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Glomerular ultrastructure in the kidney of a hibernating animal.

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Glomerular Ultrastructure in the Kidney
of a Hibernating Animal '
MARILYN L. ZIMNY AND ELMORE RIGAMER
Department o f Anatomy, Louisiana State University, School of Medicine,
N e w Orleans. Louisiana
Kidney samples were obtained from ten hibernating and ten control
ABSTRACT
13-lined ground squirrels. Citellus tridecemlineatus. The tissue was fixed in cold
buffered 2% osmium tetroxide, dehydrated i n graded alcohols and embedded i n Maraglass. Sections were made with glass knives on a Porter-Blum microtome and viewed
with RCA-EML and RCA-EMU 3 electron microscopes. Observations and comparisons
were made of the glomerular capillaries in samples from both groups. In comparison
with data on other animals reported in the literature, the basement membrane of the
renal glomerulus of the control ground squirrel is approximately twice as thick, averaging 2,000-2,500 A in width. Hibernation results i n several structural modifications
of the glomerular ultrafilter as follows: (1) the endothelial pores are decreased in
number and size; ( 2 ) the podocytic foot processes are swollen and the slit pores reduced in size; and (3) the basement membrane thickens, averaging 4,000-5,000 A in
width, and becomes irregular in contour. It is concluded that the porosity of the
glomerular ultrafilter decreases during hibernation. This aids i n explaining the decrease in the rate of urine formation which occurs during this time of lowered body
temperature and metabolic depression.
During hibernation in the 13-lined
ground squirrel the rate of urine formation
is markedly reduced as evidenced by the
cessation of urinary output and the presence of a distended urinary bladder containing 0.5-1.0 ml of urine in the animal
when sacrificed. In control animals the
average water intake is 34 ml per 24 hours
and the average urinary output is 15 ml
(40-60 ml/kg body wt/day). In hibernating animals both these values are zero.
Histological study of kidneys from these
animals revealed a pronounced congestion
of the vessels of the glomeruli and vasae
recti in kidneys from hibernating animals
(Zimny and Bourgeois, '60). Hester and
Hoffman ('64) also reported a decrease in
urinary volume during hibernation in the
13-lined ground squirrel. Furthermore,
they stated that the specific gravity and
percentage of solids changed relative to
volume. Studies of the golden mantled
ground squirrel by Pengelley and Fisher
('61) give further evidence that during
hibernation urine formation is not appreciable.
Studies of renal function during hypothermia and hibernation in the rat
and the 13-lined ground squirrel have
demonstrated that a decrease in the gloANAT. REC., 154: 87-94.
merular filtration rate following a reduction in renal blood fiow may be the main
cause for the reduction of urine flow during hypothermia. The excretory patterns
during hibernation were like those observed
in nonhibernating hypothermic animals.
Any species differences were small and
quantitative rather than qualitative in nature (Hong, '57). Lowering of the body
temperature in dogs produces a decrease
in renal blood flow, a depression of both
tubular reabsorption and secretion and a
decrease in the renal concentration process
(Hong and Boylan, '59). Induced hypothermia in rabbits results in decreases in
urine output and atration presumed to be
due to vasoconstriction within the kidney.
Decreases in concentration capacity and
percentage reabsorption of urea in these
animals were related to diminution of the
permeability of the nephrons directly affected by the cold (Anderson and Nielsen,
'55).
These studies suggest an inhibition or
reduction of glomerular filtration at reduced body temperature related in part to
decreased renal blood flow. The purpose
1 Supported by Research grant A-2027 from the National Institute of Arthritis and Metabolic Diseases,
National Institutes of Health.
ZSupported by training grant G-TI-GM 399.
87
88
MARILYN L. ZIMNY AND ELMORE RIGAMER
of this paper is to present the ultrastructural modifications found in the glomeruli
of kidneys from hibernating animals as
compared with non-hibernating controls as
evidence of another factor to be considered
in interpreting the rate of urine formation
at low body temperatures.
MATERIALS AND METHODS
OBSERVATIONS
In kidney samples from the control
ground squirrels (fig. 1 ) the wall of the
glomerular capillary shows the typical
structure consisting of an inner endothelial
layer, an outer epithelial layer and a basement membrane in between. The basement membrane is noticeably thick averaging 2,000-2,500 A in width.
In kidney samples from the hibernating
ground squirrels (figs. 2, 3, 4 ) three major
differences are noted when the structure of
the wall of the glomerular capillary is conipared with that of the controls. These differences are as follows: ( 1 ) the endothelial pores are decreased in number and
size; (2) the pedicels of the podocyte
trabeculae are swollen, consequently reducing the size of the slit pores and giving
the appearance of a continuous membrane;
and ( 3 ) the basement membrane is approximately twice as thick, averaging
4,000-5,000 A in width, and irregular in
contour. A reduction in the urinary space
of Bowman is often seen and is presumably related to the swollen podocytes. The
podocytes in these kidneys often contain
vacuoles and their mitochondria are
smaller and darker giving a condensed appearance. In view of these structural modifications it is concluded that the porosity
of the filtering membrane is decreased during hibernation.
Kidneys were obtained from ten 13-lined
ground squirrels, Citellus tridecemlineatus,
after hibernating five days in a cold room
at 3-5°C and from ten control ground
squirrels housed at 2527°C. The hibernating animals were sacrificed in the cold
room without anesthesia using instruments
cooled to the temperature of the environment. The controls were sacrificed in the
laboratory under nembutal anesthesia. At
sacrifice a kidney was quickly removed
within 30 seconds, and a thin slice of cortex placed immediately in 2% cold buffered osmium tetroxide and cut into cubes
with a razor blade. After one hour of fixation in the buffered osmium the samples
were placed in 10% buffered formalin for
30 minutes. Then the tissues were dehydrated and embedded in Maraglass (Freeman, '64). Thin sections were cut with
glass knives on a Porter-Blum microtome,
mounted on uncoated grids and stained
with lead citrate (Reynolds, '63). The sections were examined with an RCA EML
and an RCA EMU 3F.
DISCUSSION
The width of the basement membrane
This study of the ultrastructure of the
of the glomerular capillary became of particular interest during this study and con- renal glomerulus of a hibernator, the 13sequently 10-20 measurements of its width lined ground squirrel, agrees essentially
were made on electron micrographs from with the studies of Hall ('54), Rhodin
each tissue sample. Two landmarks were ('55), Pease ('55), Yamada ('55) and
used in selecting the points for measure- Mueller ('57) regarding the glomerular filment. First the foot process of the podo- ter. This filter has three components. One
cyte had to be perpendicular to the base- is endothelial and is perforated with many
ment membrane and secondly, the endo- pores. A second cellular layer is present
thelial pores had to appear in a single row on the urinary surface and is composed of
on the inner aspect of the basement mem- cells called podocytes which have branchbrane. Measurements per tissue sample ing interdigitating processes applied to the
were then averaged and the data used in basement membrane. The basement memcomparing glomerular capillaries in kid- brane appears to be the definite filter of the
neys from hibernating ground squirrels glomerulus and reportedly its thickness
with those from control ground squirrels. varies from 800 A to 1,400 A.
When compared with these data, the
In addition, these data were compared with
basement membrane measurements of glomerular basement membrane in kidother laboratory animals that are cited in neys from control ground squirrels is approximately twice as thick. Whether this
the literature.
GLOMERULAR ULTRASTRUCTURE
89
The glomeruli of the kidneys from hibersignifies a better or a poorer kidney functionally is open to speculation at this time. nating and control ground squirrels demonThe urinary output of the ground squirrel strated little to no staining reaction for oxiis relatively small, 40-60 ml/kg body dative enzymes (Zimny and Bourgeois, '60).
wt/day, and may well be related to the The glomerular ultrafilter is truly a structural filter which can physically respond
width of the basement membrane.
In the kidneys of the hibernating ground to hydration and blood pressure changes.
squirrel the thickness of the basement We were fortunate in the course of this
membrane approximately doubles when study to obtain a kidney sample from an
compared with the controls. In addition to animal entering hibernation. This animal
was placed in cold room, within 24 hours
this the endothelial pores and podocytic
urinated twice the average control volume
slit pores decrease in number and size. It
and went into a torpor. Electron microis no wonder that the rate of urine forma- scopic examination of this tissue revealed
tion diminishes. Since the urinary blad- that the renal glomerulus resembled the
der is found distended when sacrificing a control. Apparently, the ultrastructural
hibernating animal, the rate of glomerular modifications found in the glomeruli of
filtration is not completely stopped but kidneys from hibernating ground squirrels
greatly reduced.
occur during hibernation and not during
The shut down of the filtering system refrigeration prior to hibernation.
during hibernation may vary in degree
It has been established that hibernation
from animal to animal. All kidneys from is not in fact continuous. Periodic arousals
hibernating ground squirrels showed a do occur (Pengalley and Fisher, '61). AS
thicker basement membrane. In some ani- a result of a structural decrease in the
mals the endothelial lining n a y appear porosity of the glomerular ultrafilter, a decontinuous while in others a few endo- crease in renal blood flow, a decrease in
thelial pores may be seen. The same is the rate of reabsorption and other comtrue of the swollen foot processes. In all plex physiological factors the rate of urine
cases, however, the porosity of the glomer- formation is decreased during hibernation.
ular ultrafilter is decreased in the hibernat- The urinary bladder, however, distends
ing ground squirrel as compared with the slowly during this time. It could well be
control.
that distention of the urinary bladder
During hibernation in the ground squir- eventually creates an autonomic stimulus,
rel the concentrations of sodium and chlo- then mediated through the hypothalamus
ride in the urine decrease and the concen- to the pituitary which initiates a reaction.
tration of potassium increases (Zimny and This reaction in addition to the accumulaBourgeois, '60). Depression of metabolic tion of metabolic end products in the blood
activity is associated with an increase in (Fisher, '64) may help to explain the
cellular hydration and tissue swelling re- mechanism of periodic arousals.
sulting from a movement of both solute
ACKNOWLEDGMENT
and water into the tissues (Leaf, '59).
When metabolism is inhibited the energy
The technical assistance of Mrs. Addie
required for ion transport is no longer avail- Kern is gratefully acknowledged.
able. Sodium diffuses into the cell, potassium leaks out of the cell, the membrane
LITERATURE CITED
potential is reduced, chloride enters the Anderson, M., and K. C. Nielsen 1955 Studies
cell, an isotonic gain of water occurs and
on the renal function under hypothermy in
rabbits. Acta Med. Scand., 251: 191-199.
the tissue swells. This process is reversible
K. C. 1964 On the mechanism of
for a time (Leaf, '59). This effect may also Fisher,
periodic arousal in the hibernating ground
apply to the podocyte whose metabolic
squirrel. Ann. Acad. Sci. Fenn. A: IV: 72:
processes presumably maintain the nor143-1 52.
mal shapes of the pedicels and trabeculae Freeman, J. A. 1964 Cellular Fine Structure.
McGraw-Hill, New York.
(Hall, '57) and to the basement mem- Hall,
B. V. 1954 Further studies of the normal
brane whose sol-gel consistency would be
structure of the renal glomerulus. Proc. Ann.
influenced by changes in ionic gradients.
Conf. Nephrotic Syndrome, 6th Conf., pp. 1-39.
90
MARILYN L. ZIMNY AND ELMORE RIGAMER
1957 The protoplasmic basis of glomerular ultrafiltration. Amer. Heart J., 54:
1-9.
Hester, R. J., and R. A. Hoffman 1964 Excretion patterns of the hibernating ground squirrel
(Citellus tridecemlineatus). Amer. Zool., 4:
295-29 6.
Hong, S. K. 1957 Renal function during hypothermia and hibernation. Amer. J. Physiol.,
188: 137-150.
Hong, S. K., and J. W. Boylan 1959 Renal concentration operation in hypothermic dogs.
Amer. J. Physiol., 196: 1150-1154.
Leaf, A. 1959 Maintenance of concentration
gradients and regulation of cell volume. Ann.
N. Y. Acad. Sci., 72: 396404.
Mueller, C. B., A. D. Mason, Jr. and D G. Stout
1955 Anatomy of the glomerulus. Amer. J.
Med., 18: 267-276.
Pease, D. C. 1955 Fine structures of the kidney seen by electron microscopy. J. Histochem. Cytochem., 3: 295-308.
Pengelley, E. T., and K. C. Fisher 1961 Rhythmical arousal from hibernation in the goldenmantled ground squirrel, Citellus lateralis
tescorum. Can. J. Zool., 39: 105-120.
Reynolds, &. S. 1963 The use of lead citrate
at high pH as a n electron-opaque stain in electron microscopy. J. Cell Biol., 17: 208-212.
Rhodin, J. 1955 Electron microscopy of the
glomerular capillary wall. Exp. Cell Res., 8:
572-574.
Yamada, E. 1955 The fine structure of the
renal glomerulus of the mouse. J. Biophys.
Biochem. Cytol., 1 : 551-566.
Zimny, M. L. 1960 Phosphates as related to
intermediary metabolism in hibernators. Bull.
Mus. Comp. Zoo. Harv., 24: 457473.
Zimny, M. L., and C. Bourgeois 1960 Histochemical localization of some enzymes i n the
kidney of a hibernator. J. Cell. and Comp.
Physiol., 56: 93-102.
PLATES
PLATE 1
EXPLANATION O F FIGURES
92
1
The renal glomerulus of a non-hibernating control ground squirrel.
Average thickness of basement membrane is 2,863 A. Basement
membrane (BM),endothelial cell lining ( E N ) and foot processes or
pedicels of podocyte (FP). x 14,400.
2
The renal glomerulus of hibernating ground squirrel number 1. Average thickness of basement membrane is 6,299 A. Basement membrane (BM), endothelial cell lining ( E N ) and foot processes or
pedicels of podocyte (FP). x 14,400.
GLOMERULAR ULTRASTRUCTURE
Marilyn L. Zimny and Elmore Rigamer
PLATE 1
93
PLATE
GLOMERULAR ULTRASTRUCTURE
Marilyn L. Zimny and Elmore Rigamer
EXPLANATION OF FIGURES
3
The renal glomerulus of hibernating ground squirrel number 3. Average thickness of
basement membrane is 4,492 A. Basement membrane (BM), endothelial cell lining
( E N ) and foot processes or pedicels of podocyte (FP). X 14,400.
4 The renal glomerulus of hibernating ground squirrel number 7. Average thickness of
basement membrane is 5,175 A. Basement membrane (BM), endothelial cell lining
(EN) and foot processes or pedicels of podocyte (FP). X 14,400.
94
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