Scanning and Transmission Electron Microscopy

of the Rat Kidney'

RUTH ELLEN BULGER, FRANCES LEA SIEGEL
AND ROBERT PENDERGRASS
Department of Pathology, University of Maryland,
Baltimore, Maryland 21201
ABSTRACT This study utilized scanning and transmission electron micros-
copy of renal tissue to provide new information on the gross and microscopic
structure of the kidney. The luminal surface of the proximal convoluted tubule
was characterized not only by the border of microvilli, but also by crater-like
depressions, and circumferential folds. In tissue processed for scanning electron
microscopy the proximal tubular cells separated along their lateral surfaces
clearly exposing the topography of the lateral cell projections of cytoplasm which
have been generally unavailable for viewing because of their interdigitation with
adjacent cells. The various segments of the nephron were identified on the basis
of position in the kidney, general morphology, and the distribution and form of
apical microvilli, cilia, or flaps. The external surface of the papillary tip had
several parallel furrows into which the collecting ducts opened. Large plaque-
like depressions lined the papillary surface. The opposed surface of the renal
pelvis had small plaque-like depressions separated by narrow ridges. Transmis-
sion electron microscopy of plastic-embedded tissue specimens which had been
previously dehydrated by the critical point drying method demonstrated that
little damage occurred from this procedure.
Scanning electron microscopy has been
utilized repeatedly to study the renal cor-
puscle in normal and diseased states
(Buss and Kronert, '69; Buss, '70; Ara-
kawa, '70, '71; J ulkunen, '71; Barger and
Herd, '71; Fujita et al., '70, '71; and
Spinelli et al., '72a,b). The present study,
therefore, concentrated on the description
of various regions of the uriniferous tu-
bule of which only a few pictures exist in
the literature (Hagege and Richet, '70, '71;
Hornych et al., '71; Spinelli et al., '72a) as
a basis for studies of changes in renal
structure resulting from injury or disease.
Seventeen Wistar-Furth and four
Sprague-Dawley rats were utilized in these
experiments (table 1). Prior to sacrifice
the animals were anesthetized with 0.07
ml / l OO gm of body weight of sodium
pentobarbital. The kidneys were fixed by
intravascular perfusion (Griffith et al., '67),
filling the abdominal cavity with fixative
(in vivo immersion), or by injecting fixa-
MATERIALS AND METHODS
AM. J. ANAT., 139: 483-502.
tive directly into the renal pelvis using
2.5% glutaraldehyde and 2% formalde-
hyde buffered in 0.04 M potassium phos-
phate or 0.04 M sodium cacodylate (Kar-
novsky, '65). The tissues were cut into
appropriate regions to be studied and
stored in 0.1 M potassium phosphate or
0.1 M sodium cacodylate to each of which
7.5% sucrose had been added.
The tissues for scanning electron mi-
croscopy were postfked in l % osmium
tetroxide for 30-90 minutes at room tem-
perature, dehydrated with ethyl alcohol,
and immersed in two changes of amyl ace-
tate for 15 minutes each. The tissue was
subsequently dried in a Sorvall critical
point drying apparatus utilizing carbon di-
oxide (Anderson, '51). The blocks were
stored in scintillation vials in a dessicator
until mounted. Appropriately cut or broken
pieces of tissue were mounted on alumi-
num stubs utilizing conducting silver paint
(Ernest F. Fullam, Inc.). The stubs were
1 Research support by Public Health Service grant
AM 16236.
483
484 R. E. BULGER, F. L. SIEGEL AND R. PENDERGRASS
gently heated to dry the paint. They were
placed in a vacuum evaporator with a ro-
tating stage or in a sputtering device and
were coated with carbon and subsequently
with gold, or with gold or gold-palladium
alone. The specimens were studied using
the JSM-U3, the Cambridge Mark 11, or
the ETEC autoscan scanning microscope.
The tissue for transmission electron mi-
croscopy was cut into appropriate pieces,
rinsed in 0.1 M sodium cacodylate or PO-
tassium phosphate buffer containing 7.5%
sucrose, postfixed i n 2% osmium tetroxide
buffered with 0.1 M s-collidine for one
hour at O'C, stained in block for one hour
with 0.5% uranyl acetate buffered in
Veronal-acetate (pH 4.7-4.9), dehydrated
in ethyl alcohol, treated with propylene
oxide, embedded in Epon epoxy resin, cut
with an ultramicrotome, stained sequen-
tially with 7.5% uranyl magnesium ace-
tate and lead citrate, and viewed in an
AEI-6B electron microscope. Several tissue
blocks which were embedded in Epon were
fractured according to the method of
Humphreys et al. ('73) and viewed in the
scanning microscope. Tissue blocks which
had been dried using a critical point dry-
ing apparatus and in addition dried speci-
mens coated with carbon and gold were
subsequently embedded in epoxy resin by
placing the dried tissue directly in the
Epon.
Tubular identification. Slices of kidney
which extended from the cortex to the tip
of the papilla of the rat kidney were fre-
quently utilized. Low magnification pic-
tures were therefore taken and used for
the selection and subsequent location of the
exact tubule which was photographed at
higher magnification in the scanning mi-
croscope (fig. l ). This procedure provided
information useful in the determination of
which segment of the uriniferous tubule
was being studied. Occasionally there still
was a problem in distinguishing certain
segments such as late distal tubule from
the early collecting duct because both were
characterized by two surface cell types and
could occupy the same zone of the kidney.
Profiles of thin limbs were sometimes diffi-
cult to distinguish from capillaries in the
inner medulla.
.
OBSERVATIONS
The renal corpuscle. In kidney speci-
mens which had been fractured, the
glomerulus of the renal corpuscle usually
stayed intact (fig. 1) . Renal corpuscles
were seen containing the entire glom2rular
tuft or devoid of it (fig. 2). When the renal
corpuscle was empty, the urinary pole was
frequently present, supporting the hypothe-
sis that the glomerular tuft remains at-
tached at the vascular pole. In kidney spe-
cimens which had been cut after fixation,
TABLE 1
Tissue preparation
Number
of rats
Species
Number
Route of processed Number processed
fixative for scanning for transmission
Fixative Buffer application studies studies
3 Wistar-Furth 2.5% glutaraldehyde cacodylate perfusion 3 3
3 Wistar-Furth same phosphate perfusion 3 3
3 Wistar-Furth same phosphate i n vivo 3 3.
4 Wistar-Furth same phosphate injection into 4 1
4 Wistar-Furth same cacodylate injection into 4 CI
2 Sprague- same cacodylate injection into 2 0
2 Sprague- same cacodylate perfusion 2 2
and 2% formaldehyde ( 5 zones)
( 5 zones)
immersion (cortex onl y)
renal pelvis (papilla only)
renal pelvis
Dawley renal pelvis
Dawley (5 zones)
SCANNING EM OF KIDNEY 485
capillary loops were more frequently cut
open allowing one to examine the structure
of the endothelium.
The glomerular podocytes have been de-
scribed in earlier studies and our observa-
tions were in agreement with those which
have shown that adjacent pedicels invari-
ably appear to result from interdigitation
of processes of different cells.
The parietal wall of the renal corpuscle
was lined by epithelial cells which were
sparsely covered by small microvilli (fig. 2).
The microvilli were more concentrated in
areas which formed irregular meandering
bands and appeared to demarcate t he pe-
riphery of the cells. From these demarcated
areas, single cilia projected into Bowmans
space. In some preparations, bulging areas
were seen which appeared to overlie the
cell nuclei.
At the urinary pole, the microvillus
border began abruptly but with an irregu-
lar contour. A few of the microvilli in the
first row were about half the height of the
remaining microvilli (fig. 8).
The proximal tu-
bules appeared to be cut and broken open
both in cross and longitudinal sections dur-
ing specimen preparation depending on
their orientation within the specimen. The
cells most often appeared to separate
along the lateral intercellular spaces dem-
onstrating the smooth undulating lateral
ridges, typical of these cells. These ridges
were more complex in the basal and apical
regions of the cells (fig. 3). In the apical
region, the lateral contours frequently ap-
peared rounded and looked like a ball and
socket-type of interlocking. Certain cellu-
lar regions appeared to fracture across the
cytoplasm, These areas appeared more
rough, irregular, and granular. Rounded
structures of various sizes were seen in
these regions, presumably being remnants
of cell organelles (figs. 6, 7).
The lumens of the perfused kidneys
were patent but considerable irregularity
was present along their surfaces. Frequent
craters of various size were formed when
the microvilli were tipped outwards leaving
a central depression (fig. 4). Transmission
electron micrographs demonstrated areas
of irregularity in the brush border which
presumably corresponded to the crater
(fig. 5) . I n these micrographs, the cell
The proximal tubule.
membrane at the base of the crater was
either intact or artifactually broken. The
scanning microscope also demonstrated in-
frequent bulbous projections into the proxi-
mal lumen and a few tubules had ridge-
like elevations oriented circumferentially
around the luminal border. Occasional
structures appearing to be cilia projected
above the brush border into the lumen. The
microvilli extended from the apical sur-
face as parallel structures as expected from
transmission pictures. However, there were
slight differences in length of the micro-
villi and smaller irregular processes were
occasionally identified (fig. 8).
At the base of the cell, the basal lamina
and associated collagenous fibers were ir-
regularly broken. The collagenous fibers
appeared to have broken forming long
processes which assumed abnormal loca-
tions (fig. 3) . In some regions the fracture
surface appeared to form along the outer
surface of proximal tubules and interstitial
cells. Vessels could frequently be identified
in these regions (figs. 6, 7).
Thin limb of Henles loop. The tubular
structures with large lumens that were ob-
served in the inner medulla and inner
stripe of the outer medulla appeared to be
thin limbs. The walls of these thin limbs
were narrow and the cells often bulged in
the region of the nucleus and sometimes
were depressed along the cell border. Short
rounded microvilli formed irregular pat-
terns on the luminal surface and single
large cilia projected from the cell far into
the lumen (fig. l o).
Identifying the parts of
the distal tubule proved the most difficult.
Ascending thick limbs of the distal tubule
were identified by their location in the
outer medulla, the lack of dark cells (de-
scribed below), the cell height, and by the
undulating lateral ridges of the cells in the
regions where they appeared to have sep-
arated from adjacent cells (fig. 11). The
lumen of this type of tubule contained
short microvilli of irregular shape. A
single long cilium appeared to project at
frequent intervals into the lumen, In the
cortex, tubules were identified with surface
characteristics similar to those of the cells
of the ascending thick limb of the distal
tubule just described (fig. 12).
Collecting tubule. Many tubules were
Distal tubule.
486 R. E. BULGER, F. L. SIEGEL AND R. PENDERGRASS
seen in the cortex or outer medulla which
were fractured or cut in such a manner
as to be open for long distances. These
relatively straight tubules were character-
ized by the presence of two types of cells
with distinctive luminal morphology, by a
relatively simple lateral cell border con-
figuration, and by sites where two of these
tubules merged to form one tubule. The
nuclei frequently bulged into the lumen
and depressions clearly marked their lat-
eral extent (fig. 13).
The most numerous cell type lining the
collecting tubule was characterized by one
long cilium projecting into the lumen, OC-
casional crater-like structures, and small
bulbous microvilli which were uniform in
size but had an uneven distribution some-
times with fewer in the center of the cell.
The luminal outline was polygonal, From
comparison with transmission electron mi-
crographs, this cell appeared to be the
principal or light cell (fig. 15).
The second cell type lining the collecting
tubule was characterized by a luminal sur-
face covered with ridges of greater height
than the microvilli on adjacent cells. The
ridges were of various lengths and fol-
lowed a meandering path with a branching
or looping form. A few microvilli were
present as well. Cilia were not identified
projecting from this cell type. The cell out-
lines were also polygonal. From compari-
son with transmission electron micro-
graphs, this cell was identified as the in-
tercalated or dark cell of the collecting
duct (fig. 15).
The papillary t i p. The tip of the papilla
had a number of furrows that were roughly
parallel (fig. 16). Some of the furrows
followed a slightly curving course. The
large collecting ducts opened along the
base of the furrows (figs. 16, 18). The
papilla lies in the funnel-shaped renal
pelvis which was lined by an epithelium
whose surface was characterized by small
depressed plaques of various size and
shape with thin ridges separating the
plaques (fig. 17). These small irregular
shaped plaque-like depressions were ap-
proximately 1000-5000 A in diameter.
DISCUSSION
Various segments of the nephron and
the surfaces of the papilla and pelvis were
described in this paper. One could study
not only luminal topography but also lat-
eral cell contours because the cells fre-
quently separated along their lateral bor-
ders. This was particularly true of the
proximal tubule where the extensive apical-
lateral, lateral, and basal-lateral projec-
tions were easily visualized. The luminal
topography of the various tubular segments
largely substantiated previous suppositions
gained from light microscopy and trans-
mission electron microscopy although cer-
tain new features were noted. The luminal
surface of the proximal convoluted tubule
after intravascular perfusion not only had
the expected microvilli but was studded
with frequent crater-like depressions and
occasional circumferential folds. These ap-
peared to explain the presence of the ir-
regularities of the microvillus border seen
in transmission electron micrographs. I t
was not known whether these structures
were present in vivo or were caused by
fixation. The infrequent bulbous projec-
tions were probably a fixation artifact.
The elaborate luminal extensions from
dark cells of the distal tubule and collect-
ing ducts appeared as long branching
flaps or folds instead of finger-like micro-
villi. This had been surmised by transmis-
sion studies (Griffith, '67) but serial recon-
structions were never done. The pictures of
Spinelli et al. ('72) showed that an inter-
calated cell of a medullary collecting duct
had leaf-like microvilli somewhat different
from those seen on the rat intercalated
cells in this study.
The presence and distribution of single
cilia projecting into the lumen from many
cells lining the uriniferous tubules were
readily apparent. Cilia were not evident
on intercalated cells, however. Occasional
pock-like depressions were identified on
the luminal surface of collecting duct
light cells.
The most surprising finding was the
presence of long parallel furrows on the
external surface of the papilla. The col-
lecting ducts appeared to open into the
furrows. The functional significance of
these furrows is unknown. The surface
of the transitional epithelium on the
renal pelvis contained small irregular
shaped plaque-like depressions approxi-
mately 1000-5000 A in diameter which
SCANNING EM OF KIDNEY 487
were surrounded by narrow ridges. The
plaques may correspond to areas of thick
120 A bladder-type cell membrane and the
ridges to the thinner cell membrane regions
demonstrated by Silverblatt ('73).
Transmission electron micrographs of
tissue pieces which were first dried by crit-
ical point drying and then embedded in
Epon demonstrated remarkably little cell
or tissue damage. These results suggest
that the critical point drying technique may
provide a fairly faichful representation of
tissue morphology.
LITERATURE CITED
Anderson, T. F. 1951 Techniques for the preser-
vation of three dimensional structure in pre-
paring specimens for the electron microscope.
Trans. N.Y. Acad. Sci., Ser. 11, 13: 130.
Arakawa, M. 1970 A scanning electron micros-
copy of the glomerulus of normal and nephrotic
rats. Lab. Invest., 23: 489-496.
1971 A scanning clectron microscope
study of the human glomerulus. Amer. J . of
Path., 64: (2): 457-462.
Barber, V. C., and A. Boyde 1968 Scanning elec-
tron microscopic studies of cilia. Zeit. fur
ZelLforsch., 84: 269-284.
Barger, A. C., and J . A. Herd 1971 The renal
c&culation.. New England J ournal of Medicine,
284: 482-490. . -. ~~~
Buss, H. 1970 Die morphologische Differen-
zierung des visceralen Blattes der Bowmanschen
Kapsel (Raster- und durchstrahlungs-electronen-
mikroskopische Untersuchungen am Nieren-
glomerulum der Ratte). Zeit. fur Zellforsch.,
111: 346.
Buss, H., and W. Kronert 1969 Zur Struktur des
Nierenglomerulums der Ratte (Raster elek-
tronenmikroskopische Untersuchungen). Vir-
chows Arch. Abt. B Zellpath, 4: 79.
Fujita, T., J . Tokunaga and M. Miyoshi 1970
Scanning electron microscopy of the podocytes
of renal glomerulus. Arch. histol. J ap., 32:
99-113.
Fujita, T., J . Tokunaga and H. Inoue 1971 Atlas
of scanning electron microscopy i n medicine.
Elsevier Publishing Co. Amsterdam, London,
New York.
Griffith, L. D., R. E. Bulger and B. F. Trump
1967 The ultrastructure of the functioning
kidney. Lab. Invest., 16: (2): 220-246.
HagBge, J ., and G. Richet 1970 Etude par mi-
croscapie electronique a balayage de la surface
apicale des cellules du tube contour& distal du
rein de rat. C. R. Acad. Sci., Serie D, 271: 331.
1971 Microscopie klectronique 8 bala-
yage du pole urinaire des cellules tubulaires
distales chez l e Rat soumis & une acidose
gazeuse et B une alcalose mbtabolique. C. R.
Acad. Sci. Paris t 273 Serie D, p. 1818-1819.
Hornych, H., M. Beaufits and G. Richet 1971 Ef-
fets de l'angiotensine exogPne sur les capillaires
des glomtrules corticaux et juxta-m6dullaires
du Rat: Etude en microscopie blectronique B
balayage. C. R. Acad. Sci. Paris t 273 Serie D,
p. 1129-1131.
Humphreys, W. J ., T. J . Wodzicki and J . J . Paulin
1973 Fractographic studies of plastic-embed-
ded cells by scanning electron microscopy. J.
Cell Biol., 56: 876.
J ulkunen, H. 1971 Scanning electron micro-
scopic study i n scleroderma. Annales Medicinae
Experimentalis et Biologiae Fenniae, 49: 180-
185.
Karnovsky, M. J . 1965 A formaldehyde-glutar-
aldehyde fixative of high osmolality for use in
electron microscopy. J . Cell Biology, 27: 137A-
138A.
Silverblatt, F. Personal Communication.
Spinelli, F. R., H. Wirz, C. Briicher and G. Pehling
1972a Non-existence of shunts between af-
ferent and efferent arterioles of juxtamedullary
glomeruli i n dog and rat kidneys. Nephron, 9:
123-128.
Spinelli, F., H. Wirz, C. Briicher and G. Pehling
1972b Fine structure of the kidney revealed
by scanning electron microscopy. Ciba-Geigy
Limited, Basle, Switzerland.
PLATE 1
EXPLANATION OF FIGURES
1 Low magnification scanning electron micrograph of rat renal cortex.
Glomerular tufts (G) and the parietal wall of renal corpuscles (RC)
not containing glomerular tufts can be identified. The majority of the
parenchyma consists of proximal tubules (PT). The renal capsule is
on the left. x 230.
2 Scanning electron micrograph showing the cells lining t he parietal
wall of Bowmans capsule. Microvilli and cilia (C) project into Bow-
mans space. The microvilli lining the proximal tubule begins at the
urinary pole (UP). x 1,500.
488
SCANNING EM OF KIDNEY
R. E. Bulger, F. L. Siege1 and R. Pendergrass
PLATE 1
PLATE 2
EXPLANATI ON OF FIGURES
3 Scanning electron micrograph of a proximal convoluted tubule. The
microvilli (M) project into the patent lumen. The fracture plane fol-
lows the lateral intercellular space displaying the lateral projections
of the tubular cells (P). Collagenous fibers (CF) have been pulled
from their normal position in the left side of the micrograph but were
unbroken elsewhere. x 13,800.
Scanning electron micrograph of a proximal convoluted tubule (PT)
showing the crater-like structures (CS) frequently seen in the micro-
villus border (M). x 4,300.
Transmission electron micrograph of the microvilli (M) of a proximal
convoluted tubule showing irregularity in the orientation of the mi-
crovilli (arrow) which appears to correspond with the crater-like struc-
tures seen by scanning microscopy. x 19,100.
4
5
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SCANNING EM OF KIDNEY
R. E. Bulger, F. L. Siege1 and R. Pendergrass
PLATE 2
PLATE 3
EXPLANATION OF FIGURES
6, 7
Scanning electron microscopy of a stereopair taken with a tilt dif-
ference of 7" of two proximal convoluted tubules. The fracture plane
sometimes follows the lateral cell membrane displaying the lateral
projections (P). What is interpreted as cellular organelles are also
seen (arrows). The largest round structure has the appropriate size
and position to be a nucleus (N). Capillaries (Cap) can be identified.
x 3,200.
492
SCANNING EM OF KIDNEY
R. E. Bulger, F. L. Siege1 and R. Pendergrass
PLATE 3
PLATE 4
EXPLANATION OF FIGURES
8 Scanning electron micrograph of a proximal convoluted tubule micro-
villus border at the urinary pole of Bowmans capsule. In the first row
some microvilli are not full height (arrows). x 20,200.
Transmission electron micrograph of part of a proximal tubule from
a tissue specimen which had been dried by the critical point method
and subsequently embedded in Epon. I n spite of being dried and then
embedded in plastic, the morphology is well preserved indicating that
the drying procedure causes little structural alteration. Nucleus, (N);
mitochondria, (Mt); microvilli, (M). x 13,800.
Scanning electron micrograph of a thin limb segment. This tubule is
characterized by the low height of the cells i n its wall (arrows) and
by apical microvilli (M) and cilia (C). x 3,900.
9
10
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SCANNING EM OF KIDNEY
R. E. Bulger, F. L. Siege1 and R. Pendergrass
PLATE 4
PLATE 5
EXPLANATION OF FIGURES
11 Scanning electron micrograph of the medullary ascending thick seg-
ment of the distal tubule. The fracture plane has followed the lateral
intercellular space in certain regions demonstrating the lateral pro-
jections (P) which interdigitate with those from adjacent cells. The
luminal surfaces of the cell are provided with cilia (C) and micro-
villi ( M) . x 10,500.
Scanning electron micrograph of a cortical distal tubule. This tubule
has few unique features but is characterized by position in the kidney,
projections (P) of the lateral surfaces, and luminal microvilli and
cilia ( C) . x 8,300.
12
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SCANNING EM OF KIDNEY
R. E. Bulger, F. L. Siege1 and R. Pendergrass
PLATE 5
PLATE 6
EXPLANATION OF FIGURES
13 Scanning electron micrograph of the luminal surface of a collecting
tubule. Two types of surface cells are identified. The light (L) or
principal cell is characterized by luminal microvilli and cilia whereas
the dark (D) or intercalated cell is covered by irregular ridges or
flaps. x 4,500.
14 Scanning electron micrograph showing the irregular shape of the
apical extensions of the dark (intercalated) cell. X 26,400.
15 Transmission electron micrograph from the same animal as illustrated
i n figure 14 showing the surface configuration of a collecting duct
dark cell (D) and light cell (L). X 12,500.
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SCANNING EM OF KIDNEY
R. E. Bulger, F. L. Siege1 and R. Pendergrass
PLATE 6
PLATE 7
EXPLANATION OF FIGURES
16 Scanning electron micrograph of the papillary tip. The long parallel
furrows traverse the surface. The large collecting tubules open at the
base of the furrows (arrows). X 470.
High magnification scanning electron micrograph of the surface of
the transitional epithelium lining the renal sinus. The surface has
small irregular plaque-like depressions 1000-5000 A in diameter sep-
arated by narrow ridges. X 20,000.
Scanning electron micrograph of the papillary tip which was bisected
to show the collecting tubules as they open onto the surface. Two
collecting ducts (arrows) can be seen to open into a furrow (F) cut
in cross section. X 460.
17
18
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SCANNING EM OF KIDNEY
R. E. Bulger, F. L. Siege1 and R. Pendergrass
PLATE 7