation that some fibers in obvious collagen bundles outside the glomerulus may appear nonperiodic and, in cross section, ringlike. The uranium poisoned rabbit shows that large fibers can occur in the central
540
LATTA
layer, but whether exposed or newly formed is not certain at present. In summary, a large portion of the basement membrane appears to be collagen, but very little of it appears in recognizable form. Small fibrils run in the plane of the central layer like a feltwork and are either a form of collagen or glycoprotein or both. The functional reality of the outer less dense layer of the basement membrane is demonstrated by permeation with enough hemoglobin to make it appear darker than the central layer. A similar increase in density is shown by HRPO (ref. 6, Fig. 6). Because both hemoglobin and HRPO pass readily into the glomerular filtrate, the increased density is probably due to the outer layer having a looser structure than the central layer. Its structure appears to be only a little denser than that of the inner layer. The faint fibrils which run from the foot processes to the central layer probably have a supportive function. The studies with myeloperoxidase (6, 8) and catalase (8) indicate a barrier to these molecules in the epithelial slits, probably at the level of the outer limiting or slit membrane (ref. 6, Fig. 16). That this barrier allows the passage of some MPO molecules is shown by a little reaction product on some of the epithelial membranes distal to the outer limiting membrane and in the phagosomes of a few of the proximal tubules (ref. 6, Fig. 2). Permeation of the outer layer over the mesangium by hemoglobin and by HRPO (6) indicates that it is also involved in glomerular filtration.
Foot processes of the epithelium The bundles of parallel filaments frequently seen in epithelial cytoplasm and foot processes resemble filamentous bundles in smooth muscle cells. A larger filament is seen in myoid cells with inert dehydration and is thought to represent myosin which is not preserved by conventional procedures (20). Fine filaments such as those seen in the present studies are thought to be actin (20). Although the abundance of fine myoid filaments in glomerular epithelial cells is surprising because constriction of glomerular capillaries does not seem to have been described, it may be that these filaments are responsible for retraction of foot processes that is probably the basis for their loss in diseases associated with proteinuria. The roughly similar spacing that separates the foot processes from each other and from overlying epithelial cell cytoplasm in normal animals gives the impression of pieces of a jig-saw puzzle which have shrunk, leaving a gap between them. An obvious exception to this shrinkage would be the portion of the foot processes embedded in the basement membrane. The conclusion that shrinkage during fixation and embedding has occurred is hard to escape when the foot processes are examined during excretion of hemoglobin. At the time when hemoglobin is filling the inner and outer layers of the basement membrane and passing into the free portions of Bowman's space there are large relatively clear areas between most foot processes. This indicates
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541
that the sides of most foot processes were in contact in vivo while the hemoglobin was passing into Bowman's space. The presence of thin masses of hemoglobin in the center of some spaces between foot processes indicates that the contact is not perfect, or that some shrinkage of the processes occurred before the hemoglobin in Bowman's space was fixed. Similar results are also seen with H R P O (6). Much evidence indicates that the free surface of the plasma membrane of glomerular epithelial cells is covered by a polysaccharide coat, about 120 • thick (7, 14, 19). These layers have little intrinsic electron density and stain poorly with the usual stains but quite strongly with substances that stain polysaccharides. If the surfaces are usually in contact in the living animal as we believe, then the plasma membranes of most adjacent foot processes would be separated by two covering layers totaling approximately 240 A in thickness. The layers stick to the foot processes when they shrink (7, 14). An early estimate of about 100 ~ for the width of the filtration slit was of considerable physiologic interest (11), but the present evidence for a much wider slit and the presence of other structures in it introduce considerable change in the physiologic considerations. The glomerular fikrate would have to pass through the surface coats for the height of the foot processes, or some 3000-5000 ~ in most cases. This is consistent with the function suggested for the covering layers on other cells--holding water and maintaining a pathway for the diffusion of ions and small molecules (19). Between the few processes where some hemoglobin is seen, the filtrate would still have to pass through a portion of the polysaccharide coat covering the slit membrane. Even with considerable shrinkage of the foot processes, a surface coat remains covering the slit membrane (7). The evidence that hemoglobin and H R P O penetrate the surface coats while only very little myeloperoxidase is able to penetrate (ref. 6, Fig. 16), makes it appear that the covering layers have a permeability similar to that of the basement membrane at the level of the slit membrane. Thus it is difficult to say whether the slit membrane or the surface coats or both are the main filtration barrier. The thinness of the slit membrane (about 40-60 ~ ) makes it seem likely that the covering layers support the slit membrane and back it up as a filtration barrier to myeloperoxidase, catalase and larger molecules. The effective pore size here can be estimated as lying between 64 and 95 ~ , from the dimensions of hemoglobin and catalase, because the shape of myeloperoxidase is not known. Measurements of the width of the slit membrane correspond to the thickness of two surface coats filling the slit between the foot processes, or about 240 A. One might ask whether some shrinkage of the portion of the foot processes embedded in the outer layer of the basement membrane might occur with fixation and embedding. This seems not to occur to a significant extent because it would produce a decreased density or clear space in the peroxidase precipitates between the base of the foot processes. Only a faint space is seen in some places (6).
542
LATTA
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FI6. 12. Peripheral glomerular capillary wall. Diagram of the probable relationships of the components in the rat in vivo. The filtration path runs through the fenestrations (F) of the endothelium (En); the loose inner layer (IL), the compact central layer (CL), the loose outer layer (OL), and the slit membrane (SM)of the basement membrane; and the surface coat (SC) of epithelial foot processes (FP) which usually seem to be in lateral contact in vivo, to Bowman's space (BS). Epithelial cells (Ep) contain many bundles of fine myofilaments (Mf) and show a triple-layered plasma membrane (PM).
Glomerular filtration Glomerular permeability studies with a variety of molecules are consistent with theoretical curves for an isoporous membrane having cylindrical pores 70-84/k in diameter (11). Thus the conformity with the electron microscopic data is fairly close. The relative impermeability of the glomerular capillary wall to albumin and globulin is probably due to the great asymmetry of these molecules. The observation that large masses of denatured globin can work their way through the basement membrane and between foot processes led to the suggestion that the membrane acts like a thixotropic gel which liquefies under pressure (16). It may also be pointed out that relatively mild alterations produce proteinuria of serum albumin or globulin, such as intravenous injection of solutions of these proteins or passive congestion as in heart failure. The probable fine structure of the peripheral glomerular capillary wall and the process of filtration through it can now be described and illustrated (Fig. 12). Most endothelial fenestrations appear to be open in the rat. The inner less dense layer of the basement membrane seems to be a loose gel which is easily filled with hemoglobin or horseradish peroxidase molecules. Thorotrast particles and ferritin molecules
GLOMERULAR CAPILLARYWALL
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penetrate it less readily, and it may be supposed that the elongated molecules of serum albumin and globulin would also experience difficulty. The central dense layer seems to be composed of densely packed fibrils, probably both collagen and glycoprotein, many lying in the plane of the membrane, with just enough channels to allow free passage of hemoglobin molecules, but normally stopping molecules or particles over 100 • diameter. The outer layer of the basement membrane appears to be a loose gel similar to the inner layer but with more radially oriented molecules to support the foot processes. The main filtration barrier to protein molecules between 64 and 95 A in greatest dimension seems to be the outer limiting membrane and the polysaccharide coat which generally covers it and the free surface of the foot processes. As the polysaccharide coats of most foot processes are probably in contact normally in vivo, most of the glomerular filtrate and its ionized salts must pass through long distances of these covering layers. The author wishes to thank Mrs Elizabeth Sykes and Mrs Lya Cordova for very capable technical assistance. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8.
9. 10. 11.
12. 13. 14. 15. 16. 17. 18. 19.
CLEMENTI,F. and PALADE, G. E., J. Cell Biol. 41, 33 (1969). ERICSSON,J. L. E., Nephron 5, 7 (1968). FARQUHAR,M. G., in RICHET, G. (Ed.), Nephrology, pp. 358-376. Karger, Basel, 1961. FARQOHAR,M. G., WISSIG, S. L. and PALADE, G. E., J. Exp. Med. 113, 47 (1961). FURR, W. E., BOURDEAU,R. V., ROACH, H. D. and LAUFMAN,H., Surg. Gynecol. Obstet. 95, 465 (1952). GRAHAM,R. C., JR and KARNOVSKY,M. J., Y. Exp. Med. 124, 1123 (1966). GRONIOWSKI,J., BICZYSKOWA,W. and WALSKI, M., Y. Cell Biol. 40, 585 (1969). KARNOVSKY,M. J., VENKATACHALAM,M. A., GRAHAM,R. C., JR. and COTRAN, R. S., Abstr. General Sessions-Syrup., p. 28, 4th Intern. Congr. Nephrology, Stockholm, 1969. KEFALIDES,N. A. and WINZLER, R. J., Biochemistry 5, 702 (1966). KURTZ, S. M., Lab. Invest. 10, 1189 (1961). LANDIS, E. M. and PAPPENHEIMER,J. R., in HAMILTON, W. F. and Dow, P. (Eds.), Handbook of Physiology, Section 2: Circulation, Vol. II, p. 961, American Physiological Society, Washington, D.C., 1963. LATTA,H., in RICHET, G. (Ed.), Nephrology, pp. 382-397, Karger, Basel, 1961. -d. Ultrastruct. Res. 5, 364 (1961). -ibid. 6, 407 (1962). LATTA, H., MAUNSBACH,A. B. and MADDEN, S. C., Jr. Ultrastruct. Res. 4, 455 (1960). MENEVEE, M. G. and MUELLER, C. B., in DALTON, A. J. and HAGUENAU, F. (Eds.), Ultrastructure of the Kidney, p. 73. Academic Press, New York, 1967. OSVALDO,L. and LATTA, H., J. Ultrastruct. Res. 15, 589 (1966). PEASE, O. C., Anat. Rec. 121, 701 (1955). -J. Ultrastruct. Res. 15, 555 (1966).
544 20. 21. 22. 23. 24.
LATTA
-ibid. 23, 304 (1968). PERt~TZ, M. F., Harvey Lectures 63, 213 (1969). PIERCE, G. B. and NAKANE, P. K., Lab. Invest. 17, 499 (1967). R~OD~N, J. A. G., 3. Ultrastruet. Res. 6, 161 (1962). SCnROEDER, W. A., SHELTON, J. R., Sm~LTON,J. B., ROBB~RSON, B. and AVELL, G., Arch. Biochem. Biophys. 131, 653 (1969). 25. V~aNSHTZIN,B. K., BARYNrN, V. V. and GURSI¢AYA,G. V., Aeta Crystallog. Sect. A 25, S 181 (1969).
526
J. ULTRASTRUCTtJRER~SZARC~I32, 526--544 (1970)
The Glomerular
Capillary Wall s
HARRISON LATTA
Department of Pathology, School of Medicine, University of California, Los Angeles, California 90024 Received February 13, 1969 Observations of the glomerular capillary wall of rats and rabbits in normal and experimental conditions, including hemoglobin excretion, clarify the fine structure of the different components involved in the process of glomerular filtration. During excretion of large amounts of hemoglobin, the protein fills the inner and outer layers of the basement membrane, indicating a fairly loose gel structure. The lesser density of the central layer of the basement membrane and of the surface coat on the foot processes indicate rather compact structures without much space for hemoglobin to accumulate, although it must pass through both layers. The absence of hemoglobin between most foot processes when other parts of Bowman's space are filled with it, suggest that normally in vivo the surface coats between adjacent foot processes are usually in contact for the entire height of the processes. The glomerular filtrate must pass through the two surface coats filling the slit approximately 240 It wide between the foot processes. Several lines of evidence support the following conclusions. Glomerular endothelial fenestrations offer little or no barrier to filtration. The central layer of the basement membrane is a dense structure normally stopping molecules larger than about 100 It. The slit membrane and the polysaccharide surface coat beyond it appear to act as a finer filter, but are permeable to hemoglobin (64 in largest diameter) and smaller molecules. Earlier electron microscopic studies with Thorotrast (15) and ferritin (4) showed that the central layer of the basement m e m b r a n e acted as a coarse filter. M o r e recent studies with peroxidases and catalase have suggested the epithelial slits at the level of the slit m e m b r a n e as a finer filtration barrier for relatively small proteins (6, 8). In the course of experiments designed for various purposes, observations have been made which clarify the structure and function of the peripheral capillary wall. They give a new concept of foot processes in vivo with sides in contact. They emphasize the role of the slit m e m b r a n e and surface coats in filtration, and indicate that the basement m e m b r a n e in vivo really has three layers. 1 This work was supported by Research Grant No. AM-06074 from the National Institute of Arthritis and Metabolic Diseases, US Public Health Service.
GLOMERULAR CAPILLARY WALL
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MATERIALS AND METHODS The animals used in the present report have been selected from a larger number of experimental and control animals studied over the past two years. Four animals showed material in Bowman's space thought to be hemoglobin. Two rabbits were injected in the left renal artery with a blood clot fragmented in a Waring Blendor. Kidney tissue was removed and fixed after 1 hour (rabbit 35) and 24 hours. The left renal artery of rabbit 88 was clamped for 1 hour before tissue was removed for fixation. Rat 731 was given intravenously 1 ml of centrifuged Clostridium welchii type A toxin (Lederle, No. 379). When the animal became moribund after 15 minutes, renal blocks were removed for fixation. After the above animals were studied, 7 rats were injected with hemolyzed blood or crystallized hemoglobin. Two adult rats (including rat 750) were injected intravenously with hemolyzed rat blood (0.75 ml/100 g body weight) in normal saline. The kidneys were fixed within 30 minutes. One rat (749) was injected intraperitoneally with the same dose, and the left kidney was fixed after 1 hour by dripping buffered 1.5 % glutaraldehyde on the kidney in the living animal for 20 minutes. Two rats were injected intravenously with crystallized bovine hemoglobin (Mann Research Laboratories), 100 and 150 mg/100 g body weight, and after 5 minutes buffered glutaraldehyde was dripped on the kidney. No hemoglobin was found in Bowman's space in the 5 rats above, and the glomeruli seemed essentially normal. Crystalline bovine hemoglobin was then injected into two rats at the level of the renal arteries with the lower aorta clamped. One rat was given about 25 mg/100 g body weight and the other, rat 785, was given about 100 mg/100 g body weight, the aorta being clamped in addition above the celiac artery. The kidney of the latter turned dark during injection and the renal vein was clamped immediately afterward. After 10 minutes, blocks were removed and fixed in glutaraldehyde. Only rat 785 showed considerable hemoglobin in Bowman's space. Additional animals were utilized as follows. Two rabbits anesthetized intravenously or subcutaneously served as controls. Two rats (717 and 629) were immunized with small amounts of ferritin over a 3-month period, but the glomerular capillary walls appeared in the normal range. The kidneys of a Macaca mulatta adult female monkey were fixed by perfusion with glutaraldehyde, and appeared normal. (The author is indebted to Dr. Lydia Osvaldo-Decima for this material.) Rat 678 was injected intravenously with 100 mg of uranium sulfide (US~) suspended in normal saline. Five days later when the animal was moribund, it was sacrificed and renal tissue was removed. The small blocks of kidney fixed in glutaraldehyde buffered with phosphates were postfixed in osmium tetroxide, embedded in Epon, cut with diamond knives on a Porter-Blum MT 1 microtome and examined with a Siemens Elmiskop 1 at 80 k ¥ .
OBSERVATIONS
Capillary wail in control animals The o b s e r v a t i o n s o n n o r m a l a n d c o n t r o l animals are described here for c o m p a r i s o n with the animals showing h e m o g l o b i n in the filtration p a t h a n d to enable a m o r e precise d e s c r i p t i o n of structures in this path. The descriptions will a p p l y to the r a t unless otherwise stated.
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The endothelium lining the peripheral capillary wall is perforated by fenestrations (Figs. 1-3) roughly about 1000 A in diameter in the rat. Only rarely is a diaphragm seen across a fenestration, although they are frequently seen in the rat medulla (17). Occasionally junctional complexes are seen where two layers of cytoplasm come in contact (Fig. 4, arrow). The endothelial layer is often loosened from the basement membrane, suggesting that it is not firmly attached (Fig. 2). The basement membrane has three layers in the rat (Figs. 1 and 2) totaling about 1500 A (1100-1600 A) in thickness when measured from the endothelial cell to the bottom of the epithelial foot processes. The inner layer is about 200-300 A and the central and outer layers about 500-600 A thick. The inner layer is sometimes quite variable in thickness, especially in poorly fixed or abnormal states. In the rabbit the basement membrane is also usually about 1500 A thick (1200-1900 A) but the inner layer seems wider than in the rat. The central layer varies somewhat in thickness and the edges are not clearly marked so that measurements are difficult, but often each layer forms about a third of the distance from the endothelium to the base of the epithelial cell. In the monkey (Fig. 3), the thickness is much greater, about 25003000 A. The inner layer is about 200-400 ~ thick, the central layer about 2000-2400 A, and the outer layer about 400 A. The inner layer is of light staining density and shows faint fine fibrils sometimes running from the endothelial cell to the central layer. Large fairly straight fibrils about 100 A wide are seen occasionally in the inner layer in rats and rabbits, sometimes appearing to run out of the central layer. Their surface stains more heavily so that they are sometimes seen longitudinally as pairs of dense lines and in cross section as circles (Fig. 2). They are not periodic, yet they are similar to fibers forming part of large bundles of collagen fibers outside Bowman's capsule, most of which are periodic. The central layer is moderately dense after exposure to osmium, uranium, and lead stains (Figs. 1-3). At high magnifications a faint fine fibrillar pattern is seen, something like a layer of felt. The fine poorly stained fibrils about 20-50 A wide are somewhat irregular in thickness and linearity but tend to run in the plane of the layer. Infrequently a large fibril about 100 A wide m a y be found. After uranium poisoning FIO. 1. Peripheral glomerular capillary wall. The endothelial fenestrations appear open. The inner and outer layers of the basement membrane are much lighter than the central layer. The foot processes are embedded in the outer layer of the basement membrane, the outer portion of which is marked by the slit membrane. The free portions of the foot processes appear to have shrunk away from each other and from the overlying epithelial cytoplasm. See Fig. 12 for labeling of these structures. Rat 749. x 102,000. FIO. 2. Glomerular capillary wall. Poorly staining fine fibrils may be seen in the three layers of the basement membrane. Some large fibrils are seen in longitudinal and cross section (arrows). Bundles of myofilaments (Mr) cut in cross section lie in foot processes and epithelial cytoplasm. Rat 749. × 102,000. Fie. 3. Monkey glomerular capillary wall. Thecentral layer (CL) is much wider than that of the rat. Fine fibrils are seen in all three layers, x 100,000.
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a number of large fibrils were revealed in the central layer of one basement membrane (Fig. 5), similar to those sometimes seen in the inner layer. They run in the plane of the membrane and in a few places a rough periodicity is suggested. The spaces between the fibers are lighter than the usual density of the basement membrane. In the monkey the fine fibrils of the central layer are similar to those of the rat, but may be more apparent (Fig. 3). The outer layer is almost as light in staining density as the inner layer. Fine fibrils are a little more apparent here than in the inner layer and usually run from the central layer to the epithelial foot processes (Figs. 1-3). The foot processes of the epithelial cells are embedded in the outer layer for a distance of 400-500 A. In good preparations the surface of the outer layer in the slit between foot processes is usually covered with a darkly staining membrane about 40-60/~ thick, and is called the outer limiting membrane or slit membrane. Its width between the foot processes runs about 240 •. Where the foot processes of epithelial cells are embedded in the outer layer of the basement membrane they are usually wider than the free portion, which is sometimes only half the width of the base. Most foot processes are between about 3000 and 5000 ~ in height. The space between the free portions is often about 800 A wide, and a similar space often separates them from the overlying epithelial cytoplasm. The matching contours of adjacent foot processes and overlying epithelium suggest that the cytoplasm has shrunk during fixation and embedding, except for the base in the basement membrane (Figs. 1, 2, and 4). The plasma membrane of the foot processes has a triple-layered structure about 90/~ thick like that of other cells (Figs. 1-3, and 6). The surface coat is not usually revealed very well after conventional staining techniques; however, at times a layer of material 100-150 A thick may be seen on the plasma membrane and occasionally on the slit membrane (Figs. 3 and 6). Three types of fibrillar systems are clearly seen in the visceral epithelial cytoplasm in addition to the generally recognized structures. Microtubules about 200 A wide (Figs. 2 and 7) and small randomly oriented fibrils about 50-100 N wide seem more frequent in the cell body and primary processes. Bundles of parallel filaments about 50 N wide resembling myofilaments in smooth muscle are frequently seen as large masses in the larger portions of the cytoplasm (Fig. 7) and as smaller bundles in foot processes in longitudinal or cross section (Figs. 1-3, and 6). They often fill the outer portion of the foot processes so that the latter are club-shaped in cross section (Figs. 1 and 2).
Capillary wall during hemoglobin excretion In the animals injected intravenously or intraperitoneally with hemolyzed blood or hemoglobin, no definite masses of hemoglobin were seen in the blood plasma, the
FIG. 4. Tangential section of glomerular capillary. The endothelium shows attachment bodies (arrow). The foot processes fit together like pieces of a jig-saw puzzle that has shrunk. Most of them contain bundles of myofibrils. Rat 750. x 22,000. FIG. 5. Large fibrils ( ~ 100 ~ ) are found in the central layer of the glomerular capillary basement membrane of a rat poisoned with uranium. In cross section the profiles are ring-like (arrow). The density of the matrix between these fibrils is less than usual. Rat 678. × 100,000.
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basement m e m b r a n e or B o w m a n ' s space. Clamping the aorta before and the renal vein after intra-aortic injection of hemoglobin did enable moderate to large amounts of this material to be f o u n d in B o w m a n ' s space (rat 785). The greatest concentrations of hemoglobin in the filtration path f r o m plasma to B o w m a n ' s space were f o u n d in 3 animals. One, rabbit 35, was injected with a fragmented clot which was hemolyzing because loss of hemoglobin f r o m red cells was obvious in the supernatant of the injected suspension as well as in electron micrographs. The glomeruli of another, rabbit 88, showed focal necrosis an h o u r after clamping the renal artery. A third animal, rat 731, was injected with Clostridium welchii toxin. H e m o g l o b i n was the only apparent available substance in these animals which could increase the density of material in the capillary wall to the extent observed. All three animals showed high concentrations of h e m o g l o b i n in the capillary blood plasma, in the inner and outer layers of the basement membrane, and in B o w m a n ' s space (Figs. 8-11). The inner and outer layers were usually nearly uniformly filled, but sometimes they were not. The hemoglobin was occasionally more concentrated in the inner layer in the region of the filtration slit (Figs. 8 and 9). In the slits between adjacent foot processes hemoglobin was usually either absent or present in low concentration in small irregular masses of light density. Sometimes thin dense masses were seen midway between the foot processes and some of these were seen to connect with the large masses filling B o w m a n ' s space. The relatively clear space between the f o o t processes was a b o u t 600-800 • wide, which was several times width of the surface coat (about 120 fli). Over the free portions at the top of foot processes covered by dense concentrations of hemoglobin there was a narrower relatively clear space which was one to two times as thick as the surface coat (Figs. 8 and 9). The bundles of myofilaments at the top of the foot processes seemed to be associated with the lesser shrinkage there. These observations suggested that, except for the surface coat, the clear space was artifactual and that there was little space in the surface coat for FIG. 6. Surface coat on triple-layered plasma membrane of foot processes. It is somewhat wider than the plasma membrane. Rat 629. x 118,000. FIG. 7. Large bundles of myofilaments (Mr) in visceral epithelium. Microtubules (T) and randomly oriented fibrils (F) are also seen. Rat 717. x 32,000. FIG. 8. Hemoglobin masses in peripheral capillary, inner (/) and outer (O) layers of basement membrane and in Bowman's space. The relatively small amounts between adjacent foot processes indicate that most of them were in contact in vivo. Rat 731. x 88,000. FIG. 9. Hemoglobin masses in mesangial matrix (M) and outer (O) layer of basement membrane. The hemoglobin in Bowman's space is separated from the foot processes by a relatively clear space which is one to two times the width of the surface coat. Rat 731. x 86,000. FIG. 10. Hemoglobin masses in inner and outer layers of a rabbit glomerular basement membrane. Rabbit 35. x 87,000. FIG, 11. Hemoglobin extends from capillary lumen (C) through basement membrane to Bowman's space (BS). A pyknotic epithelial cell has contracted dark foot processes which alternate with the foot processes of a more normal cell. The relatively clear space over the contracted processes is about the thickness of the surface coat. Rabbit 88. x 58,000.
1
I
538
LATTA
hemoglobin. The looser portions of mesangial matrix and the outer layer of the basement membrane over the mesangial region were also penetrated by hemoglobin (Fig. 9). In the glomerulus of rabbit 88 some epithelial cells had died after clamping of the renal artery and the foot processes were quite shrunken and dense (Fig. 11). These foot processes alternated with less shrunken foot processes, probably from a different epithelial cell. Hemoglobin filled most of the space between these foot processes which apparently shrank before fixation and embedding. These processes were not separated from the mass of hemoglobin in Bowman's space by a large clear space, but they were surrounded by a thin relatively clear space which was just about the thickness of the surface coat. This also suggested that the surface coat was compact enough to keep hemoglobin molecules from increasing its density appreciably. DISCUSSION The observations on normal and control animals confirm and emphasize certain findings in earlier studies (3, 4, 10, 15, 18). After intravenous injection of hemoglobin, it does not ordinarily appear in concentrated form in the glomernlus (2). What concentrated it in the present studies? The three animals with the highest concentrations probably had hemolyzing red cells in their glomerular capillaries. In addition local glomerular filtration was probably greatly reduced or absent. The main features of the reaction to Clostridium welchii type A toxin (primarily lecithinase) are hemolysis and a shock-like state (5). Emboli of clot fragments and clamping of the renal artery or aorta would reduce glomerular filtration to negligible levels. The layers of the glomerular capillary wall will be discussed in order.
Endothelium A variety of large molecules and particles pass through endothelial fenestrations in the rat and mouse without apparent difficulty, and have been observed by electron microscopy in the basement membrane. These include Thorotrast particles about 200 /k in diameter (15), ferritin [about 100 fit (4) or 110 ]i (1) in diameter], catalase [mol. wt. about 232,400 (24); dimensions approximately 95 × 80 × 70 ]l (25)], myeloperoxidase [mol. wt. about 160,000 (8)], hemoglobin [tool. wt. 64,500; dimensions 64 × 55 × 50 A (2•)], and horseradish peroxidase [mol. wt. about 40,000 (6); tool. dia. about 50 it (1)]. Diaphragms with central knobs have been described in the mouse glomerulus (23), but they do not seem to present a significant barrier to the peroxidase molecules (6). In a detailed study of their permeability, diaphragms in the capillaries of mouse
GLOMERULAR CAPILLARY WALL
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intestiDe presented little barrier to horseradish peroxidase although they did slow the passage of ferritin (1). Diaphragms are rarely seen in the rat glomerulus under the same conditions where they are well preserved in the medulla (17). Even if they should prove to be more frequent in the rat glomerulus than has hitherto been described, they apparently offer little barrier to the passage of Thorotrast (15), ferritin (4), or hemoglobin molecules.
Basement membrane Earlier work showing Thorotrast particles of about 140 ~ diameter (15) and ferritin (4) in the inner less-dense layer of the basement membrane indicated that this layer was functionally significant and probably had the structure of a loose gel. The filling of this layer with hemoglobin in the present studies and with horseradish peroxidase (HRPO) and myeloperoxidase (MPO) (6) reinforces this conclusion. The central dense layer of the normal basement membrane has been shown to stop the passage of Thorotrast (15) and ferritin (4) particles of about 100 A diameter. The observations that the central dense layer does not increase much in density at the time when the inner and outer light layers are filled with hemoglobin in these studies or with horseradish peroxidase (6) indicates that this layer is molecularly fairly dense and penetrated by relatively few channels narrower than about 100 A and wider than 64 A. (The channels or pores do not need to be cylindrical: they could be elliptical or slit-like in cross section.) Biochemical (9) and immunologic studies (22) indicate that a large portion of the glomerular basement membrane is collagen and that the remainder is composed of glycoproteins. Both of these proteins occur as elongated molecules. The observation of faintly stained fine fibrils in the plane of the central layer in the present studies with Epon embedding confirms earlier evidence with methacrylate embedding (3, 4, 10). Epon is generally considered superior to methacrylate in maintaining fine structure. Some caution regarding the presence and size of fibrils in the earlier observations was rightly advised (3, 4); but other considerations based on birefringence, artifactual layering, and a structural need to resist the force of the blood pressure suggest that elongated molecules run in the plane of the central layer (12). The large fibers in the inner layer have not been noted frequently in these studies or by others (4). The failure to observe a definite periodicity in them or any other structure in the basement membrane, even when a collagen periodicity was revealed in the mesangial region (13) indicates that, if collagen is present, it is not in a stainable form or the staining is blocked by some other substances, possibly mucoproteins. This idea is strengthened by the observation that some fibers in obvious collagen bundles outside the glomerulus may appear nonperiodic and, in cross section, ringlike. The uranium poisoned rabbit shows that large fibers can occur in the central
540
LATTA
layer, but whether exposed or newly formed is not certain at present. In summary, a large portion of the basement membrane appears to be collagen, but very little of it appears in recognizable form. Small fibrils run in the plane of the central layer like a feltwork and are either a form of collagen or glycoprotein or both. The functional reality of the outer less dense layer of the basement membrane is demonstrated by permeation with enough hemoglobin to make it appear darker than the central layer. A similar increase in density is shown by HRPO (ref. 6, Fig. 6). Because both hemoglobin and HRPO pass readily into the glomerular filtrate, the increased density is probably due to the outer layer having a looser structure than the central layer. Its structure appears to be only a little denser than that of the inner layer. The faint fibrils which run from the foot processes to the central layer probably have a supportive function. The studies with myeloperoxidase (6, 8) and catalase (8) indicate a barrier to these molecules in the epithelial slits, probably at the level of the outer limiting or slit membrane (ref. 6, Fig. 16). That this barrier allows the passage of some MPO molecules is shown by a little reaction product on some of the epithelial membranes distal to the outer limiting membrane and in the phagosomes of a few of the proximal tubules (ref. 6, Fig. 2). Permeation of the outer layer over the mesangium by hemoglobin and by HRPO (6) indicates that it is also involved in glomerular filtration.
Foot processes of the epithelium The bundles of parallel filaments frequently seen in epithelial cytoplasm and foot processes resemble filamentous bundles in smooth muscle cells. A larger filament is seen in myoid cells with inert dehydration and is thought to represent myosin which is not preserved by conventional procedures (20). Fine filaments such as those seen in the present studies are thought to be actin (20). Although the abundance of fine myoid filaments in glomerular epithelial cells is surprising because constriction of glomerular capillaries does not seem to have been described, it may be that these filaments are responsible for retraction of foot processes that is probably the basis for their loss in diseases associated with proteinuria. The roughly similar spacing that separates the foot processes from each other and from overlying epithelial cell cytoplasm in normal animals gives the impression of pieces of a jig-saw puzzle which have shrunk, leaving a gap between them. An obvious exception to this shrinkage would be the portion of the foot processes embedded in the basement membrane. The conclusion that shrinkage during fixation and embedding has occurred is hard to escape when the foot processes are examined during excretion of hemoglobin. At the time when hemoglobin is filling the inner and outer layers of the basement membrane and passing into the free portions of Bowman's space there are large relatively clear areas between most foot processes. This indicates
GLOMERULAR CAPILLARY WALL
541
that the sides of most foot processes were in contact in vivo while the hemoglobin was passing into Bowman's space. The presence of thin masses of hemoglobin in the center of some spaces between foot processes indicates that the contact is not perfect, or that some shrinkage of the processes occurred before the hemoglobin in Bowman's space was fixed. Similar results are also seen with H R P O (6). Much evidence indicates that the free surface of the plasma membrane of glomerular epithelial cells is covered by a polysaccharide coat, about 120 • thick (7, 14, 19). These layers have little intrinsic electron density and stain poorly with the usual stains but quite strongly with substances that stain polysaccharides. If the surfaces are usually in contact in the living animal as we believe, then the plasma membranes of most adjacent foot processes would be separated by two covering layers totaling approximately 240 A in thickness. The layers stick to the foot processes when they shrink (7, 14). An early estimate of about 100 ~ for the width of the filtration slit was of considerable physiologic interest (11), but the present evidence for a much wider slit and the presence of other structures in it introduce considerable change in the physiologic considerations. The glomerular fikrate would have to pass through the surface coats for the height of the foot processes, or some 3000-5000 ~ in most cases. This is consistent with the function suggested for the covering layers on other cells--holding water and maintaining a pathway for the diffusion of ions and small molecules (19). Between the few processes where some hemoglobin is seen, the filtrate would still have to pass through a portion of the polysaccharide coat covering the slit membrane. Even with considerable shrinkage of the foot processes, a surface coat remains covering the slit membrane (7). The evidence that hemoglobin and H R P O penetrate the surface coats while only very little myeloperoxidase is able to penetrate (ref. 6, Fig. 16), makes it appear that the covering layers have a permeability similar to that of the basement membrane at the level of the slit membrane. Thus it is difficult to say whether the slit membrane or the surface coats or both are the main filtration barrier. The thinness of the slit membrane (about 40-60 ~ ) makes it seem likely that the covering layers support the slit membrane and back it up as a filtration barrier to myeloperoxidase, catalase and larger molecules. The effective pore size here can be estimated as lying between 64 and 95 ~ , from the dimensions of hemoglobin and catalase, because the shape of myeloperoxidase is not known. Measurements of the width of the slit membrane correspond to the thickness of two surface coats filling the slit between the foot processes, or about 240 A. One might ask whether some shrinkage of the portion of the foot processes embedded in the outer layer of the basement membrane might occur with fixation and embedding. This seems not to occur to a significant extent because it would produce a decreased density or clear space in the peroxidase precipitates between the base of the foot processes. Only a faint space is seen in some places (6).
542
LATTA
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FI6. 12. Peripheral glomerular capillary wall. Diagram of the probable relationships of the components in the rat in vivo. The filtration path runs through the fenestrations (F) of the endothelium (En); the loose inner layer (IL), the compact central layer (CL), the loose outer layer (OL), and the slit membrane (SM)of the basement membrane; and the surface coat (SC) of epithelial foot processes (FP) which usually seem to be in lateral contact in vivo, to Bowman's space (BS). Epithelial cells (Ep) contain many bundles of fine myofilaments (Mf) and show a triple-layered plasma membrane (PM).
Glomerular filtration Glomerular permeability studies with a variety of molecules are consistent with theoretical curves for an isoporous membrane having cylindrical pores 70-84/k in diameter (11). Thus the conformity with the electron microscopic data is fairly close. The relative impermeability of the glomerular capillary wall to albumin and globulin is probably due to the great asymmetry of these molecules. The observation that large masses of denatured globin can work their way through the basement membrane and between foot processes led to the suggestion that the membrane acts like a thixotropic gel which liquefies under pressure (16). It may also be pointed out that relatively mild alterations produce proteinuria of serum albumin or globulin, such as intravenous injection of solutions of these proteins or passive congestion as in heart failure. The probable fine structure of the peripheral glomerular capillary wall and the process of filtration through it can now be described and illustrated (Fig. 12). Most endothelial fenestrations appear to be open in the rat. The inner less dense layer of the basement membrane seems to be a loose gel which is easily filled with hemoglobin or horseradish peroxidase molecules. Thorotrast particles and ferritin molecules
GLOMERULAR CAPILLARYWALL
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penetrate it less readily, and it may be supposed that the elongated molecules of serum albumin and globulin would also experience difficulty. The central dense layer seems to be composed of densely packed fibrils, probably both collagen and glycoprotein, many lying in the plane of the membrane, with just enough channels to allow free passage of hemoglobin molecules, but normally stopping molecules or particles over 100 • diameter. The outer layer of the basement membrane appears to be a loose gel similar to the inner layer but with more radially oriented molecules to support the foot processes. The main filtration barrier to protein molecules between 64 and 95 A in greatest dimension seems to be the outer limiting membrane and the polysaccharide coat which generally covers it and the free surface of the foot processes. As the polysaccharide coats of most foot processes are probably in contact normally in vivo, most of the glomerular filtrate and its ionized salts must pass through long distances of these covering layers. The author wishes to thank Mrs Elizabeth Sykes and Mrs Lya Cordova for very capable technical assistance. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8.
9. 10. 11.
12. 13. 14. 15. 16. 17. 18. 19.
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-ibid. 23, 304 (1968). PERt~TZ, M. F., Harvey Lectures 63, 213 (1969). PIERCE, G. B. and NAKANE, P. K., Lab. Invest. 17, 499 (1967). R~OD~N, J. A. G., 3. Ultrastruet. Res. 6, 161 (1962). SCnROEDER, W. A., SHELTON, J. R., Sm~LTON,J. B., ROBB~RSON, B. and AVELL, G., Arch. Biochem. Biophys. 131, 653 (1969). 25. V~aNSHTZIN,B. K., BARYNrN, V. V. and GURSI¢AYA,G. V., Aeta Crystallog. Sect. A 25, S 181 (1969).