Ultrastructure of three-dimensionally localized distal nephron segments in superficial cortex of the rat kidney

JOURNAL

OF ULTRASTRUCTURE

AND

MOLECULAR

STRUCTURE

RESEARCH

9,

169-187 (1988)

Ultrastructure of Three-Dimensionally Localized Distal Nephron Segments in Superficial Cortex of the Rat Kidney JENS

Department

of

D~RUP

Cell Biology, Institute of Anatomy,

University of Aarhus, DK-8000 Aarhus C, Denmark

Received April 7, 1988 The ultrastructure of superlkial distal nephron segments was analyzed after precise localization of tubule cross sections using computer-assisted three-dimensional reconstructions. Five systems of tubules, each with three interconnected distal tubules, were reconstructed and the lengths of the post macula densa segment of the distal straight tubule (DST), the distal convoluted tubule (DCT), the connecting tubule (CNT), and the initial collecting tubule (ICT) were determined. Each cortical collecting duct (CCD) was in continuity with only one tubule in contact with the renal capsule. In three of the five reconstructions, the two nonsubcapsular tubules fused and had a common connection to the subcapsular tubule. The length, between the macula densa (MD) and the confluence, of subcapsular tubules (2.68 + 0.15 mm) significantly exceeded the length of tubules not in contact with the renal capsule (2.05 + 0.10 mm). This difference was mainly due to a longer ICT in subcapsular tubules. Subcapsular tubules always contacted the renal capsule in the early DCT and often again in the ICT. Cells in the early DCT showed more microvilli on the luminal surface and more infoldings of basolateral membranes than cells in the late DCT. The ultrastructure of intercalated cells (I cells) varied within a range of different manifestations and the ultrastructural variation of I cells was similar in all the analyzed tubule segments. Connecting tubule cells and principal cells were similar in ultrastructure in all tubule segments and cortical levels analyzed. 0 1988 Academic Press, Inc.

INTRODUCTION

The distal nephron segmentsin the renal cortex comprise the distal straight tubule (DST), the distal convoluted tubule (DCT), the connectingtubule (CNT), the initial collecting tubule (ICT), and the cortical collecting duct (CCD) (for reviews see Kaissling, 1982; Madsen and Tisher, 1986). Each nephron segment contributes in its own specific way to the transportfunctions of the tubule, and hormones and diuretics affect thesefunctions differently depending on the segment(Morel et al., 1976;Wright and Giebisch, 1978;Wadeet al., 1979;Morel et al., 1982;Materson, 1983;S&latter et al., 1983; Kaissling et al., 1985;Stanton, 1985; Hays et al., 1986;Bonvalet et al., 1987;Ellison et al., 1987;Kashgarianet al., 1987).Important functional information on distal nephron segments has particularly been obtained through micropuncture and microperfusion studies (Malnic et al., 1963;

Burg and Stoner, 1973;Field et al., 1984; Levine, 1985;O’Neil and Hayhurst, 1985; Giebisch, 1986).The anatomical organization of the distal tubule andits segmentshas been dealt with in extensive microdissection studies (Peter, 1927;Oliver, 1968)but without reference to the ultrastructure of the tubule segments. In many previous qualitative electron microscope studies the segmentswere identified with the aid of adjacent semithin sections for light microscopy (Griffith et al., 1968;Kaissling, 1977; Welling et al., 1981;Larsson, 1982).The segmental ultrastructure of one single three-dimensionally (3D) reconstructed distal nephron was analyzed qualitatively by Crayen and Thoenes(1978).Qualitative and quantitative structural information has beenacquiredon individual cell types in the distal nephron (Wade et al., 1979;Stanton et al., 1981;Madsenand Tisher, 1983,1984; Dorup, 1985a; Verlander et al., 1987). However, quantitativeultrastructural infor-

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mation on individual cell types combined with information on their precise location along the distal nephronhas not beenpublished. In the present study the threedimensional course of superficial distal nephron segmentswas analyzed with the aid of the method for computer-assisted3D reconstruction(Dorup et al., 1983),in order to obtain information on the length and cellular composition of individual segments and to combine this information with ultrastructural information on the individual cell types in the distal nephron. Particular emphasiswas placedon the structural characterization of subcapsularnephrons,which are available for micropuncture, and on a comparison between these nephrons and superficial nephronsnot accessibleto micropuncture. The present study provides a quantitative structural basis for studies dealing with the functions of the different distal nephron segments. MATERIALS

AND METHODS

Animals

Five female Wistar rats (stock, Mol:WIST) weighing 19&230 g were studied. The animals were kept on standard rat pellets and had free access to tap water. The same animals were used as controls in previous studies (Dotup, 1985a, 1985b). Preparation

for

Light

and

Electron

Microscopy

The preparation for light and electron microscopy was the same as that in previous studies (Dotup, 1985a). Briefly, the kidneys were perfusion-fixed with 2% glutaraldehyde in 0.1 M sodium cacodylate buffer through the abdominal aorta. Superficial cortical tissue was excised in rectangular, 1 x 1 x 3-mm blocks including the renal capsule at one end. All blocks were post-fixed in 1% osmium tetroxide, stained en bloc with 0.5% uranyl acetate, dehydrated in alcohol, and embedded in Epon. Semithin sections were stained with toluidine blue for light microscopy. Electron microscope analysis was performed after reembedding and resectioning of the 4-urn semithin sections (Maunsbath, 1978). Ultrathin sections were stained with uranyl acetate and lead citrate and analyzed in a JEOL 1OOB or 1OOCX electron microscope. Perfusion-fixation, osmium tetroxide fixation, en bloc staining, embedding, and section staining were carried out as previously described (Maunsbach, 1966).

Identl$cation of Cell Distal Nephron

Types

and

Segments

in the

The distal nephron, from the beginning of the distal straight tubule (also termed the thick ascending limb of the loop of Henle, TAL) to the end of the cortical collecting duct contains a complex distribution of five different cell types: distal straight tubule cells (DST cells), distal convoluted tubule cells (DCT cells), connecting tubule cells (CNT cells), intercalated cells (I cells), and principal cells (P cells) (Kriz et al., 1978; Kaissling, 1982; Dotup, 1985a; Madsen and Tisher, 1986). DST cells were identified as low cells with a centrally placed nucleus. Basolateral interdigitations were extensively developed and the basolateral membranes showed a close association to mitochondria. Often the interdigitations extended almost to the apical cell membrane (Kaissling, 1982; Kone et al., 1984). DCT cells, CNT cells, I cells and P cells were identified as described in detail earlier (Stanton et al., 1981; Kaissling, 1982; Derup, 1985a; Madsen and Tisher, 1986). All of these cells could be positively identified on the basis of ultrastructural characteristics on sections passing through the center of the cells. When the section passed through the periphery of the cells the identification of the cells was sometimes more difftcult than when the cells were cut centrally. However, even these peripherally sectioned cells could usually be identified on the basis of their cytological criteria. The identification of segments in the distal nephron was based on the cell types present in any given segment. Identification was based exclusively on TEM micrographs. The resolution of light microscopy was insufftcient to distinguish consistently between, i.e., the late DCT and the CNT and between the CNT and the ICT. This became obvious in the present study since the same tubule cross sections were examined first in the light microscope and later, after reembedding and resectioning, in the electron microscope. The segment identification was based on which cell type (DST, DCT, CNT, or P cells) had the highest volume density, determined by point counting, in the tubule cross section. The volume density was used rather than the numerical density of each cell type since the numerical density cannot be measured on single micrographs. The post macula densa segment was considered a part of the distal straight tubule. It was composed of a homogeneous population of DST cells (Kaissling et al., 1977). The transition from the DST to the distal convoluted tubule was sharp and was easily identified by the change from the low DST cells to the tall DCT cells. The distal convoluted tubule was identified as that part of the tubule in which DCT cells predominated (had the highest volume density). The early DCT was identified as that part of the DCT where no I cells were present whereas the late DCT was characterized

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FIG. 1. Schematic drawing of the five systems of distal tubules reconstructed and analyzed. The renal capsule is indicated by the horizontal straight line. The reconstructions are oriented perpendicular to the renal capsule. Distal straight tubules are marked by numbers indicating the tubule levels. The position of the macula densa is indicated by dots. Asterisks indicate points of confluence with tubules not reconstructed. In (a, c, and d) there are small arcade portions after the confluence of level 2 and 3 tubules. In (b and e) no arcades are present.

by the appearance of some I cells and some CNT cells in the tubule wall. The connecting tubule was identified as that tubule segment in which CNT cells predominated (had the highest volume density). The cortical collecting duct was identified as that tubule segment in which P cells predominated (had the highest volume density). The ultrastructures of the tubule before and after the confluence of two distal tubules were similar. The initial collecting tubule is referred to as that part of the cortical collecting duct which is positioned before the confluence with another cortical collecting duct whereas the cortical collecting duct after the confluence is referred to as the cortical collecting tubule. The identification of cell types and segments described here is in good general agreement with the recently published standard nomenclature for structures of the kidney (Kriz et al., 1988). Only at one point was there a small difference. In the present study, the CNT was defined as that tubule segment where CNT cells had the highest volume density compared to DCT cells and P cells whereas in the standard nomenclature, the beginning of the CNT is defined as that point where the first CNT cell occurs. The method for segment identification used in the present study allows for a closer correlation to the function of the segment, since it is likely that the function is related to the predominating cell type in the segment. In addition, since a small fraction of, i.e., one single CNT cell may be very difficult to detect on an electron micrograph or in the electron microscope, the present method for identification of distal nephron segments may be easier to use. Three-dimensional reconstructions. One tissue block was analyzed from each of the five animals.

From each block, a series of 250 semithin (4-pm) sections was cut parallel to the renal capsule. The renal capsule was always included in the beginning of each series. Every second semithin section was photographed in the light microscope and printed at a final magnification of x 200. Using the photographs of the semithin sections, the macula densa of the most superficial glomerulus in the center of the block was found. The distal tubule of this nephron (level 1 tubule) was then traced retrograde along the DST to about 1 mm from the renal capsule. The same tubule was also traced forward from the macula densa to the contluence with another distal tubule. This other tubule (level 2 tubule) in turn was traced backward to the macula densa and further down the DST. The collecting duct after the confluence was traced to a distance of about 1 mm from the renal capsule. A third tubule (level 3 tubule) branching either from the collecting duct or from the level 2 tubule was also traced. Thus, the three most superficial tubules, branching from one collecting duct, were traced in each animal (Fig. 1). Subsequently, each of the live systems of three tubules was reconstructed using computer-assisted 3D reconstruction (Dotup et al., 1983). The results of the reconstruction program consisted of stereoplots of the reconstructed nephrons generated by the computer and drawn on a computer plotter (Figs. 2 and 3). Moreover, the computer generated a fde containing coordinate information for each tubule cross section on each semithin section with associated information on the distance from the macula densa along the tubule axis. Since the length of the nephrons varied considerably, many cytological parameters were related not only to the actual distance from the macula densa but also to the position along the tubule axis expressed as

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a percentage of the total distance from the macula densa to the confluence of two tubules (Table I). Morphometry Inner and outer tubular diameters were measured on each of the reconstructed nephrons using the photomicrographs of the semithin sections. Only tubules cut approximately perpendicular to the tubule axis were measured. In tubule cross sections where the profile of the tubule was not perfectly round, the mean inner and outer diameters were estimated as the diameter of two circles with the best tit to outer and inner tubule profiles. Measurements were made for every 100-200 pm along the tubule axis. The epithelial height was calculated as (D,-D,)/2 where D, is the outer diameter and Q is the inner diameter of the tubule. The tubular waN volume per micrometer tubule length was calculated as n(D,* - D,2)/4. Ultrathin sections of the reconstructed nephrons were obtained with intervals of approximately 200 pm along the tubule axis starting with the late part of the distal straight tubule and ending with the first part of the cortical collecting duct. Whole tubule cross sections of the reconstructed nephrons were recorded at approximately X 2000 magnification and printed at X 6000. Point counting was performed using a square test lattice with points with distances of 1 cm. Volume densities of individual cell types were calculated as Vi/V, where Vi is the number of points falling on one particular type of cell and V, is the total number of points falling on all the cells of the tubule wall (Weibel, 1979). Intercellular spaces were not included. However, intercellular spaces amount to less than 5% of the tubule wall and thus they had a negligible influence on the calculations below. The total volume of cell types (Table II) between the macula densa and the confluence was calculated as 1 c

(Lj ’ TWVi . VVi),

where i refers to one of the following live parts ofthe tubule described as a percentage of the total length

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of the tubule from the macula densa to the confluence: i = 1 corresponds to tubule segments positioned within the fmt 7% of the total tubule length, i = 2 refers to tubules from 7 to 25%, i = 3 from 2.5 to XI%, i = 4 from 50 to 75%, and i = 5 from 75 to 100% of this length. Li is the length (in g.m) of the given tubule part, TWV, is the tubular wall volume and Vvi is the volume density of the given cell type. in the segment. The summation was performed for each of the five cell types for each of the 15 analyzed tubules. The volume of the tubule lumen (Table III) was calculated as

2

(Li ’ IT * (DJ2)2),

where i takes on the same values as above and Di is the mean inner diameter of the tubule in the given segment. Statistical Evaluation Morphometric parameters were computed and statistically evaluated using the BMDP statistical software package (Dixon, 1983). Differences between tubule segments and levels (see Materials and Methods for explanation) were tested first with an analysis of variance (ANOVA) with the segment or level as a within factor. In situations where the ANOVA reached significance (P < 0.05) the differences between segments were tested using Wilcoxon’s signed ranks test. P values below 0.05 were considered statistically significant. Data are expressed as means * SE. RESULTS

Branching of Nephrons and Relation of Superficial Distal Nephron Segments to the Renal Capsule The branching and the relation to the renal capsule for all five reconstructed nephrons are shown schematically in Fig. 1. Two of the reconstructions are shown as

FIG. 2. Stereoplot, drawn by a computer plotter, of one of the five 3D reconstructed systems of distal tubules. The plot was rotated 4” around the y axis from lefi to right. The tubule levels (see Materials and Methods) are indicated by the numbers (I-3) on the distal straight tubules. The unmarked tubule is the cortical collecting duct. After the confluence of the level 2 and 3 tubules, a small arcade portion was present before the confluence with the level 1 (subcapsular tubule). At the uppermost part of the drawing the tubule makes contact with the renal capsule. The initial collecting tubule of the level 1 nephron approaches the renal capsule although no direct contact is made. Magnification x 100. FIG. 3. Stereoplot of a system of three distal tubules where no arcade formation was found. That is, tubules from levels 1 and 2 fuse to make up the cortical collecting tubule, which in turn receives confluence from the level 3 tubule (and possibly other tubules as well). Tubule levels are indicated by numbers on the distal straight tubules. The unmarked tubule is the cortical collecting duct. Magnification x 100.

174

JENS D@RUP TABLE I LENGTH (IN mm) OF DISTAL NEPHRON SEGMENTS AS RELATED TO THEIR DISTANCE FROM THE RENAL CAPSULE Nephron level Nephron segment

I 2 * ‘k

II

Post macula densa segment Distal convoluted tubule Connecting tubule Initial collecting tubule

0.19 1.23 0.51 0.71

0.03 0.10 0.04 0.12

Macula densa to confluence

2.68 + 0.15

0.12 1.16 0.43 0.40

+ f f 2

III 0.01 0.08 0.03 0.10

0.15 1.07 0.53 0.33

+ f f t

P

All nephrons 0.03 0.08 0.06 0.04

0.15 1.15 0.49 0.48

+ + f f

0.02 0.05 0.03 0.07

N.S. N.S. N.S. I > 11,111

P < 0.05 2.11 * 0.11

1.99 + 0.12

2.26 f 0.11

I > 11,111

P < 0.05 Note. See Materials and Methods for explanation of nephron levels. Values are means + SE, n = 5. For all nephrons, the value for each animal was calculated as the mean of the three tubule levels. P values refer to statistical comparison between means of nephron lengths.

stereoplots in Figs. 2 and 3. Subcapsular tubules (level 1 tubules) always connected directly to the collecting duct whereas level 2 tubules in three cases fused with a level 3 tubule in an arcade which in turn connected to the collecting duct (a, c, d in Figs. 1 and 2) and in two cases (b and e in Figs. 1 and 3) fused with a level 1 tubule. In the three tubules forming arcades, the lengths along the tubule axis from the first to the second confluence were 22, 209, and 287 pm, respectively, Only one distal tubule, in continuity with the collecting duct, made contact with the renal capsule and would thus be accessible for micropuncture. The first contact of these five subcapsular nephrons with the renal capsule was in the distal convoluted tubule at a distance of 582 + 109 p,rn from the macula densa. One of the live subcapsular tubules made two contacts with the

renal capsule at 199 and 496 pm from the macula densa, respectively (Fig. 3). All five subcapsular tubules approached the renal capsule once more in the initial collecting tubule before the first confluence with another distal tubule in such a way that three initial collecting tubules made direct contact with the capsule whereas in two tubules the closest distances to the capsule from the tubule axis were 95 and 112 pm, respectively (Figs. 2 and 3). The cortical collecting tubule (after the confluence) never approached the renal capsule. Comparison between Tubules Located Different Distances from the Renal Capsule

at

Three different tubule levels (levels l-3), each with increasing distances from the re-

TABLE II TOTAL VOLUME (IN urn3 x ld) OF DISTAL NEPHRON CELL TYPES BETWEEN MACULA DENSA AND CONFLUENCE Cell type Nephron level

DST cells

DCT cells

CNT cells

I cells

P cells

AlI cells together

Level 1 Level 2 Level 3

67 + 16 64 k 16 46 + 8

1080 +- 50 960 f 93 805 f 78

289 + 66 267 k 42 169 f 55

235 f 25 165 f 16 126 f 23

246 f 49 130 k 49 112 rt 24

1920 + 140 1590 f 50 1260 + 30

Note. See Materials and Methods for explanation on nephron levels and methods of calculation. Values are means f SE (n = 5 animals). Differences between levels for individual cell types were not statistically significant when tested with an analysis of variance. For the total values Level 1 > Level 2 > Level 3. P < 0.05 for both comparisons.

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VOLUME OF TUBULE LUMEN (IN PICOLITERS) BETWEEN MACULA DENSA AND CONFLUENCE Distance in % of distance from MD to confluence Nephron .Level Level Level .-

level 1 2 3

Mean l-3

<7

7-25

25-50

SO-75

75-100

O-100

25 k 5 22 2 3 20 2 3

70 f 14 52 +- 8 48 * 7

147 4 29 104 t 19 94 ‘- 9

192 k 14 153 2 24 138 f 10

160 f 34 123 + 18 121 2 10

594 2 69 454 k 61 422 2 25

22 + 2

57 f 6

115 k 13

161 2 11

135 f 13

490 + 36

Note. Values are means rf: SE (n = 5 animals). The values for “<7%” corresponds approximately “7-25%” to the early DCT, “25-50%” to the late DCT, “50-75%” to the CNT, and “75-100%” Differences among the three levels were not statistically significant.

nal capsule, were analyzed (see Materials and Methods). The distance from the macula dense to the first confluence with another distal tubule (Table I) for subcapsular (level 1) tubules, 2.68 * 0.15 mm (n = 5), was significantly longer than for nephrons not in contact with the renal capsule, 2.11 2 0.11 mm for level’2 nephrons and 1.99 ? 0.12 mm for level 3 nephrons (P < 0.05 for both comparisons). The lengths of the post macula densa segment, the distal convoluted tubule, and the connecting tubule were not significantly different in the three tubule levels. The initial collecting tubule, however, was significantly longer in level 1 nephrons than in level 2 and level 3 nephrons (Table I). Values for outer and inner tubular diameters, epithelial height, and tubular wall volume were not significantly different in the three analyzed tubule levels (Fig. 4). Qualitatively, there were no differences in the ultrastructure of each of the five cell types when comparing cells from the three tubule levels. Structure of Distal Nephron Segments in the Superficial Renal Cortex

The epithelial height, outer and inner tubular diameters, and the tubular wall volume per micrometer tubule length are shown in Fig. 4. Volume densities of the cytoplasm (including the nucleus) of the five cell types as related to the distance from the macula densa in each of the five subcapsular tubules are shown in Fig. 5. Figure 6 shows the volume density of each

to the DST, to the ICT.

cell type expressed as the mean of the three tubule levels. The distance from the macula densa was measured as a percentage of the total distance from the macula densa to the confluence in each tubule. The mean length of the post macula densa segment was 0.15 + 0.02 mm corresponding to 7% of the distance from the macula densa to the first confluence with another distal tubule. The mean length of the distal convoluted tubule in the superficial renal cortex was 1.15 + 0.05 mm (n = 5 animals, each represented by the mean of three tubules). The epithelium in the early part of the distal convoluted tubule was significantly higher (8.8 ? 0.5 km) than that in the post macula densa segment (5.0 +- 0.2 km), P < 0.001, Fig. 4. The distal convoluted tubule in its course generally described four to seven sharp convolutions of 180” or more. There were no obvious patterns in the arrangement of distal convolutions and no differences among the three tubule levels analyzed. In some tubules, the convolutions extended for a long distance through the cortical tissue. In other tubules multiple convolutions were present in the close vicinity of the glomerulus. The length of the connecting tubule in the superficial renal cortex averaged 0.49 ? 0.03 mm (n = 5 animals, each animal represented by the mean of three tubules). In midcortical and in juxtamedullary nephrons, the CNT is known to be considerably longer and to be part of the arcades (Potter, 1972; Kaissiing, 1982). In the present study

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04 0

25

15

Epithelial

125

100

75

50

150

Height

I

01 0

25

800,

25 Percent

100

75

Tubular

0

04

50

75 50 of Distance

Wall

from

125

150

Volume

M.D. to

COnilUWlCe

____ -_. from

M.D. Cm)

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of superficial nephrons, however, the CNT in all the analyzed tubules were short and ended before the confluence with another tubule. The length of the initial collecting tubule averaged 0.48 k 0.07 mm (Table I). In the first small part of the ICT, CNT cells were gradually replaced by P cells and thereafter only P cells and I cells were present in the tubule wall. The inner tubular diameter increase gradually from 13 km in the early DCT to 20 pm in the CCD. The epithelial height and the tubular wall volume were lower in the ICT and CCD than in the DCT and the CNT. The increase in inner tubular diameter corresponds to an increase in volume of the tubule lumen from 1.33 x lo5 km3/mm tubule length in the early DCT to 3.14 x lo5 pm3/mm tubule length in the CCD. Segmental Ultrastructure of Cells in the Superficial Distal Nephron Distal straight tubule cells. The epithelium of the post macula densa segment of the distal straight tubule (Fig. 7) consisted of DST cells similar in ultrastructure to the epithelium of the distal straight tubule immediately before the macula densa. The ultrastructure of epithelial cells in the post macula densa segment was characterized by extensive infoldings and invaginations of basal and lateral cell membranes. These invaginations often extended into the apical cytoplasm. Especially in the basal cytoplasm, basolateral membranes were closely associated with long, rod-shaped mitochondria. The nucleus was positioned centrally in the cells. Further details on the ultra-

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structure of the post macula densa segment have been described earlier (Kaissling, et al., 1977; Kone, et al., 1984). Distal convoluted tubule cells. DCT cells were generally higher than DST cells (Figs. 4, 7, 8) and the transition from the distal straight tubule to the distal convoluted tubule was sharp and distinct. The ultrastructure of the epithelial wall varied along the course of the distal convoluted tubule (DCT). The early DCT consisted of a homogeneous epithelium of DCT cells (Fig. 8). The apical surfaces were covered with numerous small microvilli. The cell nucleus was positioned near the luminal surface. Infoldings and interdigitations of basolateral membranes were well developed and the intercellular spaces were narrow (Kaissling, 1982; Dot-up, 1985a). The late DCT was composed of DCT cells as well as CNT cells and I cells (Figs. 9-14). DCT cells in the late DCT showed fewer apical microvilli, and infoldings and interdigitations of basolateral membranes were less developed than those in the early DCT. Intercalated cells. I cells were first observed at a distance of 1.03 + 0.04 mm from the macula densa (range 0.84-1.34 mm, n = 15). The volume density of I cells increased from about 10% in the late DCT to 30% in the ICT and the first part of the cortical collecting duct (Fig. S).The ultrastructure of I cells varied considerably from cell to cell even within the same tubule cross section (Figs. 9, 11-13). I cells were often more electron dense than neighboring cells (Fig. 12). However, I cells with an electron density of the cytoplasm similar to neighboring DCT, CNT, or P cells were also found (Fig.

FIG. 4. Outer and inner diameters, epithelial height, and tubular wall volume related to the tubule level and to the distance from the macula densa expressed as a percentage of the total distance from the macula densa to the first confluence of two tubules. That is, the confluence corresponds to 100%. ((- + -) level 1; (0) level 2; (.x.) level 3.) Single observations are means of observations in the intervals: O-7% (post macula densa segment of DST), 7-25% (roughly the early DCT), 25-50% (roughly the late DCT), 50-75% (roughly the CNT), 75-W% (roughly ICT), and MO-125% and 125-150% (CCD). The differences among levels 1 to 3 were not statistically significant in any of the analyzed segments. FIG. 5. Volume density of individual cell types relative to the total tubule wall and related to the distance along the tubule from the macula densa measured in micrometers. The curves represents one level 1 (subcapsular) tubule from each of the five animals analyzed. (x) DST cells; (0) DCT cells; (- + -) CNT cells; (-) I cells; (*O.) P cells. Arrows indicate levels of confluence with another distal tubule.

JENS D@RUP

T TT DST cells

40

50

60

70

J

DCT cells

CNT cells P cells

0

Percent of distance from M.D. to confluence FIG. 6. Volume density (in %) of individual cell types in the total tubule wall related to the distance along the tubule expressed as a percentage of the total distance from the macula densa to the confluence with another distal tubule. Each animal was represented by the mean of the tubule levels (l-3). Each column represents the mean of the five animals analyzed. Bars indicates SE (n = 5). The arrow points to the level of the confluence with another tubule (100%).

13). The nucleus in I cells were usually round and positioned either in the basal or in the central part of the cytoplasm. The basal cytoplasm, in all I cells, contained a palisade-like arrangement of basolateral infoldings with no obvious structural relation to mitochondria. Extensions from the basal cytoplasm of I cells were often found to project laterally under neighboring CNT cells (Fig. 15). Where I cells were in contact with P cells (Fig. 16) or rarely with other I cells (Fig. 12) the cellular contacts consisted of finger- or plate-like interlocking projections. I cells may be positively distinguished from neighboring cells by the characteristic

appearance of vesicles and microplicae/ microvilli in the apical part and of the palisade-like arrangement of membrane infoldings in the basal part of I cells (Kaissling, 1982; Madsen and Tisher, 1986). The variation in ultrastructure of intercalated cells was particularly pronounced for the apical cytoplasm. Some I cells showed a narrow apical cell pole with few intermediate (diameter 80-200 nm) vesicles, many small (40 nm) vesicles in the apical cytoplasm, and few short microprojections from the apical surface (Fig. 11). Other typical I cells contained large (>200 nm) vesicles along with numerous intermediate vesicles and a few small vesicles. Typically these I cells

FIG. 7. Electron micrograph from the post macula densa segment of the DST 70 pm after the macula densa. The epithelium consists of low DST cells. The luminal membrane is covered with numerous microvilli. At the asterisk the basal cytoplasm is cut tangentially. Mitochondria are closely packed in the basal cytoplasm. Magnification X 4000. Fro. 8. Electron micrograph from the early DCT 460 urn from the macula densa. Only DCT cells are present. The nucleus is positioned in the luminal part of the cytoplasm. Lateral cell borders are difficult to distinguish because of the elaborate basolateral interdigitations. Magnification x 4000.

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FIG. 9. Electron micrograph from the late DCT 1210 urn from the macula densa. To the left on this micrograph only DCT cells are present. To the right, intercalated cells and CNT cells (C) are present. The nuclei in CNT cells are more rounded and more centrally placed than those in DCT cells. Magnitication x 2000. FIG. 10. Electron micrograph of a CNT cell and a DCT cell from the late distal convoluted tubule 1010 pm from the macula densa. The CNT cell (to the left) is lower than the DCT cell. The lateral membranes of the two cells show extensive interdigitations. Magnitkation x 6000.

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also showed a narrowing of the apical cytoplasm (Fig. 13). Other I cells were characterized by an extensive development of the apical membrane into plate-like microplicae and a relatively wide apical cell pole. The number of intermediate vesicles in thesecells was generallysmall. The number of mitochondria varied considerablybetween I cells. Although someI cells showed a cytoplasm packed with mitochondria, none of the above-mentionedappearances of I cells were consistently rich in mitochondria and it should be noted that the mean volume density of mitochondria in I cells is significantly lower than that of DCT cells and not significantly different from that of CNT and P cells (Dorup, 1985a). The ultrastructure of I cells showed the same variation in the late DCT, the CNT, the ICT, and the CCT. Critical examination of I cells in all the analyzed segmentsrevealed all possibleintermediary forms, and thus a distinction between two or more structurally different types of I cells was impossible. Connecting tubule cells. CNT cells intermingled with DCT cells in the early part and with P cells in the late part of the CNT. Although the ultrastructure of CNT cells varied from cell to cell, no consistentqualitative differences were found when comparingthe ultrastructure of CNT cells in the early and in the late part of the CNT. CNT cells were generally lower than DCT ceils. The nucleuswas positioned centrally in the cells (Figs. 9 and 10).Both in the late DCT and in the CNT, interdigitations of basolateral membranes of neighboring CNT and DCT cells were extensively developed. Principal cells. The height of P cells decreasedslightly from the ICT to the CCD (Fig. 4). However, P cells in the late part of the CNT, in the ICT, and in the CCD showed the same general ultrastructural characteristics(Fig. 13).The cell shapewas simple, compared to DCT and CNT cells. The nucleuswas positioned centrally in the cells. The luminal surface showed only a few microvilli. Lateral cell membranes

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showedfew interdigitations with neighboring cells. Basal infoldings showedno structural associationwith mitochondria. DISCUSSION

The present study describes the threedimensional structure of distal tubules in the superficial renal cortex and relates ultrastructural findings in distal nephron segments to the precise location in the nephron. It is noteworthy that only one distal tubule, branching from each cortical collecting duct, made contact with the renal capsule,and that the first contact was consistently made by the early distal convoluted tubule at a point whereonly DCT cells were present in the tubule wall. Two hundred to six hundred micrometers after the contact with the capsule, however, both I cells and CNT cells appearedin the epithelium. In some subcapsulartubules, another direct contact with the renal capsule was made in the initial collecting tubule (ICT, before the confluence with another distal tubule). These findings imply that in microperfusion studies of distal tubules, the perfusion pipet will typically be placed in the early DCT andthe collection pipet in the ICT. In between these two pipets, four structurally and functionally different cell types will be present, which may complicate the interpretation of results of micropuncture studies on these segments. In a recent study, however, Velazquez et al. (1987)have demonstratedthat in a subpopulation of subcapsulartubules, similar to one of the five subcapsular tubules analyzed in the present study, the early distal convolutedtubule makestwo contactswith the renal capsulein addition to the contact made by the ICT. In these tubules microperfusion of a segment containing only DCT cells is possible. Another observationwhich may be relevant when interpreting results from micropuncture studies was that the lengths and cellular compositions of segmentsin subcapsulartubuleswere significantly different from those in nonsubcapsulartubules only

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with respectto the length of the initial collecting tubule, which was longerin the subcapsulartubules. This finding indicates that results from structural analysesof subcapsular tubules may be consideredrepresentative for all superficial tubules with the exception of the length of the ICT. The total length of the tubule segmentsfrom the macula densa to the confluence of two distal tubules, which was determined to 2.68 + 0.15 mm in subcapsulartubules by computer-assisted 3D reconstruction in the present study, only slightly exceededthe length of the same segmentsfound in a recent investigation as measured on casts made by injection of Microfil or latex into the tubule lumen (2.1 ? 0.1 mm in one and 2.3 + 0.1 mm in another group of tubules, Velazquez et al., 1987).The slight differencein length may be relatedto differences in breed and sex of the animals analyzed. The structural organizationof both luminal and basolateral membranes of DCT cells was more complex in the early than in the late part of the distal convoluted tubule. For CNT cells, no consistent differences were found between the segment and different parts of segmentsanalyzed. However, the connecting tubule in the superficial tubules analyzed in the present study was very short comparedto connectingtubules deeper in the cortex. Therefore, the possibility cannot be excluded that differencesexist in the ultrastructure of CNT in the deep cortex.

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Intercalatedcells were present in the tubule wall from the distal convoluted tubule to the cortical collecting duct. Recently it has been shown that I cells both in the medulla (Madsen and Tisher, 1983; 1984)and in the cortex (Dot-up, 1985b;Verlander et al., 1987)respondstructurally to changesin the acid/base balance. It has been concludedthat I cells both in the medulla andin the cortex participate in the maintenanceof the acid/basehomeostasis.It is well established that in control animals I cells exhibit different structural appearances(Crayen andThoenes,1978;Kaissling, 1982;Dorup, 1985a).On the basisof the structural heterogeneity of I cells, Verlander et al. (1987) describetwo structurally different types of I cells in the cortical collecting duct in both the outer and the inner cortex: one (type A), similar to I cells in the medullary collecting duct and in the connecting tubule, andanother(type B), with a darker staining cytoplasmic ground substance, an extensive vesicular compartment including vesicles in the basal cytoplasm, and an apical band of vesicle free cytoplasm. The presence of two cytochemically different types of I cells hasbeensuggestedon the basis of the detection of the anion transporting glycoprotein band-3 in the basolateral membranesof a subpopulationof I cells in the renal cortex (Schuster et al., 1986; Holthofer et al., 1987; Verlander et al., 1988).Regionaldifferencesin the affinity of I cells to lectins have suggestedthat I cells

FIG. 11. Electron micrograph from the initial collecting tubule 1530 urn from the macula densa. A principal cell (P) positioned between two intercalated cells is shown. Note the difference in ultrastructure of the two I cells. The I cell to the left contains many small vesicles in the apical cytoplasm and its apical cell membrane is narrowed. In contrast, the I cell to the right contains many intermediate size vesicles and a few large apical vesicles. Note the lack of structural association between mitochondria and basolateral membranes in both cell types. Magnification x 5000. FIG. 12. Part of the tubule wall from the point of confluence between two distal tubules. This confluence was 1890 pm from the macula densa of a level 2 tubule and 1726 pm from the macula densa of a level 3 tubule. Two neighboring intercalated cells are seen. In the contact zone, thin plate-like projections extend from each cell. Magnification x 5000. FIG. 13. Part of the tubule wall from the cortical collecting duct, 160 pm after the confluence of two distal tubules. The cells are low and the number of cytoplasmic organelles is small. The intercalated cell show a narrowing of the apical cytoplasm which contains numerous intermediate size vesicles. The arrows points to the tight junctions between the I cell and the neighboring P cells. Magnitication x 5000.

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in the cortex and outer stripe of the outer medulla may be cytochemically different from I cells in the inner stripe of the outer medulla and in the inner medulla (Brown et al., 1985). In addition, recent evidence from an in vitro microperfusion study suggests that conversion of one configuration of I cells to another may occur during acute metabolic acidosis (Satlin and Schwartz, 1988) an observation which is analogous with the decrease in ultrastructural heterogeneity of I cells found in acute metabolic acid/base changes in vivo (Dot-up, 1985b). The present results on the ultrastructure of I cells that were exactly located in the nephron segments were examined in order to detect possible evidence for two or more ultrastructurally distinct types of I cells. Configurations of I cells similar to those described by Verlander et al. (1987) were observed in the present study, in the late DCT, the CNT, the ICT, and the CCD. However, intermediary forms, which exhibited some characteristics of the abovementioned A form and some of the B form, were also observed. Moreover, the same spectrum of manifestations of I cells was observed in all the analyzed segments. On the basis of ultrastructural criteria, the present study therefore does not permit a clear distinction between two different types of I cells in any of the segments analyzed. One explanation for the apparent discrepancy in observations in the present study and in the study of Verlander et al. (1987) may be that different parts of the cortex have been examined. In the present study, all the analyzed tubules were precisely located and identified by 3D recon-

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structions and the I cells in the present study were never located in the medullary rays. In the study of Verlander et al., parts containing medullary rays were preferred for both TEM and SEM. Since collecting ducts typically enter the medullary rays about XXI-1000 p.m after the confluence of two distal tubules, the study of Verlander et al. probably excluded almost all of the superficial tubules analyzed in the present study. Identification of the homogeneous epithelium of the DST and the early DCT is usually very easy on TEM micrographs. This also applies to the collecting duct where P cells and intermingling I cells may be easily identified. However, the segments in between, the late distal convoluted tubule, the connecting tubule, and the initial collecting tubule, may be difficult to distinguish particularly in those nephrons that are not visible from the renal surface or positioned in the medullary rays. The present study demonstrates that segment identification may be made by identification of the cell types present in the segment and that consistent identification of all of the cell types is possible provided that TEM is used. In a recently published standard nomenclature for structures of the kidney (Kriz er al., 1988) the connecting tubule is considered to begin with the first occurrence of a CNT cell. In the present study, the segment identification was based on which cell type (DST, DCT, CNT, and P cells) had the highest volume density, estimated by point counting, in the tubule cross section. The present method for segment identification probably permits a more precise correlation to the function of the seg-

FIG. 14. Electron micrograph of a section through the base of three DCT cells from the late DCT, 1080 urn from the macula densa. The elaborate interdigitating cell processes are closely associated with mitochondria. The arrow indicates the point at which the three cells meet. Magnification x 15 000. FIG. 15. Basal part of the cytoplasm of CNT cells positioned 1450 urn from the macula densa. A cytoplasmic process from an intercalated cell extends between the CNT cells. The cross section of this process has a star-like appearance with plate-shaped projection. These thin plates contain no mitochondria. Magnification x 20 000. FIG. 16. Cytoplasmic process from an intercalated cell under a neighboring principal cell. The electron micrograph is from the point of confluence between two distal tubules. Plate-shaped projections interdigitate with the P cell. Projections, however, are fewer and smaller than those in Fig. 1.5.

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ment, since it is likely that the function is closely related to the cell which predominates in the segment. In addition, a small fraction of, i.e., a CNT cell may be di&uIt to identify, and, therefore, the present method for segmentidentification may be easierto use. In most tubule cross sections it is obvious which cell type occupies the largest area, and the segmentidentification may thus be established without point counting. This method of identification allows for analysis of randomly sampledsections, also without the need for 3D reconstructions, and thereforemay be applied to all areasof the renal cortex, including areas not visible from the renal surfaceand areas between the medullary rays. The present study, for the first time, reports volume densities of the five distal nephron cell types related to the distance along the tubule axis from the macula densa. When these results are combined with those of previous studies on morphometrical parameters on individual cell types, estimates of absolute parameters may be calculated. As an example, the total volume of mitochondria in DCT cells per subcapsulartubule may be calculatedas 1.08 x lo6 pm3 * 0.287 p,m3/pm3 - (1 - 0.284km3/pm3) = 2.17 x 10’ p,m3,

where235x lo3 brn3is the volume of I cells between the macula densaand the conlluence(Table II), 0.44 km2/pm3is the surface density of the luminal surfaceof I cells, and 0.254um3/p,m3is the volume density of the nucleusin I cells (Dotup, 1985a).The correspondingvalue for DCT cells is 376 x lo3 pm2, for CNT cells 56 x lo3 pm2, for P cells 62 x lo3 pm2, and for DST cells 31 X lo3 pm2 (the surfacedensity of the luminal surface on DST cells from Kone et al., 1984).The sum of all five cell types above yields a total inner surface area of the tubule between the macula densa and the contluenceof 602 x lo3 km2. The precautions taken in the previous study (Dorup, 1985a)concerningcomponent biased sampling apply to the above absolutevalues as well as to the surface densities. Another sourceof error stems from the fact that the surface densities used were sampled from the superficial 500 urn of the renal cortex without regardto the segmentand thus represent the mean of all segments,whereas the total volumes of each cell type, in the calculations above, were from subcapsular tubules only. However, qualitative observations in the present study indicate that the mean surfacedensitiesfrom the superficial cortex are representativefor the subcapsulartubules. I thank Professor Arvid B. Maunsbach for encouragement and constructive discussions throughout this study. Helle Bergmann, Inger Kristoffersen, and Poul Boldsen provided skillful technical assistance. This investigation was supported by the Danish Medical Research Council (12-6707 and 12-7184).

where 1.08x lo6 pm3is the total volume of DCT cells in subcapsulartubules(Table II), 0.287km3/p.m3is the volume density of mitochondria in the cytoplasm of DCT cells (excludingthe nucleus),and0.284um3/Fm3 REFERENCES is the volume density of the nucleusin DCT BONVALET, J.-P., PRADELLES, P., AND FARMAN,N. cells (Dorup, 1985a).Similarly, the total lu(1987)Amer. J. Physiol. 253 (Renal, Fluid Electrominal surface of intercalated cells before lyte Physiol. 22), F377-F3g7. the confluence, for subcapsular tubules, BROWN, D., ROTH,J., ANDORCI, L. (1985) Amer. J. Physiol. 248 (Cell Physiol. 17), C348-C356. may be calculatedas BURG,

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