JOURNAL OF ULTRASTRU~~URE
RESEARCH 92, 101-l
18 (1985)
Ultrastructure of Distal Nephron Cells in Rat Renal Cortex JENS D~RUP Department of Cell Biology, Institute of Anatomy,
University of Aarhus, DK-8000 Aarhus C, Denmark
Received October 2, I985 Distal nephron segments in the rat renal cortex contain distal convoluted tubule cells (DCT cells), connecting tubule cells (CNT cells), intercalated cells (I cells), and principal cells (P cells). The present study was carried out to expand present knowledge on the ultrastructure of these cells. The cells were sampled from superficial cortex and analyzed by electron microscopy. Several morphometric parameters were determined and statistical comparison between cell types was performed. Significant structural differences between the cell types were demonstrated. DCT cells showed the highest volume density of mitochondria whereas the amplification of basolateral membranes was higher in CNT cells than in I and P cells. The surface density of the membrane that bounds intermediate vesicles in the apical cytoplasm was twofold higher in I cells than in the other cell types. The morphological differentiation found in the present study adds to available evidence indicating a functional differentiation between the cell types and provides a reference for structurefunction correlations in these cells. o 1985 Academic press. IX.
It is well established that the cortical distal nephron of mammalian kidneys can be divided in the distal convoluted tubule, the connecting tubule and the cortical collecting tubule and that these segments contain four structurally distinct cell types: distal convoluted tubule cells (DCT cells), connecting tubule cells (CNT cells), intercalated cells (I cells) and principal cells (P cells) (Rhodin, 1958;Myersetal., 1966;TisheretaZ., 1968; Morel et al., 1976; Kriz et al., 1978; Crayen and Thoenes, 1978; Tisher, 198 1; Kaissling, 1982). There is also substantial evidence that these cell types have different functions. Thus, sodium reabsorption has long been attributed to DCT cells (Malnic et al., 1963; Hierholzer et al., 1965; Windhager, 1979). In a recent micropuncture study, potassium secretion was not demonstrable in the distal convoluted tubule, which is composed of mainly DCT cells, whereas potassium secretion was observed in addition to sodium reabsorption when the puncture site was in a subsequent segment containing CNT, P, and I cells (Stanton et al., 198 1). Moreover, in vitro perfusion studies have shown that segments lined exclusively by DCT cells are insensitive to mineralocorticoids (Gross et al., 1975) while CNT cells are thought to
be sensitive to these hormones since they respond structurally to desoxycorticosterone acetate (DOCA) by increasing the basolateral membrane area (Kaissling and Le Hir, 1982). P cells, like CNT cells but unlike neighboring I cells, have been shown to respond structurally to potassium adaptation (Stanton et al., 198 1) or prolonged treatment with DOCA (Wade et al., 1979; Stanton et al., 1985) indicating that P cells are active in potassium secretion. I cells have been studied in the medullary collecting tubule where they respond structurally to potassium depletion (Stetson et al., 1980) and respiratory and metabolic acidosis (Madsen and Tisher, 1983, 1984) by increasing the area of luminal plasma membrane, and it has been suggested that these cells play a role in potassium reabsorption and hydrogen ion secretion in the renal medulla. In the cortex, however, I cells have been reported to be structurally unaffected by potassium depletion (Toback et al., 1976; Stanton et al., 198 1). Several recent investigations have illustrated the usefulness of morphometric methods for structure-function correlations in these four cell types (Wade et al., 1979; Stetson et al., 1980; Stanton et al., 198 1; 101 0022-5320/85 $3.00 Copyright 0 1985 by Academic Press, Inc. All rights of reproduction in any form reserved.
102
JENS D(dRUP
Kaissling and Le Hir, 1982; Madsen and Tisher, 1983, 1984; Stanton et al., 1985; Kaissling et al., 1985). The present investigation was designed to provide additional morphometric data allowing comparison of structural parameters in the four cell types in the rat distal convoluted, connecting, and collecting tubules. Particular emphasis was placed on quantitation of areas of luminal and basolateral plasma membranes and of the vesicle compartment in the apical cytoplasm, since these membranes are probably involved in important transport processes. The results extend present information regarding the structural differences between the four cell types and provides a morphometric basis for experimental studies on the cells. MATERIALS
AND METHODS
Animals Ten female Wistar rats weighing 190-230 g were studied. All animals were kept on standard rat pellets and had free access to tap water. Five of the animals which were used for the quantitative studies and which also served as controls for the acid/base experiment reported separately (Dsrup, 198 5), received by gastric tubing 2 ml of tap water at 1. 3. and 5 hr prior to anesthesia. Fixation for Light and Electron Microscopy, The animals were anesthetized by intraperitoneal injection of 0.5 mg/kg body weight of sodium pentobarbital. Fixation of the kidneys was performed within 20 min after start of anesthesia by retrograde perfusion of 2% glutaraldehyde in 0.1 M sodium cacodylate buffer through the abdominal aorta. The kidneys were excised and kept in the same fixative until further processed after 24 hr or more. Superficial cortical tissue was excised in 1 x 1 x 3-mm blocks oriented in the corticomedullary direction and including the renal capsule at one end. Blocks for qualitative analysis of outer medullary tissue were excised from two animals. All blocks were postfixed in 1% osmium tetroxide in Veronal acetate buffer, stained en bloc with 0.5% many1 acetate in maleate buffer at pH 5.2, dehydrated in alcohol, and embedded in Epon 8 12. Survey sections, 1 wrn in thickness, and 300- to 700-A ultrathin sections were cut from the superficial cortex parallel to the kidney surface not more than 500 pm from the surface. The semithin sections were stained with toluidine blue for light microscopy. Ultrathin sections were stained with uranyl acetate and lead citrate and analyzed in a JEOL 1OOB or 1OOCX electron microscope. For details
and references regarding perfusion-fixation, osmium tetroxide fixation, en bloc staining, embedding, and section staining see Maunsbach (1966). Ident&ation Qf Ceil l:vpcs in Distal Convoluted, Connecting, and Collecting Tubules Several structural characteristics allowing the identification of the four cell types studied here have been described (Rhodin, 1958; Myers et al., 1966; Griffith et al., 1968; Tisher et al., 1968; Crayen and Thoenes, 1978; Kaissling and Kriz, 1979; Kaissling, 1982; Stanton et al., 1981; Welling et al., 1981, 1983). In general agreement with these previous studies. the cells in this work were identified as follows. Distal convoluted tubule cells (DCT crlls) exhibit extensive interdigitations of basal and lateral membranes. Mitochondria are closely associated to these membranes. Typically, the nucleus, which is slightly flattened, is positioned near the luminal cell membrane. On the luminal surface there are short and stubby microvilli. Connecting tubule cells iCNT cells) like DCT cells exhibit extensive basolateral interdigitations and infoldings. However, basolateral membranes are not always associated with mitochondria (Crayen and Thoenes, 1978; Kriz, 1978; Kaissling, 1982). The nucleus is usually round and is positioned centrally in the cell. Microvilli on the luminal surface are less frequent and usually more slender than on DCT cells. Intercalated cells (I cells) in the rat cortical nephron are distinguished from the other three cell types by the abundance of vesicles in the apical cytoplasm and the presence of microplicae on the luminal surface. The frequency of microplicae and apical vesicles, however. varies considerably even between I cells in the same tubule cross section (Griffith et al., 1968; Kaissling, 1982). The basal cytoplasm in the 1 cell is characterized by a low palisade-like arrangement of basal infoldings without obvious association to mitochondria. Principal cells lP cells) are characterized by a centrally placed nucleus and few cytoplasmic organelles. Mitochondria are smaller than in CNT and DCT cells and usually not associated with basolateral membrane infoldings. Lateral interdigitations are sparsely developed and the luminal surface show few small microvilh. Segmental distribution of cell types. It is now well established that the beginning of the distal convoluted tubule in the rat nephron is composed exclusively of DCT cells and that the cortical collecting duct is composed of P cells with intermingling I cells (Crayen and Thoenes, 1978; Tisher. 198 1; Kaissling, 1982). The distribution of cell types in the connecting and initial collecting tubule in rat, however, is not yet fully described. In the present study the different cell types were sampled without regard to the segment. In a subsequent study the possible segmental variations between the cell types wilt be considered (Dsrup, in preparation).
ULTRASTRUCTURE
OF RAT DISTAL
Morphometry One tissue block was analyzed from each of the 5 animals. Each section was systematically analyzed starting at the upper left comer. In each tubule cross section the examination started at the upper left comer and proceeded clockwise around the tubule wall. Only cells which were sectioned approximately perpendicular to the basement membrane and which contained a centrally sectioned nucleus were included in the analysis. This systematical sampling method introduces a bias on morphometric parameters on structures, that are not homogeneously distributed in the cells, such as the nucleus (component biased sampling, see Weibel, 1979). However, this sampling method was necessary to allow positive identification of cell types prior to the morphometric analysis (see also Discussion). The first cell of each type that Nfilled the criteria was chosen and only one cell of each type was recorded per tubule cross section. Micrographs containing whole cell profiles were recorded at a primary magnification of x 5000 and analyzed at a final magnification of x 15 000 for determination of the volume density of the nucleus, the volume density of mitochondria, and the surface density of luminal and basolateral cell membranes. For evaluation of the vesicle compartment in the apical cytoplasm micrographs were recorded at a primary magnification of x 16 000 and analyzed at x 48 000. These micrographs were taken from the same cells that were recorded at the lower magnification. Exact magnifications were calculated with the aid of a calibration replica with 2 160 lines per millimeter. From each animal three DCT cells, three CNT cells, three P cells, and three I cells were recorded in the electron microscope irrespective of the segment. All morphometric analyses were made using the curvilinear test system of Merz (1967) both for intersection counting and point counting. The diameter of the semicircles on the test lattice was 1 cm, corresponding to 0.67 pm at the lower and 0.21 Frn at the higher magnification. Points with distances of 1 cm were used for volume density of mitochondria whereas coarse points spaced 2 cm were used for the nucleus and the cytoplasm. Unless otherwise stated the morphometric formulas specified in the following were derived from Weibel(1979). The volume density of the nucleus was calculated at V, = P,J(P, + PA where P,, is the number of coarse points on the nucleus and PC is the number of coarse points on the cytoplasm (excluding the nucleus). The cell area was estimated from A = D2(P, + PJ where D is the distance between coarse points which corresponded to 1.34 pm at the actual magnification. The volume density of mitochondria in the cytoplasm was calculated as V, = P,,,/P, .4 where P, is the number of fine points falling on mitochondria. The surface density of luminal plasma membranes was calculated as the sum of the surface density of microvilli (DCT, CNT, and P cells) or microplicae (I
NEPHRON
103
cells), S, = K,.2.ZIL,, and the surface density of lumitral plasma membrane between microvilli or plicae, Sv = r/2.NL,, where K, is the correction factor for section thickness effect (see below), Z is the number of intersections between cell surface and testlines, and L, is the total testline length over the cytoplasm. For the plasma membrane between microvilli or microplicae the above formula takes into account that this membrane is oriented approximately perpendicular to the section plane on sections perpendicular to the basement membrane. The surface density of basolateral membranes was calculated using the formula Sv = 2’ I/L,. The boundary length of luminal and basolateral cell surfaces was calculated as B(A) = 1.d where I is the number of intersections between cell surface and testlines and d is the diameter of the semicircles which corresponded to 0.67 pm. The amplication factor for luminal membranes was calculated as the surface density of the luminal membrane divided by the surface density of an imaginary surface following the main course of the luminal cell surface and indicated on the micrographs as a line drawn through the base of microvilli or microplicae. The surface density of this imaginary surface was calculated as Sv = lr/2. I/L, since it was perpendicular to the section plane. The amplification factor of the basolateral membranes was obtained by dividing the surface density of basolateral membranes by the surface density of a plane parallel to the basement membrane. The latter surface density was obtained from the formula Sv = r/2. I/L,. The vesicle compartment in the apical cytoplasm was analyzed at a final magnification of x 48 000. All micrographs were oriented with the luminal membrane parallel to the image frame by rotation of the grid in the electron microscope. The apical pole of the nucleus was placed in the center of the image frame. The luminal plasma membrane was always included. The area to be analyzed was arbitrarily limited by a line drawn parallel to the luminal plasma membrane and tangential to the nucleus and by the width of the image frame which corresponded to 5.8 pm at the magnitication used. Profiles of the following four different populations of vesicles could be distinguished in the apical cytoplasm. (1) Intermediate vesicles between 80 and 200 nm in diameter. (2) Small vesicles averaging about 50 nm in diameter, and always below 80 nm. (3) Large vesicles above 200 nm in diameter. (4) Tubular structures with maximum diameter larger than 1.5 times the minimum diameter. The number of profiles per square micrometer of apical cytoplasm on the micrographs (numerical density) was calculated as NA = N.d2/4.P, where N is the number of profiles, PC is the number of coarse points on the apical cytoplasm, and d is the distance between
104
JENS DBRUP
fine points on the grid which corresponded to 0.22 pm on the section. This was done for all four types of profiles in I cells but only for intermediate profiles in DCT, CNT, and P cells. Since circular profiles on the micrographs may represent cross sections of tubular membrane structures, a clear distinction between tubular and spherical structures was not possible for each individual profile. However, since tubular profiles represent only 4% of all profiles in I cells (see Results) it is a reasonable approximation to estimate the surface density as if circular profiles represent spherical vesicles. The surface density of intermediate vesicles in the apical cytoplasm was therefore determined as S, = K, 2.I/L, and as Sy = 2. I/L, for the large vesicles in 1 cells. The correction factor for the section thickness effect, K,, was computed as specified below. The expected relative standard error estimates (RSEJ (Cochran, 1953) of volume densities of mitochondria was calculated as RSE = \/ (1 -- V,)lP,. V,) where I’, is the volume density of mitochondria and PT is the total number of test points per animal. This error was below 10%. The relative standard error of the surface density measures was calculated by the formula RSE = 2/m (Hilliard, 1976) where S, is the surface density and LT is the total testline length over the micrographs. This error was below 10% for basolateral membranes and for intermediate apical vesicles whereas for luminal membranes and for large apical vesicles il ranged from 15 to 25%. Correction factors for sectlon thickness ejtict. Since dimensions of the smallest structural elements analyzed (intermediate vesicles and microvilli and microplicae) were close to the thickness of the ultrathin sections, the effect of section thickness on their surface density estimates could not be neglected (Holmes, 1927). The section thickness was determined for all sections used for morphometry. On most sections, small section folds were recorded and the section thickness measured according to Small (1968). These micrographs were recorded at a magnification of x 20 000 and repro-. duced at x 60 000. On a few sections, no section folds were found and the section thickness was then approximated by comparison with adjacent, measured sections. The section thickness ranged from 300 to 700 A with an average of 495 A. Correction factors for section thickness effect depend on the shape and size of the structural elements analyzed (Weibel and Paumgartner, 1978). Ve.sicle dzameters were measured on micrographs of the apical cytoplasm with a final magnification of x 48 000 by placing a transparent overlay with a series oftest circles of known diameters over the micrographs (Weibel. 1979). The spacing between the test circles corresponded to 10 nm. Only profiles ofintermediate vesicles with a maximum diameter smaller than 1.5 times the minimum diameter were measured. To exclude peripherically cut vesicles only profiles limited by a sharply
defined membrane at the entire circumference were measured. This procedure also reduced interference from tubular structures since most tubular structures cut obliquely exhibit fuzzy membranes at two opposite places of their circumference. All intermediate circular profiles from the cells meluded in the morphometric analysis at the magnification of x 48 000 were measured provided they fulfilled the above criteria. For calculation of correction factors the average diameter was calculated in each animal for each of the four cell types. Since the section thickness was close to the dimensions of intermediate vesicles, the estimate of the surface density of intermediate vesicles was affected by the fact that very small caps (truncated vesicles) could not be detected on the micrographs. Therefore the following formula taking into account the effect of truncation was applied KS&) =
2[pm
TT + sin I-
+ 2gi
where p = ddd, do is the diameter of the smallest visible cap which was approximately 40 nm, d = the diameter of the vesicles, g is the relative section thickness = section thickness/vesicle diameter (Weibel and Paumgartner, 1978). The height and minimum diameter of all sharply delineated microvilli and the thickness of microplicae were measured on the same micrographs as those used for the morphometric analysis (final magnification x 48 000). The three-dimensional organization of microvilli and microplicae is difficult to deduce from observations of transmission electron microscope micrographs. However, the fact that on I cells profiles of microplicae were elongated and usually continuous with the luminal cell membrane in the plane of the section which was not always the case for microvilli on DCT, CNT, and P cells, indicates that the surface specializations on I cells are actually plicae. Furthermore, scanning electron microscope observations of rat distal nephron cells (Evan et al.. 1980; Hagege et al., 1974) show that luminal membrane specializations on I ceils are leaf-like plicae with a length of about 1 pm whereas they are roughly cylindrical on DCT, CNT. and P cells. In calculations of correction factors for surface density estimates the plicae were equated with disks with a mean diameter of (length i height)/2 where the height was measured on the micrographs (Table I) and the length estimated from observations by scanning electron microscopy (Evan e! a/., 1980; Hagege et al., 1974). It should be noted that the more critical dimension for the purpose of calculation of correction factors is the thickness of the disks (Weibel, 1979). The following formula was used for the calculation of correction factors for microplicae 6(6/2 t 1) K&s,) = 6(6/2 + 1) + g(6 + 2hj
ULTRASTRUCTURE
OF RAT DISTAL
NEPHRON
105
325
275 250. II 2 :
225. zoo
‘; a
175
2 2
150
al 2
125.
2
100 DCT cells 75 CNT cells
25 0 Apical Membranes
Apical Membranes
Basolataral
Basolateral
Membranes
Membranes
Yitochondria
Apical Vesicles
FIG. 1. Morphometric parameters in distal convoluted tubule cells (DCT cells), connecting tubule cells (CNT cells), intercalated cells (I cells) and principal cells (P cells). S, indicates surface densities, V, volume densities and B boundary lengths. Measures on apical vesicles includes only intermediate vesicles. All values are relative to values in DCT cells and represent averages from five animals. Bars indicate SEM.
where 6 is the shape coefficient = diameter/thickness of disks, and g is the relative section thickness, section thickness/diameter of tubules (Cahn and Nutting, 1959). For microvilli the following formula, assuming a tubular shape, was used
US”) = x+g(1
x +2x/7r)
where h is the shape coefficient = length/diameter of microvilli. Both formulas depend on random orientation of the structural elements relative to the section plane. Although both microvilli and microplicae often bend in a variety of directions, the fact that they always protrude from the cell surface implies that this condition is not fulfilled on sections cut perpendicular to the basement membrane. True random sectioning of the cells, which would be desirable from a stereological point of view, was not possible in the present study because of the need to identify the cell types. However, as pointed out by Weibcl ( 1979) “it is often preferable to use correction factors based on an imperfect model, rather than to be misled by accepting uncorrected data at their face value.” Correction factors were computed separately for each animal and cell type. They ranged from 0.52 to 0.72 for intermediate apical vesicles, from
0.54 to 0.79 for microvilli on DCT, CNT, and P cells and from 0.85 to 0.9 1 for microplicae on I cells. Statistical evaluation. Morphometric parameters were computed and statistically evaluated with the BMDP statistical software package (Dixon, 1983) using a CDC Cyber 825 computer. Differences between cell types were tested by a two-way analysis of variance (ANOVA). When the ANOVA reached significance (P i 0.05) comparisons between cell types were performed using paired t tests with adjustments made for multiple comparisons (Bonferroni test, see Miller, 196 1). Variances on morphometric parameters were compared using an Ftest. Pvalues below 0.05 were considered statistically significant. Data are expressed as means + SEM. RESULTS
The quantitative results reported below in Table I and Fig. 1 were based on the 5 animals which received three doses of 2 ml tap water prior to the analyses (see Materials and Methods). The qualitative observations originated from all 10 animals. There were no ultrastructural differences between cells in the two groups.
0.24 k 0.03 22.8 2 1.8 1.7 +- 0.1 3.54 i 0.46 244 i 35 20.1 +- 3.0 4.71 xic 1.02 1.30 k 0.22 244 + 24 98 2 4 134 +- 8
k 0.07 k 0.9 -+ 0.2 + 0.74 + 14 k 1.7 t 1.04 + 0.24 I 49 k 3 i 2
399 -I 41 76 + 6 121 If- 4
9.31 + 0.57 2.67 2 0.20
1.94 2 0.03 83 -+ 7 9.5 * 0.7
0.44 k 0.08 22.8 I 4.9 2.9 + 0.3
73.4 + 5.8 25.4 k 1.5 25.7 k 0.5
I cells
207 2 24 97 + 3 120 ” 6
4.42 k 0.38 1.38 +- 0.21
2.74 2 0.31 119 + 26 10.4 It 2.0
0.32 t 0.03 18.5 t 2.0 1.4 k 0.1
70.0 + 11.5 21.4 ?z 3.0 16.7 -?r 2.7
P cells
CELLS),
I > CNT, P: P < 0.05; DCT > p: P < 0.01 DCT > P > I: P < 0.05; DCT > I: P < 0.01 N.S.
I > CNT, DCT, P: P < 0.05 I > CNT, DCT, p: P i 0.05
N.S. CNT > I: P < 0.05 CNT > I: P < 0.05; CNT > P: P <: 0.05
N.S. N.S. DCT > CNT: P c: 0.05
N.S. N.S. DCT > CNT: P < 0.01; DC-l- 1 I: P < 0.05
Statistical significance
(CNT CELLS),INTERCALATEDCELLS(I RATS?
(P < 0.05) cell types were compared with paired t tests with adjustments made for multiple comparisons (Bonferroni
a Values are means f SEM of five animals. Statistical evaluation was performed by two way analysis of variance (ANOVA). When the ANOVA reached significance test). All P values in the table are from the Bonferroni test. h For details on the method used for these measurements, see Materials and Methods.
108.9 t 8.6 19.0 i 1.5 26.0 i 0.3
CNT cells
TABLE I (DCT CELLS),CONNECTINGTUBULECELLS (P CELLS)IN RENAL CORTEX FROM CONTROL
* 9.9 f 3.8 k 0.1
DCI cells
AND PRINCIPALCELLS
PARAMmERs OFDISTALCONVOLUTEDTUBULECELLS
81.2 Cell area, pm’ Volume density of nucleus, IO-* pm3/pm3 28.4 Volume density of mitochondria, 10m2gm3/pm3 28.7 Luminal membrane 0.47 Surface density, pm2/prn3 Boundary length, pm 27.2 AmpliEcation factor 2.8 Basolateral membrane 3.14 SurEtce density, pmz/rm 127 Boundary length, pm 16.6 Amplification factor Intermediate vesicles in apical cytoplasm 3.38 ProEles, number/~m2 Surface density, pm2/pm3 0.91 Dimensions used for calculation of correction factorsb Height of microplicae/microvilli, nm 395 Thickness of microplicae/microvilli, nm 123 Diameter of intermediate vesicles, nm 117
----l__llll--
MORPHOMETRK
ULTRASTRUCTURE
OF RAT DISTAL
NEPHRON
107
Ultrastructureof Distal ConvolutedTubule Cells (DCT Cells)
Ultrastructure of Connecting Tubule Cells (CNT Cells)
In distal convoluted tubule cells, the nucleus was usually flattened and positioned near the luminal cell membrane (Fig. 2). Microvilli on the luminal surface were more frequent than on CNT and P cells (Fig. 3) and they were significantly thicker than on I and P cells (Table I). The amplification factor for the luminal membrane, which is a measure of the degree of convolution of the membrane, was significantly larger in DCT cells than in CNT cells, 2.8 + 0.2 versus 1.7 + 0.1 (P < 0.05) whereas the boundary length of the luminal membrane was not significantly different in DCT and CNT cells. Infoldings and interdigitations of the basolateral membrane were well developed in DCT cells (Fig. 4). In the basal part of the cells they were closely associated with rodshaped mitochondria. The volume density of mitochondria was significantly larger than in CNT and I cells (Table I). Four different types of vesicles could be distinguished in the cytoplasm of both DCT, CNT, I, and P cells (Table II). Intermediate vesicles,80-200 nm in diameter were predominant in the apical cytoplasm of all four cell types. Their mean diameter in DCT cells was 177 +- 2 nm. Usually no cytoplasmic coat was found on the membrane of these vesicles but a few intermediate vesicles were coated or partially coated. B,oth coated and uncoated vesicle membranes of intermediate vesicles were similar in thickness and structure to the plasma membrane. Occasionally small vesicleswith a mean diameter of 50 nm were continuous with intermediate vesicles. These small vesicles were usually coated. Large vesicles(>200 nm) and tubularprofileswere infrequent in DCT cells. Unlike in CNT cells apical vesicles were often located in the small microvilli on the luminal surface. Rough endoplasmatic reticulum was present both in the apical and basal cytoplasm but like in I cells, CNT cells, and I? cells it was not prominent.
Ultrastructural characteristics of CNT cells varied considerably even from cell to cell in the same tubule cross section. Thus, some CNT cells showed resemblance to DCT cells while neighboring cells resembled P cells to some extent. In particular, the arrangement of basolateral membranes and their degree of association to mitochondria varied considerably between CNT cells. The nucleus was oval to round and usually positioned in the center of the cells (Fig. 5). Microvilli on the luminal surface were slender and infrequent (Fig. 6). Lateral cell processes showed extensive interdigitations with neighboring CNT, DCT, or P cells, whereas lateral processes only interdigitated to a limited extent with I cells. Basolateral membrane interdigitations and infoldings were generally well developed in CNT cells and often there were two or more membrane infoldings between two mitochondria at the base of the cells (Fig. 7). The amplification factor for basolateral membranes was higher in CNT cells than in I and P cells (Table I). The surface density of basolateral membranes, however, was not significantly different from DCT, I, and P cells. The volume density of mitochondria was significantly lower than in DCT cells. Golgi complexes were often located at the lateral aspects of the nucleus. The frequency of intermediate vesicles was lower than in I cells but similar to the frequencies in DCT and P cells. Small coated vesicles, large vesicles and tubular structures were very infrequent.
Ultrastructureof Intercalated Cells (I Cells) I cells like CNT cells varied considerably in ultrastructure. Some cells showed a dark staining cytoplasmic ground substance whereas others with typical characteristics of I cells showed a light staining cytoplasm. The nucleus was usually positioned in the basal part of the cells (Fig. 8). Some I cells
108
JENS DQRUP
showed a large cell surface in contact with the tubule lumen whereas others exhibited a narrowing of the apical cytoplasm and a small contact area toward the tubule lumen. The mean value for boundary length of luminal membrane was not significantly different from the other three cell types. The variance in boundary length, however, was significantly larger in I cells than in both DCT and CNT cells (120 pm2 in I cells versus 4 Km2 in DCT cells and 16 pm2 in CNT cells, P < 0.05 for both Ftests). There were only few protrusions from the lateral surfaces of I cells and interdigitations with neighboring cells were infrequent (Fig. 9). The basal surface showed true infoldings of the plasma membrane often with a palisadelike arrangement in basal microvilli or plates and without structural association with mitochondria. Mitochondria in I cells were shorter than in DCT and CNT cells and they were evenly distributed in the cytoplasm. The volume density of mitochondria was lower than in DCT cells but it was not significantly different from the values in CNT and P cells (Table I). Golgi complexes were prominent in the cytoplasm lateral to the nucleus. As in the other three cell types, the apical cytoplasm of I cells contained four different types of vesicles (Fig. 10, Table II). Serial sectioning demonstrated that the vast majority of profiles represent spherical vesicles (Fig. 11). Vesicles near the luminal cell surface were often continuous with the lumen whereas vesicles positioned deeper in the cytoplasm were shown to be closed structures. Some intermediate vesicles were continuous with small coated vesicles. The es-
timated surface density of the membrane that bounded intermediate vesicles in the supranuclear cytoplasm as defined under Materials and Methods was 2.67 -t 0.20 pm2/pm3 and there was an average of 9.3 1 +0.57 profiles/pm2 of sectioned apical cytoplasm. The other three types of apical vesicles were relatively infrequent. Large vesicles, with diameters above 200 nm showed 0.24 -+ 0.05 profiles/pm2 and had an estimated surface density of 0.30 + 0.05 pm2i pm3. Small vesicles with diameters below 80 nm and tubular structures showed 0.2 1 + 0.03 profiles/pm2 and 0.39 -t 0.11 profiles/ Fm2, respectively. Large vesicles were usually found in the apical cytoplasm near the nucleus (Fig. 10). I cells from the outer medullary collecting tubule (Fig. 12) showed the same four types of vesicles as cortical I cells and the frequency of each vesicle type appeared similar in cortical and medullary I cells. Ultrastructure
of Principal Cells (P Ceils)
Principal cells showed a more simple cell shape and fewer cytoplasmic organelles than the other three cell types (Fig. 13). The nucleus was oval to round and positioned in the center of the cells. Microvilli on the apical surface were short and few (Fig. 14). The arrangement of basal and lateral surfaces was similar to the arrangement in I cells. Thus, there were few laterally interdigitating processes and the basal infoldings showed a palisade-like arrangement without association to mitochondria (Fig. 15). The amplification factor of basolateral membranes was significantly lower than in CNT cells (Table I). Mitochondria were few and rod-
FIG. 2. Electron micrograph of distal convoluted tubule cell (DCT cell) and part of intercalated cell (I). The DCT cell shows extensive basolateral interdigitations with associated mitochondria. The cytoplasm of the I cell stains darker than that of the DCT cell. Lateral interdigitations between the DCT cell and the I cell are few. x 10000. FIG. 3. Apical cytoplasm from DCT cell. Intermediate vesicles are located both in the apical cytoplasm and within the stubby microvilli. x 40 000. FIG. 4. Basal cytoplasm from DCT cell. The structural association between mitochondria and basolateral membranes is very close. x 40 000.
ULTRASTRUCTURE
OF RAT
DISTAL
NEPHRON
109
110
JENS D0RUP TABLE I1
SEMIQUANTITATIVE CELLS
(DCT
Vesicle type Small Intermediate hse Tubular
CELLS),
ANALYSIS
OF VESICLE
CONNEC-TING
TUBULE
Diameter
TYPES IN THE APICAL
Coat
200 nm -*
CYTOPLASM
OF DISTAL
CELLS (CNT CELLS), INTERCALATED CELLs (P CELLS)
DCT cells
++/-
t/- ~
DISCUSSION
The present study details structural differences between the four cell types in the rat distal convoluted, connecting, and cortical collecting tubules and provides a morphometric characterization of each cell type including statistical comparison of several structural parameters in the cells. The study for the first time reports surface densities of the membrane that bounds apical vesicles. The observation that many morphometric -
CELLS),
AND
TUBULE PRINCIPAL
---
I cells
P cells
t
t
+
t++
+ ++
+ -t
+- t i i-
Note. +: <2 profiles/pm2; f+: 2-5 profiles/pm*; + t +: >5 prohleslpm? uncoated, +/ - - : usually uncoated, occasionally coated or partially coated. * Maximum diameter larger than 1.5 times minimum diameter.
shaped. Golgi complexes and lysosome-like bodies were not prominent. The apical cytoplasm in P cells, like in DCT cells and CNT cells showed few intermediate vesicles and small vesicles, tubular profiles and large vesicles were infrequent.
CONVOLUTED
(I
CNT cells
ii
+/-+,- ~..
CELLS
+ + ++/-:
-t +
usually coated, rarely
parameters differ significantly between the cell types supports previous qualitative investigations describing the existence and characteristics of the four cell types and expand available evidence indicating a different fimctional significance of each cell type. The thickness of microvilli and microplicae on the luminal surface and the diameter of vesicles in the apical cytoplasm of the cells was close to the thickness of the ultrathin sections which introduces a systematical overestimation of the uncorrected morphometric data (Holmes, 1927). The present data were corrected for this error, thus reducing the estimates of surface density of the membrane that bounds intermediate vesicles by a factor of 0.52472, depending on the section thickness, which _
FIG. 5. Connecting tubule cell (CNT cell) and part of an intercalated cell (I). The nucleus in the CNT cell is more round and the luminal membrane is more smooth than in DCT cells, compare with Fig. 2. x 10 000. FIG. 6. Apical cytoplasm from CNT cell. Apical microvilli are few and slender. Intermediate vesicles (arrows) are occasionally continuous with small coated vesicles (arrowheads). x 40 000. FIG. 7. Basal cytoplasm from a CNT cell. The structural association between mitochondria and basolateral membranes is less close than in DCT cells. x 40 000. FIG. 8. Intercalated cell (I cell). The nucleus is positioned in the basal cytoplasm. The apical cytoplasm contains numerous intermediate vesicles and a few large vesicles positioned close to the apical pole of the nucleus. The mitochondria are evenly distributed in the cytoplasm. x 10 000. FIG. 9. Lateral interdigitations between I cell (I) and P cell (P). Interdigitating processes are short and the intercellular space is somewhat distended. x 18 000. FIG. 10. Apical cytoplasm from I cell. Intermediate vesicles (80-200 nm in diameter) are abundant (arrows). Arrowhead points to a small coated vesicle. x 40 000. FIG. 11. Series of six consecutive sections, each about 50 nm thick, from the apical cytoplasm of an I cell. Numbers 1 to 5 indicate intermediate vesicles which can be traced through the serial sections. Number 6 indicates an invagination from the luminal surface. Most intermediate vesicles are spheres. x 32 000. FIG. 12. Apical cytoplasm from I cell from the outer medulla. The structure of intermediate vesicles is similar in medulla and in cortex (compare with Fig. 10). x 40 000.
ULTRASTRUCTURE
OF RAT DISTAL
NEPHRON
111
112
JENS
DORUP
ULTRASTRUCTURE
OF RAT
DISTAL
NEPHRON
113
114
JENS D0RUP
FIG. 13. Principal cell (P cell) and part of neighboring I cell (I). The P cell shows few organelles and few basolaterai membrane infoldings and interdigitations. x IO 000. FIG. 14. Apical cytoplasm from a P cell. The luminal surface is smooth and the appearance of apical vesicles is similar to that in DCT cells and CNT cells. x 40 000. FIG. 15. Basal cytoplasm in a P cell. There is no structural association between mitochondria and plasma membrane. x 40000.
ULTRASTRUCTURE
OF RAT DISTAL
was estimated for each section (Weibel and Paumgartner, 1978). The corresponding correction factors for microvilli on DCT, CNT, and P cells ranged from 0.54 to 0.79 and for microplicae on I cells they ranged from 0.86 to 0.91. The corrections imply that numerical values in the present study are lower than corresponding uncorrected morphometric results which should be considered before comparing the present results with uncorrected data. True random sectioning which is desirable from a purely stereological point of view was not possible because ofthe need to identify each cell type positively prior to the morphometric examination. Therefore, the present data, which were obtained from sections oriented approximately perpendicular to the basement membrane and passing close to the center of the cells, overestimates the central part of the cells relative to the cell periphery (component biased sampling, see Weibel, 1979). This sampling bias can be expected to especially affect the estimates of the volume density of the nucleus (nuclear bias) resulting in overestimation of this parameter. Similar component biased sampling has been applied in most previous studies on these cells because of the needs to identify the cell types (Stanton et al., 198 1; Kaissling and Le Hir, 1982; Madsen and Tisher, 1983, 1984). Volume densities of mitochondria and surface densities of basolateral membranes may have been slightly underestimated by the present morphometric method since both mitochondria and basolateral membranes are slightly less concentrated in the cytoplasm below the nucleus as compared to the cell periphery. However, qualitative evaluation of the micrographs indicated that this bias can be expected to be of minor importance for mitochondria and basolateral membranes since the inhomogeneity of the distribution in the cells was small. Moreover, in all four cell types, apical vesicles and microvilli/plicae were evenly distributed in central and peripheral parts of the apical surface and cy-
NEPHRON
115
toplasm and quantitative data on these structures are therefore considered to be representative for the apical region of the cell. Volume densities of mitochondria and surface densities of luminal and basolateral membranes were estimated with the cytoplasm, excluding the nucleus, as reference volume. This was done because the area of the nucleus varied considerably from micrograph to micrograph which would introduce a large variation in the densities had the whole-cell profile been used as reference volume. To compare the present results with previous results where cell profiles were used as a reference volume the present data should be decreased by multiplication with 1 - V,(Nu), where I/,(Nu) is the volume density of the nucleus (see Table I). Morphometric values on luminal and basolateral membranes in cortical I cells and P cells have been reported previously (Wade et al., 1979; Stanton et al., 1981; Zalups et al., 1985; Stanton et al., 1985). Corresponding results in CNT cells have only been reported in one previous investigation (Stanton et al., 1981). In DCT cells surface densities of luminal and basolateral membranes were reported by Larsson (1982) Stanton et al. (198 l), Zalups et al. (1985) and &&sling et al. (1985). The results of the present study, taking into consideration the differences in morphometric methods applied (see above), are in good general agreement with these previously published results. The appearance of the apical cell pole in I cells may be highly variable. An I cell may show a narrow apical cell pole whereas another I cell, even in the same tubule cross section, may show a large area in contact with the tubule lumen. Also the content of vesicles in the apical cytoplasm and the density of the cytoplasmic ground substance may vary considerably. It has therefore been suggested, on the basis of TEM observations, that different types of I cells may exist in the cortex (Crayen and Thoenes, 1978;
116
JENS
Kriz et al., 1978; Kaissling, 1982; Vetlander et al., 1985). Stetson et al. (1980) suggested on the basis of studies of freeze-fracture replicas that medullury I cells can be divided into two populations; one with and one without high-density rod-shaped particles in the luminal membrane. The present study demonstrates a very large variation in the ultrastructure of cortical I cells. However, the density of the cytoplasmic ground substance varied continuously and the values for the boundary length of the luminal membrane and the number of apical vesicles did not fall in two or more distinct groups. Thus, there is no evidence in the present results for the existence of two or more types of cortical I cells. The function of apical vesicles in distal nephron cells is still not finally established although some evidence has been presented. Apical vesicles are particularly prominent in I cells and it has been suggested that in medullary I cells they may be involved in transport of membrane from the cytoplasm to the luminal cell surface in potassium depleted animals (Toback et al., 1976; Stetson et al., 1980) and in acidotic animals (Madsen and Tisher, 1983, 1984). In DCT cells in the rat renal cortex it has been observed that cationized ferritin injected into the tubule lumen is taken up in apical vesicles (Christensen et al., 1981; Madsen et al., 1982). Furthermore, cortical I cells take up peanut lectins bound to the luminal surface and to some extent transport the lectins into apical vesicles (Le Hir et a/., 1982) indicating that these vesicles are of endocytotic origin. Serial sectioning of the apical cytoplasm in cortical I cells in the present investigation (Fig. 11) revealed that intermediate vesicles, which are usually uncoated, are sometimes continuous with the luminal surface, an observation which is also consistent with an endocytotic/exocytotic function. Deeper in the apical cytoplasm some intermediate vesicles were continuous with small coated vesicles similar in structure to coated Golgi vesicles and some were continuous with large vesicles.
D0RUP
it is noteworthy that invaginated vesicles found by Madsen and Tisher (1983, 1984) in medullary I cells were virtually absent in cortical I cells and that tubular profiles were less frequent (4% of all profiles) in cortical than in medullary I cells (16-l 9% of all profiles, Madsen and Tisher, 1983). It is presently not possible to determine to what extent differences between apical vesicles in cortical and medullary I cells are related to functional differences and to what extent differences in preparation of the tissue are involved. However, the present results. describing four different populations of apical vesicles in cortical I cells are generally consistent with the qualitative description of Griffith et al. (1968) who showed that most vesicles in cortical I cells are round and that some, but not all, are coated. Moreover, when outer medullary tissue from the same tissue blocks, also used for the analysis of cortical tissue in the present study, was analyzed in the electron microscope a similar structure of apical vesicles in I cells was found (Fig. 12). The basal cytoplasm in DCT and CNT cells is mainly occupied by long mitochondria associated with infoldings and/or interdigitations of basolateral membranes. In DCT cells, cytochemical studies have shown that basolateral membranes contain the enzyme Na,K-ATPase (Ernst, 1975). The close association between mitochondria and basolateral membranes in transporting epithelia is generally considered to be associated with ATP-consuming reabsorption of sodium. The volume density of mitochondria was lower in CNT cells than in DCT cells and the structural association of basolateral membranes and mitochondria was closer in DCT cells than in CNT cells. These structural differences between DCT cells and CNT cells, in particular the difference in association between mitochondria and basolateral membranes suggest that DCT cells may have a larger structural basis for membrane associated transport processes, such as sodium reabsorption, than CNT cells. In I cells, the content of mitochondria was
ULTRASTRUCTURE
OF RAT DISTAL
lower than in DCT and CNT cells and mitochondria were distributed evenly in the basal and apical part of the cytoplasm. This distribution of mitochondria in I cells may indicate that energy consuming processes take place in the apical cytoplasm and possibly at or close to the luminal plasma membrane. I thank Professor Arvid B. Maunsbach for his helpful advice and encouragement throughout this study. Hanne Weiling, Inger Nielsen, Helle Bergmann, and Poul Boldsen provided skillful technical assistance. This investigation was supported by the Danish Medical Research Council, Carl og Ellen Hertz’s legat, and the P. Carl Petersen Fond.
117
B., AND PSCZOLLA, M. (1978) in H. G., AND ULLRICH, K. J. (Eds.), New Aspects of Renal Function, pp. 67-78, Excerpta Medica, Amsterdam. LARSSON, L. (1982) in SPITZER, A. (Ed.), Developmental Renal Physiology, pp. 15-23, Masson, New York. LE HIR, M., KAISSLING, B., KOEPPEN,B. M., AND WADE, J. B. (1982) Amer. J. Physiol. 242 (Cell Physiol. ll), Cl 17-Cl20. MADSEN, K. M., HARRIS, R. H., AND TISHER, C. C. (1982) Kidney Znt. 21, 354-361. MADSEN, K. M., AND TISHER, C. C. (1983) Amer. J. Physiol. 245 (Renal Fluid Electrolyte Physiol. 14), F670-F679. MADSEN, K. M., AND TISHER, C. C. (1984) Lab. Invest. KRIZ,
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