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Scanning electron microscopy of basolateral surfaces of rat renal tubules isolated by sequential digestion.

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THE ANATOMICAL RECORD 213:121-130 (1985)
Scanning Electron Microscopy of Basolateral
Surfaces of Rat Renal Tubules Isolated by
Sequential Digestion
DAVID B. JONES
Department of Pathology, State University of New York Upstate Medical Center,
Syracuse, NY 13210
ABSTRACT
Renal tubular cells and segments isolated by a trypsin, pepsin,
pronase E digestion procedure were studied with scanning electron microscopy. The
basal and lateral surfaces of S1, S2, S3 proximal tubular (PT) segments, descending
and ascending thin limbs of Henle (TL), distal ascending thick limb of Henle, or
distal straight tubule (DST) and distal convoluted tubule (DCT) segments, connecting tubules (CNT), and collecting ducts (CD) were identified and characterized. The
basal processes of the S1 and S2 PT cells were fan shaped, were oriented in a
circumferential direction, and terminated in microvilli at the basement membrane.
S3 PT cells had microvillous basal processes mainly on the lateral edges of the cells.
The basal processes of DST and DCT were similar to PT in orientation but terminated on the basement membrane with flattened, thin attachments. The long-loop
descending TL and the ascending TL exhibited distinctive interdigitating cell processes. TL segments with simple contours were present in smaller numbers and
were characteristic of short-loop descending limbs. CNT showed some cells with
basal surfaces resembling DCT cells and others resembling CD cells. Both cortical
and medullary CD segments exhibited intercalated cells with round basal contours
and a sparse pattern of basal infolding clefts. The cortical CD principal cells revealed
a much more elaborate mosaic of plicae, clefts, and microvilli than those of the
medullary CD. These observations extend the previous knowledge gained from
transmission electron microscopy and assist in the interpretation of that knowledge.
The complex structure of the basolateral surfaces of
renal tubules has been recognized since the early days
of transmission electron microscopy (TEM) in the rabbit,
rat, dog, human, and other species by such investigators
as Rhodin (19631, Bulger (1965), Maunsbach (1966),
Tisher et al. (19661, Osvaldo-Decima (1973), Kaissling
and Kriz (19791, and Bulger et al. (1979) among others.
Attempts to conceptualize three-dimensional models of
tubular cells from TEM images were carried out by
Bulger (1965), Waugh et al. (19671, Maunsbach (19731,
Welling and Welling (1976), and Welling et al. (1978).
Visualization of the complex basal surfaces of renal tubular cells by scanning electron microscopy (SEMI unencumbered by basement membrane was first carried out
on the rabbit proximal tubules by Evan et al. (1976,
1978)using a n HC1 and collagenase digestion procedure.
Subsequently the connecting and collecting tubules of
the rabbit (Welling et al., 1981, 19831, and some tubules
of the dog, rat, and frog were studied using this method
(Hay and Evan, 1979; Evan, 1981). Jones (1982),using a
trypsin digestion procedure on heavily fixed canine kidney, was able to isolate renal tubular segments free of
basement membrane from all parts of the nephron for
SEM studies. Jones (1983) had poor success with the
trypsin and the HC1-collagenase procedures using human and rat renal tissues; therefore he developed a
sequential digestion process using trypsin, pepsin, and
0 1985 ALAN R. LISS, INC.
pronase E which proved to be very successful with these
tissues. This technique permits viewing of all surfaces
of well-preserved dissociated cells and permits an improved understanding of their three-dimensional shape.
The rat is one of the more popular animals for experimental study of normal and diseased renal tubules.
There have been many SEM studies of normal rat renal
tubules including Bulger (19651, Bulger and Trump
(19661, Maunsbach (1966), Osvaldo and Latta (1966),
Waugh et al. (1967), Schwartz and Venkatachalam
(19741, Crayen and Thoenes (1975), and innumerable
TEM observations on abnormal rat renal tubules. SEM
studies of normal rat renal tubules have been mainly
limited to the appearance of the luminal surfaces (Andrews and Porter, 1974), Bulger et al. (19741, Allen and
Tisher (19761, and Burke (1976).
The author initiated the present study of the basolateral surfaces of renal tubules of normal rat kidneys in
order to form the basis of comparison with those changes
in cell shape caused by gentamicin-induced acute renal
failure (Jones and Elliott, 1984).
MATERIALS AND METHODS
Kidneys of ten female Sprague-Dawley rats and three
male Fisher rats were perfusion fixed with 1.25%glutarReceived January 28, 1985; accepted April 29, 1985.
122
D.B. JONES
aldehyde in 0.1 M cacodylate buffer, pN 7.4 (about 460
mOsm). Sex and strain conferred no recognizable differences in the present study. A cannula was introduced
into the exposed abdominal aorta under anesthesia. Perfusion was started at 160 mm Hg, after which the superior mesenteric artery and the subdiaphragmatic aorta
were ligated, and the renal vein was opened to permit
the blood and perfusate to escape freely. Perfusion was
continued until about 100 ml of fixative was delivered.
Some tissue was prepared for transmission electron microscopy by treatment with l%osmium tetroxide, dehydration with ethanol, followed by propylene oxide and
embedding in a n Epon 812-Araldite 506 mixture (Mollenhauer, 1964). Some tissue was processed using Karnovsky’s ferrocyanide reduced osmium procedure
(Karnovsky, 1971)to emphasize the basal processes.
The Sequential Digestion Procedure (Jones, 1983)
Renal tissue was postfixed in 10% buffered pH 7.4
glutaraldehyde for 8 weeks a t room temperature or 2
weeks at 38°C. This was found to be necessary to protect
the cells against digestion damage. After thorough
washing, slices of kidney were treated for 24 hours each
at 38°C in a saturated solution of porcine trypsin 1-250
(United States Biochemical Corp., Cleveland, OH) in
equal parts of 0.1 M phosphate buffer, pH 7.4, and 0.1 M
cacodylate buffer, pH 7.4, saturated pepsin solution,
1:10,000 (Sigma Chemical Co., St. Louis, MO), in phosphate buffer brought to pH 2.0 by addition of HC1, and
in a 0.1% filtered solution of bacterial protease type XIV,
pronase E (Sigma Chemical Co., St. Louis, MO). Additional time in the trypsin solution was used if sample
preparations exhibited inadequate removal of basement
membrane.
Slices of kidney were rinsed with buffer and then
placed in buffer under a dissecting microscope. The outer
renal cortex was gently teased with two dissecting
needles so that a cloud of loosened tubules and glomeruli
were released into the buffer, and these were pipetted
into a centrifuge tube. Teasing in fresh buffer and pipetting were repeated with the inner cortex next, then with
the outer medullary stripe, and finally with the inner
medulla and papilla. After the sediment of tubules was
rinsed twice with buffer it was postfixed in 1%osmium
tetroxide for half a n hour a t 60°C and rinsed with several changes of buffer to remove excess osmium
tetroxide .
Circular glass coverslips were dipped in warm 5%
gelatine, allowed to dry, and this was repeated. Two or
three drops of the sediment of tubules was pipetted onto
the center of the coverslips and the sediment was allowed to settle for about a minute. Using strips of torn
filter paper placed at the edge of the fluid drop the buffer
was carefully drawn off and the coverslip was promptly
placed in 80% ethanol. Dehydration with 95% and absolute ethanol was followed with several changes of acetone and critical-point drying with liquid COz in a
Sorvall critical-point dryer. The coverslips were fastened
to aluminum stubs with conductive cement, sputtercoated with gold-palladium and examined with a Hitachi 520 scanning electron microscope.
Samples of the sediment of these preparations were
embedded in a n Epon 812-Araldite 506 mixture by conventional methods and were prepared for TEM to compare with untreated renal tubules prepared as described
above.
RESULTS
General Observations
The sequential digestion procedure depends on the
synergistic effect of successive attack on the peptide
bonds of the basement membranes by the three enzymes
resulting in relative sparing of the cellular structures.
Aliquots of the isolated, treated tubules were examined by TEM and compared with perfusion-fixed, untreated tubules. The digestion treatment results in
removal of most of the basement membrane but trace
granular remnants remain. The prolonged aldehyde fixation and heavy osmification produces a granular image
by TEM (a common, well-recognized artifact). Cellular
organelles and plasma membranes are quite well preserved, but there is some enlargement of the spaces
between cells and the spaces between some tubular basal
processes (Fig. 1). Prolonged enzymatic digestion may
result in two artifacts: formation of tiny plasma membrane blebs and occasional tiny holes in the plasma
membrane. The blebs appear to have formed from the
cell to which they are attached or in many cases to have
formed during the teasing of the tubules and subsequently to have adhered to the cell surface. While these
artifacts may be seen in conventionally prepared TEM
images they clearly increase a s digestion time increases.
The various segments of the nephron are identified by
1) separating samples of tubules on the basis of where
certain segments are found within the kidney, 2) examining the luminal and lateral surfaces of a tubular segment by looking at the open end of a segment, 3)
comparing the structure of basal processes with both
TEM and SEM, and 4) relying on recognizable transitions from one segment to another (Bowman’s capsule
to S1 segment of the proximal tubule, or S3 of the proximal tubule to the descending thin limb of Henle).
In the description of the basolateral surfaces of various
cell types presented below lateral processes or ridges
refer to the larger cell extensions which arise from the
sides of cells and interdigitate with adjacent cells. Basal
processes and basal microvilli refer to downward extensions of the cell body toward the basement membrane.
Basal infoldings or clefts refer to invaginations or folds
into the cell body which may sometimes be lined by
stubby microvilli. Certain tubular segments are composed of cells whose basal processes exhibit specific orientation in reference to the axis of the tubule. This
characteristic orientation of processes, whether circumferential (around the tubular axis), random, or radial
from the cell center was determined from intact tubular
segments and not individual cells. Some cell clusters
exhibit a loosening of the complex basal processes resulting from release of the compressing effect of adjacent
cells and basement membranes. This permits looking
into the basal labyrinth and recognizing the organization of the basal processes and microvilli.
The Proximal Tubules
The proximal tubules of the outer renal cortex consist
only of S1 and S2 convoluted tubular segments so that
samples taken from that area permitted recognition of
each of these segments by comparing cell body height,
the height of brush border microvilli (Maunsbach, 19661,
and the pattern of basal processes using TEM and SEM.
With this isolation procedure small clusters or single
cells from these segments may be identified and exam-
RAT RENAL TUBULAR BASOLATERAL SURFACES
123
ined on all surfaces. Both S1 and S2 convoluted tubular erally are devoid of basal villi except at the cell edges
cells exhibit the shape of a truncated cone with a curved (Fig. 8).
base against the basement membrane. Generally the
cells are somewhat wider laterally or circumferentially
The Thin Limbs of Henle’s Loop
than along the tubular axis.
Isolation of the thin limbs was somewhat more diEcult than the other portions of the nephron. This is due
The Structure of the Basal Processes of the Proximal
to the meshwork of medullary interstitial cells, which
Convoluted Tubules
tend to bind the tubules and vessels together, and to the
The basal process is a fan-shaped or triangular process delicate nature of these structures. It was found that
whose widest part is nearest the basement membrane. gentle teasing in the direction of the radial orientation
These processes arise from the basal surface of the cell of these structures was necessary to obtain good prepabody and are oriented in a circumferential direction. On rations. Recognition of the various types of thin limbs is
the lateral aspect of the cell body these processes make made more difficult by the concurrent presence of seg
up the lateral ridges. The lateral ridges of proximal ments of isolated arterial vasa recta and capillarylvenconvoluted tubules originate just below the zonula occlu- ous vasa recta. The arterial vasa recta can be readily
dens and fan out toward the basement membrane and recognized by adherent smooth muscle cells. The capilare much more prominent on the circumferential sides larylvenous vasa rectra are recognizable by tenting of
than the axial sides of the cells (along the course of the the endothelium into short processes, presumably caused
tubule). These processes interdigitate with each other by points of attachment to the adjacent collagenous
and may fuse partially with adjacent processes so that stroma and by the diaphragm covered fenestrations of
when viewed from the basal aspect they may assume Y- the endothelial cells.
shaped or V-shaped configurations. This basic structure
Three types of thin limb segments have been described
of basal processes is much more easily observed and under- for the rat from TEM studies (Kriz et al., 1972; and
stood in the dog and human proximal convoluted tub- Schwartz and Venkatachalam, 1974).The complex interules where their structure is less obscured by basal digitating cells of the long-loop descending thin limb
microvilli (Jones, 1982, 1983). Near the rat basement and the complex interdigitating cells of the ascending
membrane most of the processes branch into two to thin limbs were easily found. The simplified cells particthree layers of short microvilli. Some of the basal pro- ularly characteristic for the descending limb of the short
cesses directly contact the basement membrane with loops were found with greater difficulty.
broad, circumferentially directed, flat attachments.
The complex descending thin limb segments are comThese broad processes often join with the smooth area posed of cells with interdigitating processes which are
beneath the nucleus.
mainly directed in a circumferential orientation. The
basal surfaces of these processes as well as the main cell
The Patterns of Basal Surfaces of S l and S2 Segments
body are smooth except for a few invaginations lined by
The majority of the basal surface of S1 segments is microvilli. Between these interdigitating processes are
covered with a labyrinth of randomly oriented short found many stubby microvilli and clefts which greatly
microvilli arising from the basal processes. A few large, increase the basolateral surface area (Fig. 9). In some
smooth processes with a circumferential orientation are segments these zones of microvilli are sparse and the
also present along with an area beneath the nucleus interdigitating cells resemble somewhat those of the
ascending thin limb but for the circumferential orientawhich is devoid of processes (Figs. 2-4).
S2 segments have highly developed circumferentially tion of processes. It is the impression of the author on
arranged basement membrane cristae or ridges when the basis of observing a few very long segments that the
observed with TEM (Waugh et al., 1967)which separate highly complex cells of the descending long-loop thin
the basal processes and their microvillous extensions limb gradually change to the simpler pattern as they
into rows directed in a circumferential orientation (Figs. descend into the inner medulla.
The basal surfaces of the simplified cells of the short5-7). The basal processes of the S2 segment may contact
the basement membrane without further branching into loop thin limbs exhibit polygonal cell contours of barely
microvilli, particularly at the cell edges. Here one is perceptible serrated cell junctions. These basal surfaces
able to recognize the medially and laterally directed often exhibited a few shallow pits, small plicae, wrinextensions of the basal process. Some tubular segments kles, or grooves (Fig. 10).
The basal surfaces of the cells of the ascending thin
show transitional patterns between the S1 and S2 types
with circumferential indentations resulting from base- limb reveal striking interdigitation of smooth, flattened
ment membrane cristae. The process-freearea of plasma lateral processes which are radially oriented about each
membrane beneath the nucleus tends to be much smaller cell body in contrast to the circumferential orientation
in the 52 segments. Samples taken from the inner cortex of the cells of the complex descending thin limbs. The
contain many convoluted S2 segments and straight S3 closely applied interdigitating processes of the ascending thin limb are outlined by very stubby microvilli (Fig.
segments.
The S3 segment cells are simpler in shape than the S1 11).
and S2 cells but have the tallest brush border microvilli.
The Ascending Thick Limb (Distal Straight Tubule)
The lateral surfaces of the cells are quite smooth and
the lateral ridges, which are a reduced copy of the proxAliquots taken from the outer medullary strip contain
imal convoluted tubular cells, terminate in a fringe of many segments of the ascending limb of the distal
short microvilli around the basal edge of the cell. The straight tubule. These can be recognized by the straight
basal cell surface is composed of shallow furrows and nature of the segments, the sparse luminal microvilli,
ridges oriented in a circumferential direction and gen- and the characteristic orientation of the basal processes.
D.B. JONES
Fig. 1, Transmission electron micrograph of a n 52 segment of proximal tubule illustrating the effects on the morphology of sequential
digestion and heavy impregnation with osmium tetroxide. Observe the
relatively well-preserved architecture with dense staining, the absence
of tubular basement membrane on the basal surface, and some enlargement of the intercellular spaces. x 3,200.
Fig. 2.S1 segment of proximal convoluted tubule. Scanning electron
micrograph demonstrating the circumferential orientation of the larger
basal processes and the random orientation of the short basal villi.
Direction of tubular axis (arrow). x6,OOO.
Fig. 3. S1 segment cell, basal and lateral view. Scanning electron
micrograph showing the luminal brush border microvilli (MI, lateral
processes (arrows),larger basal processes, and basal villi. ~8,000.
Fig. 4.S1 segment, basal surface. Scanning electron micrograph of
an isolated cell with basal processes frayed apart and demonstrating
the origin of basal microvilli from the base of larger basal processes.
x20,000.
RAT RENAL TUBULAR BASOLATEKAL SURFACES
125
Fig. 5. S2 segment of proximal tubule, basal view. Scanning electron
micrograph illustrating the highly circumferential orientation of the
basal processes and basal villi. x 7,000.
graph showing the luminal brush border microvilli (M)and the circumferentially oriented basal processes extending toward both the center
and edge of the cell. x 10,000.
Fig. 6. S2 segment, same tubule. Scanning electron micrograph
revealing at high magnification the parallel troughs or depressions in
the basal villi which correspond to the cristae of the tubular basement
membrane. ~20,000.
Fig. 8. S3 segment of proximal tubule, basal and lateral view. Scanning electron micrograph the tall luminal brush border microvilli (MI,
the simpler shape of the lateral surfaces of these cells, the fringe of
microvilli at the cell edges, and the basal surface with shallow circumferentially directed ridges and sparse infoldings. x 5,000.
Fig. 7. S2 segment, basal and lateral view. Scanning electron micro-
126
D.B. JONES
Fig. 9. The descending thin limb of Henle’s loop, long-loop nephron
type. Scanning electron micrograph revealing the complex larger processes which exhibit a predominantly circumferential orientation and
many stubby smaller processes occupying a considerable area between
them (large arrow). Some basal infoldings lined by microvilli may be
seen on the basal surface of the larger processes (small arrow). X5,OOO.
Fig. 10. The descending thin limb of Henle’s loop, short-loop nephron
type. The simple shape of these cells is outlined by simple cell junctions
(arrow). ~ 6 , 0 0 0 .
Fig. 11. The ascending thin limb of Henle’s loop, basal surface.
Scanning electron micrograph demonstrating the complex, radially
oriented, interdigitating processes of these cells. The cell junctions are
delineated by stubby microvilli. x 5,000.
Fig. 12. The ascending thick limb of Henle’s loop. A scanning electron micrograph of the end of a tubular segment (tubular lumen, L)
demonstrating the fan-shaped basal processes with circumferential
orientation. x 7,000.
127
RAT RENAL TUBULAR BASOLATERAL SURFACES
As with the proximal convoluted tubules, the basic unit
of the basal surface is the circumferentially oriented
triangular basal process. Like the proximal tubules, the
cells have a greater circumferential dimension than axial. They exhibit prominent lateral ridges on the circumferential side, while on the axial side the
circumferentially directed triangular processes are readily observed (Fig. 12). Most of the basal surface of the
cells is covered by similar, closely packed circumferentially directed basal processes which may be partially
joined to adjacent processes. As the processes contact the
basement membrane they widen slightly and form a flat
contact area (Figs. 13, 14). The small area under the
nucleus may be devoid of processes. The ascending thick
limbs in the inner cortex become larger in diameter and
the basal processes are a little less perfectly aligned in
a circumferential direction. The area of the macula densa
was not identified in these studies.
The Distal Convoluted Tubule
The distal convoluted tubules are readily recognized
not only by being in the outer cortical aliquots and by
their convoluted configuration, but also by the different
pattern of luminal microvilli and a random swirling
pattern of the basal processes (Fig. 15).These segments
tend to be a little larger in diameter than those of the
distal straight tubules.
The Connecting and Collecting Duct Tubules
Examination of the cortical aliquots of tubules revealed two types of tubules of the collecting duct system.
One type (A), which exhibits on the luminal surface
intercalated cells with plicae and principal cells with
prominent microvilli, is identified as a connecting tubule (Crayen and Thoenes, 1975; Kaissling, 1982). The
second type (B) is identified as a cortical collecting duct
and shows intercalated cells and principal cells with
rather sparse, stubby, luminal microvilli. The lateral
surfaces of both types look quite similar with moderate
numbers of plicae and short microvilli. The basal surface
of the connecting tubule principal cells are covered with
short, ridgelike basal processes resembling those of the
distal convoluted tubule but having a more random pattern. Relatively long, thin, basal processes formed a
fringe about the basal surface of these cells (Fig. 16).
The basal surface of the cortical collecting duct principal
cell reveals areas of microvillous processes and areas of
slitlike basal infoldings. Areas under the nucleus and
some other areas may be devoid of basal infoldings.
Microvilli form a stubby fringe about the edge of the cell
(Fig. 17). The cells in both segments with much simpler
basal surfaces with moderate numbers of infoldings and
processes were felt to be intercalated cells on the basis
of TEM correlations.
Examination of the medullary aliquots from the inner
medullary stripe and papilla revealed many medullary
collecting ducts which tend to be somewhat larger in
diameter than those of the cortex. The intercalated cells
on the basal surface could be recognized by their more
circular configuration in contrast to the pentagonal or
hexagonal basal shape of the more common principal
cells. The intercalated cells exhibit stubby microvilli a t
the basal edges but much of the basal surface is smooth
except for moderate numbers of invaginating clefts. The
principal cells show a n irregular pattern of clefts and
infoldings as well as smooth areas but the surface is less
complex than in the cortical collecting ducts. The areas
of microvillous pattern are not seen. The lateral edge of
the basal surfaces of the principal cell exhibits stubby
microvilli which interdigitate with adjacent cells (Fig.
18).
DISCUSSION
Early three-dimensional models of rat proximal tubules based on TEM thin sections were proposed by Bulger (1965), Waugh et al. (1967) and Maunsbach (19731,
and these helped in the understanding of structuralfunctional relationships. Welling and Welling (1976) developed improved models of the shapes of rabbit proximal tubular cells using computer-assisted analysis of
TEM step sections of tubules. This technique was repeated on rabbit ascending thick limb tubules (Welling
et al., 1978). SEM observations were added to the analysis of shape of rabbit collecting tubular cells by Welling
et al. (1981) and of rabbit cortical connecting tubules
and collecting ducts by Welling et al. (1983).The comprehensive TEM analysis of the rabbit kidney by Kaissling
and Kriz (1979) and the rat kidney in health and electrolyte adaptation by Stanton et al. (1981),Kaissling (19821,
and others have resulted in a high level of correlation of
structure with function in renal tubules.
Scanning electron microscopic studies of the luminal
and lateral surfaces have been carried out on rat renal
tubules by Andrews and Porter (19741, Bulger et al.
(19741, Burke (19761, and Allen and Tisher (1976). Evan
(1981) used his HC1-collagenase isolation procedure to
study the renal tubular cells of the rat. He described a n
early proximal tubule of the rat possessing numerous
folds or plates that do not divide into basal villi. He
states that these plates run parallel to each other but
perpendicular to the long axis of the tubule. The present
author feels that these observations concerning the absence of basal villi in proximal tubules are probably in
error since transmission electron microscopic studies of
many investigators demonstrate basal villi in both S1
and S2 proximal tubular segments and the present paper clearly shows these basal microvilli. It is probable
the improved resolution of the present isolation procedure makes this demonstration possible.
The importance of the complex basal labyrinth is emphasized by the extreme simplification of the basal surfaces of S1 and S2 tubules at the peak of gentamicininduced acute renal failure in the rat and the return
toward normal complexity with recovery demonstrated
by Jones and Elliott (1984).
Excellent studies have been carried out with the transmission electron microscope elucidating the ultrastructure of the thin limbs of Henle in the rat (Osvaldo and
Latta, 1966; Bulger and Trump, 1966; Schwartz and
Venkatachalam, 1974; among others). The present study
confirms the general shape of the cells of the segments
of the thin limbs, the simple cells of the descending short
loops, and the more complex cells of the other segments.
The present observations support the proposed passive
role of the thin limbs of Henle's loop in water-electrolyte
flux (Schwartz and Venkatachalam, 1974).However, the
author and others have been impressed by the complex
basolateral surfaces of the cells of the descending long
loops (Bulger and Dobyan, 1982, 1983).The high level of
carbonic anhydrase activity described by Lonnerholm
128
D.B. JONES
Fig. 15. Distal convoluted tubular cell, scanning electron micrograph
Fig. 13. Scanning electron micrograph of the basal surface of two
cells from the ascending thick limb of Henle’s loop. The basal processes of basal surface. The basal processes are coarser, less uniform, and
are quite regular in shape and are circumferentially oriented. ~ 4 , 0 0 0 . exhibit a less regular circumferential orientation when compared with
those of the ascending thick limb. ~ 8 , 0 0 0 .
Fig. 14. The basal processes of a cell from the ascending thick limb
Fig. 16. Connecting tubule, scanning electron micrograph of the
of Henle’s loop. This view of the loosened processes at the edge of this
isolated cell reveals the roughly triangular shape of these processes basal surface. Some cells exhibit basal processes which resemble those
with a long, thin, flattened surface contacting the basement mem- of distal convoluted tubular cells (A) while others resemble collecting
duct cells (B). ~ 4 , 0 0 0 .
brane. x 12,500.
RAT RENAL TUBULAR BASOLATERAL SURFACES
129
Fig. 17. Cortical collecting duct, scanning electron micrograph of
basal surface. A principle cell with complex villous basal processes (PI
lies next to the simpler basal surface of an intercalated or dark cell
(D). ~7,000.
Fig. 18. Medullary collecting duct, scanning electron micrograph of
the basal surface. Observe the oval contour and the simple basal
surface of the dark cell (D) and the mildly complex surface of the
principle cells (P) of this segment. ~ 3 , 5 0 0 .
and Ridderstrale (1980) and Dobyan and Bulger (1982)
plus the demonstration of a considerable concentration
of basolateral Na', K' ATPase by Biemesderfer et al.
(1985) suggest a possible dynamic role for this segment.
While similarities were noted between the structure of
rat thin limbs and those of the mouse by Dieterich et al.
(1975), and Psammonys by Barrett et al. (1978a,b),there
appear to be distinctive differences.
The basal processes of the rat ascending thick limb
and distal convoluted segment appear to be comparable
to those of the dog and human. The proximal convoluted
segments of the rat exhibit more complex microvillous
branching of the basal processes than the dog or human
(Jones, 1982, 1983). The author felt that he could identify characteristic cells in the rat connecting tubule segments which matched the type 2 intermediate cells
(connecting tubule cell) described with TEM by Crayen
and Thoenes (1975) and by Kaissling (1982). Since Kaissling (1982)has pointed out that intercalated cells in the
rat may also be seen in the terminal portion of the distal
convoluted tubule the author tried to separate distal
tubule and connecting tubule by other criteria, such as
the basal processes and prominent lateral processes
characteristic of distal convoluted tubular cells. TEM
studies have demonstrated prominent alterations of basolateral surfaces of parts of the distal nephron in adaptation to electrolyte handling (Stanton et al., 1981;
Kaissling, 1982). It is hoped that the presently described
tubular isolation technique may be of some use in fur-
ther documentation of such effects.
Many authors have made a strong distinction on the
basis of TEM studies between cells having basal interdigitations or true basal infoldings. In view of SEM and
TEM observations of the basal surfaces of renal tubules
of normal dogs (Jones, 1983), a human donor kidney
(Jones and Elliot, 19841, and the present study of rats,
the author feels that such a distinction may not be
completely accurate. Interdigitation of lateral processes,
basal processes, and microvilli between adjacent cells
occurs at the lateral edges of these cells only. The basal
processes which extend down from the cell body and
their microvillous branches interdigitate with one another, particularly in the proximal convoluted tubular
cells. With TEM sections this self-interdigitation may
resemble the interdigitation of lateral processes with
adjacent cells. This self-interdigitation has been misinterpreted to be processes of adjacent cells extending
deeply under the observed cell. The present technique
permits gentle isolation of cells without breaking off of
processes and permits careful examination of both lateral and basal surfaces by both TEM and SEM. These
studies have failed to reveal deeply interdigitating processes arising from adjacent cells. Other cells such as
collecting duct cells may have short or long clefts, often
lined by microvilli, but such cells also have true basal
processes. It is only by combined observation with TEM
and SEM that the three-dimensional organization of the
basal surfaces can be understood.
130
D.B. JONES
LITERATURE CITED
43:377(Abstract).
Kaissling, B., and W. Kriz (1979) Structural analysis of the rabbit
Allen, F., and C.C. Tisher (1976) Morphology of the ascending thick
kidney. Adv. Anat. Embryol. Cell Biol., 56:l-123.
limb of Henle. Kidney Int., 9%-21.
Kaissling, B. (1982) Structural aspects of adaptive changes in renal
Andrews, P.M., and K.R. Porter (1974)A scanning electron microscopic
electrolyte excretion. Am. J. Physiol., 243Renal Fluid Electrolyte
study of the nephron. Am. J. Anat., 140:81-116.
Physiol. 12):F211-226.
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