вход по аккаунту


Histochemical correlates for differences in functional activity of kidneys from active and cold-stored summer bats (Myotis lucifugus).

код для вставкиСкачать
Histochemical Correlates for Differences in Functional
Activity of Kidneys from Active and Cold-stored
Summer Bats (Myotis hcifugus)'
Department of Pathology, Albert Einstein College of Medicine,
New York, New York
Fourteen female Myotis lucifugus from a summer colony were adapted
to laboratory conditions for periods up to 60 days. Animals were then divided and exposed to the following conditions: ( I ) active bats exposed to normal summer laboratory temperature and humidity; (11) active bats exposed to normal summer laboratory
temperature but low humidity; (111) cold-stored bats; (IV) cold-stored bats exposed
to low humidity. All cold-stored bats entered a state of apparent deep hibernation,
carrying out characteristic reflexes upon being awakened. Their thyroid glands showed
significant loss of secretory activity,
Staining for ATPase activity (method of Wachstein and Meisel) on formalinfixed sections of kidney showed that there was increased infolding as well as increased enzymic activity of the plasma membranes of the proximal convolutions from
all cold-stored bats. Proximal convolutions from kidneys of active bats showed decreases in the degree of invagination of the plasma membranes with apparently expanding basal lamellae. Acid phosphatase activity (Gomori) was confined to "phagosomes" i n the proximal convolutions of active bats; cold-stored bats showed no activity.
Staining with the periodic Schiff method showed irregular staining of the brush border
of tubules from active bats; cold-stored animals showed a regular brush border with
this method. The results suggest that the histochemical methods employed reveal differences in kidney function between the active and cold stored animals.
An apparent decrease in plasma volume
has been described for hibernating mammals such as the little brown bat, Myotis
lucifugus (Kallen, '60), and the thirteenlined ground squirrel (Hong, '58). Such a
shift in water also reflects a state of relative dehydration on the part of animals in
undisturbed hibernation when compared
with animals which are active and have
ready access to water. Hong ('58) presented evidence that decreased plasma volume filtered by the kidney accompanies a
decrease in renal blood flow during hibernation but additional quantitative information on body fluids during hibernation
appears to be lacking (Riedesel, '60).
The functional adaption of the kidney
to hemodynamic alterations in the hibernating animal could be reflected in morphological alterations or adaptations within the nephron. To date, however, no
investigations have been undertaken to investigate this possibility. The purpose of
this study was to visualize structural and
functional changes accompanying the
shifts in water described by the above physiological investigations using specific histo-
chemical methods. For this, we employed
kidneys from active and artificially coldstored bats (Myotis Zucifugus) including
some animals exposed to decreased humidity.
Fourteen female Myotis lucifugus from
a laboratory stock colony captured near
Ringwood, New Jersey during July, '62,
were employed for this study. For initial
adaptation to laboratory conditions, all
animals were maintained in a semi-darkened cage for 60 days. They were fed ad
libitum on a meal-worm and Stuart Formula mixture (Stuart Company, Pasadena,
California) and water. During this period,
the normal room temperature fluctuated
from 20-32°C; the relative humidity varied
from 65-90%.
1 Supported by grants RG-5483 and A-3605 from
the United States Public Health Service (R. M. Rosenbaum).
* Recipient of a Sergei Zlinkoff Scholarship from
the City College of the City University of New York.
Supported in part by a grant from the National
Science Foundation (grant 10833) to Dr. William
Etkin, whose guidance and stimulation is gratefully
To begin the experiments, animals were
divided into four groups (see table 1) and
placed for four weeks in individual circular “adaptation” cages 14 inches in diameter and three and one-half feet high. Following this, seven animals (groups I11 and
IV, table 1 ) were placed in a cold-box at
15°C for another week and given food and
water ad libitum. These animals remained
completely active. The seven remaining
bats (groups I and 11) were kept in cages
exposed to room temperature and humidity
(table 1).
For the final phase of the experiment,
all animals from groups I11 and IV were
removed from the 15°C cold-box and
placed at 4-6°C for one to three weeks.
Under these conditions the relative humidity ranged from 60-70%. During the first
week, animals were offered food and water
ad libitum and were active. When food
was removed, all the animals were observed to enter a n apparent state of deep
For exposure to decreased humidity, four
deeply hibernating animals (group IV)
were removed from 4-6°C and immediately placed in a cage covered with Saranwrap except for a series of holes in the
base which itself contained a calcium sulfate - calcium chloride drying mixture.
In these cages, the relative humidity never
exceeded 44% at normal room temperature. Animals were exposed to these conditions for one to four hours. Active bats
were exposed to room temperature and
humidity by a similar procedure (group
11). Control animals consisted of bats in
adaptation cages exposed to room temperature and humidity only (group I)
and cold-stored animals (group 111).
Histochemical methods. Bats were
killed with chloroform. At autopsy, the
thyroid gland and both kidneys were immediately fixed. For enzyme studies, kidneys were fixed for no longer than 24
hours in cold calcium-formalin (Baker)
and in alcohol-formol-acetic acid while
the thyroid was placed in fonnol-sublimate (10% formol -10 ml; saturated
HgCL -90 ml).
For demonstration of acid phosphatase
activity, the lead salt method of Gomori
(’53) was used employing frozen sections
with 40 minute incubation in 8-glycerophosphate-lead nitrate medium (pH 5.2)
at 37°C. ATPase activity was visualized
by the method of Wachstein and Meisel
(’57). Control sections employed heat inactivation (100°C for 30 min) or omitted
the substrates. DPNH diaphorase (DPNH
tetrazolium reductase) activity was visualized essentially by the method of Novikoff
and Masek (’58) employing the tetrazolium salt Nitro BT. Control sections omitted DPNH as substrate.
Parallel kidney sections fixed in alcoholformol-acetic acid were stained overnight
in a mixture of 2 X
M methylene
blue and lo-’ M eosin at pH 6.0 and with
the Schiff reagent following periodic acid
Alterations in environmental temperature and humidity in
four mouvs of summer bats. Myotis lucifugus
no. ani-
Av. temp.
( I ) active
(11) active,
(111) cold-stored 3
Adaptive exposure
Final exposure
Av. humid.
Exp. time
Av. temp. Av. humid. Exp. time
1 4
(IV) cold-stored,
1 4
Animals not exposed to decreased humidity were maintained under “adaptive” conditions until
the time of sacrifice.
Criteria of hibernation. Several criteria
were employed to establish that coldstored animals had entered a state of
deep hibernation. Three of the seven bats
exposed to 4°C assumed the characteristic
hanging position during the first week of
exposure to this temperature. Upon awakening from deep hibernation, all of the
cold-stored animals exhibited the typical
movements described by Eisentraut ('34),
including primitive stretching and biting
reflexes. Although we took no body temperatures, the fact that all our animals
were quiescent and survived exposure to
4°C for up to three weeks, suggested that
they assumed internal temperatures closely approximating the ambient temperature (Hock, '51).
Thyroid gland. Previous investigators
have described inhibition of the thyroid
gland during natural hibernation in mammals (Adler, '20; Deane and Lyman, '54).
In our experiments, the thyroid glands
from those bats subjected to cold-storage
possessed external follicles with a markedly flattened epithelium. The epithelium
of the internal follicles from these animals was more variable, although all the
cells appeared flattened and many showed
small nucleocytoplasmic ratios (fig. 1 ).
Thyroid follicles from normal bats had a
higher epithelium indicative of increased
glandular activity (fig. 2 ) .
Basophilia and acidophilia. Observations on the binding of acid and basic
dyes were made on kidneys from all of
the animals. At the molar concentration
and pH of the dye bath we employed,
an intense basophilia could be detected
throughout the cytoplasm of the proximal
convoluted tubules from cold-stored bats.
The least cytoplasmic basophilia was observed in proximal convoluted tubules
from active bats. Active and cold-stored
bats exposed to decreased humidity
showed variable amounts of basophilia
between the two extremes. Aside from
staining of erythrocytes, no marked acidophilia could be detected with our method.
Periodic acid Schiff reaction. Brush
borders of the proximal tubular epithelium from kidneys of cold-stored bats
stained intensely regardless of the degree
of humidity to which the animals were
exposed. Staining was uniform especially
when compared with brush borders from
active bats (compare figs. 3 and 4). In
active animals, the Schiff positive reaction
was less intense and the brush borders
stained irregularly (fig. 4).
Adenosine triphosphatase. In all sections of kidneys studied, infolding plasma
membranes of proximal convoluted tubules showed deposition of reaction product indicative of ATPase activity. Enzymic
activity was associated with membranous
structures extending from the basement
membrane toward the luminal surface of
the cells (figs. 5-8). Activity was present
regardless of whether the animals were
exposed to decreased humidity or not.
However, in cold-stored bats, infolding
membranes frequently reached to the luminal aspect of the tubular epithelium
and showed strong activity (figs. 5 and
7). In active bats, infolding plasma membranes extended from the basement membrane of the proximal convoluted tubule
approximately midway toward the luminal surface of the cell and showed less
staining for enzymic activity (figs. 6 and
8 ) . The spaces between adjacent infolding membranes appeared swollen (fig. 8).
In all sections, variable amounts of reaction product were deposited in the brush
border and basement membrane while the
tubular lumen showed heavy deposition of
reaction product (figs. 5-8). Some of
this localization of the metal-salt reaction
product could be attributed to enzymic
activity in the brush border and basement
membrane. Control sections, employing
heat inactivation or omission of substrate,
revealed that a false-positive deposition of
reaction product occurred, especially in
the lumen. Rosenbaum ('62) has discussed sites of such false-positive reactions with respect to the binding of metalsalt reaction products by tissues or tissue
products showing high acidity such as
would be present in the lumen of the
proxim a1 convolutions.
Acid phosphatase. The cytoplasm of
the proximal tubules from kidneys of all
the active bats we studied possessed
a dense concentration of droplets that
stained for acid phosphatase activity (fig.
9 ) . Activity was present regardless of the
degree of humidity to which the animals
were exposed. In proximal convoluted tubules of cold-stored bats, few if any droplets staining for acid phosphatase activity
could be detected (fig. 10).
DPNH diaphorase (DPNH-tetrazolium
reductase). We could detect no differences in localization of enzymic activity
between kidney tubules from any of the
animals studied. Intense staining for mitochondrial activity within tubules occurred throughout the kidney and there
was virtually no staining elsewhere(fig.
11). These results agreed with observations pointing to the rich mitochondrial
population within the proximal convolution visualized by essentially similar methods (Novikoff, '60).
In this study it was first of all necessary
to produce a state of deep hibernation in
our animals. It must be emphasized that
we are, at present, not certain to what
extent the physiological condition of our
cold-stored animals can be compared with
naturally deeply hibernating bats. Our
criteria for establishment of a deep hibernating condition were based on observations of other investigators dealing with
naturally and artificially hibernating mammals, including bats (Adler, '20; Eisentraut, '34; Deane and Lyman, '54).
Evidence for changes in water balance
and plasma volume during hibernation
and hypothermia comes from studies on
the little brown bat, Myotis lucifugus (Kallen, ' 6 0 ) ; the 13-lined ground squirrel,
Citellus tridecemlineatus (Hong, '57), the
hamster (Lyman et al., '57) as well as
other species (Riedesel, '60). However,
virtually no morphological study has appeared which might contribute to an understanding of the ability of the kidney
cells to cope with such alterations. In our
study, therefore, we hoped that possible
functional differences between kidney tubules from animals in artiiicially induced
deep hibernation and active summer bats
could be demonstrated and that these differences might be emphasized by exposure
to decreased humidity.
The most striking observation in our
study was the marked difference between
cold-stored and active bats in the appearance of infolding plasma membranes at
the base of the proximal convoluted tubule. Cold-stored bat kidney tubular cells
showed a greater infolding of the plasma
membrane with greater ATPase activity.
The value of the method of Wachstein
and Meisel ('57) for visualizing ATPase
activity, especially in plasma membranes
of proximal convoluted kidney tubules,
was shown by Spater et al., ('58). The
pattern of staining for ATPase activity in
tubular epithelium of the cold-stored bat
may reflect something like the closely
packed plasma membranes and close contact of basal lamellar structures in poorly
functioning rat kidneys as visualized with
electron microscopy (see text fig. l A ,
Ruska et al., '57). Similarly, the localization of ATPase activity in tubular epithelium from active bats may reflect an
extension of basal lamellar structures and
widening of the intercellular compartments similar to that described for the
actively functioning rat kidney (Ruska
et al., '57). Ruska et al., ('57) and
Rhodin ('58) among others, consider that
the lamellar structures at the base of the
proximal convoluted tubule have a resorptive function with the intercellular space
serving as a pathway for fluid resorbed at
the brush border. It is not yet possible
to state whether our morphological findings in the bat can be equated with poor
functional capacity. Indeed, it is possible
that in the cold-stored bat resorption
might be increased efficiently to compensate for a decreased circulation (Hong,
Some physiological data has been presented by other investigators which appear related to our morphological observations. Hong ('58) has shown a decreased
glomerular filtration rate in hypothermic
rats and hibernating ground squirrels. If
a similar decreased filtration can be expected in the cold-stored bat, then increases in the degree of invagination of
infolding plasma membranes could reflect an increased efficiency for resorption
of fluid passing from the glomerulus into
the tubular lumen, through the proximal
tubular epithelium via the brush border
and thence through the intercellular lamellae to adjacent capillaries. In active
mammals which possess a greater plasma
and blood volume (Hong, '58; Kallen, '60)
and a greater quantity of glomerular filtrate (Hong, ’ 5 8 ) , decreases in the infolding of basal lamellae of the proximal convoluted tubule such as we described may
be a mechanism associated with less of a
need for resorptive efficiency.
It is not yet possible more than to speculate on whether alterations of the basal
lamellae such as we described are the result of direct mechanical expansion caused
by increased hydrostatic pressure of reabsorbed fluid from the cell into the lamellar spaces (Ruska et al., ’57), or an active
movement of plasma membranes mediated
by intrinsic metabolic activity as suggested
by Hall (’57).
Our observations on other histochemical differences between proximal tubules of active and cold-stored bats may not
be directly related to phenomena revealed
with ATPase activity. The presence of
numerous mitochondria situated between
the interdigitating extensions of cells of
the proximal convolution has been described by Rhodin (’58). In our material,
staining for DPNH-tetrazolium reductase
activity presumably revealed no differences in localization of primarily mitochondrial-associated enzyme. Although acid
phosphatase activity was present in tubules from all animals, it was always diminished in tubules from cold-stored bats
while intense activity was present in kidneys of active animals. In rat kidneys,
similar appearing cytoplasmic bodies, originally termed “phagosomes” (Straus, ’61),
possess acid phosphatase activity identifiable by biochemical methods (Straus, ’57).
These have been shown to contain acid
phosphatase by histochemical methods
(Novikoff, ’60; Miller, ’62). No differences
in enzymic activity were detected in active
bats, regardless of whether they were exposed to decreased humidity or not but
the hibernating bats showed less activity.
We presume that differences in the number of such intracellular inclusions possessing enzymic activity refers to differences in protein uptake, since our deeply
hibernating animals did not feed.
The non-enzymic histochemical methods
we employed in this study showed other
differences between active and cold-stored
animals. The disruption of the brush
border in the proximal convoluted tubule
stained with periodic acid Schiff reagent
was so consistent in active bats that we
consider it to show a real difference in this
region. A “disrupted” brush border may
reflect a loose arrangement of the microvilli due to spaces produced by increased
water resorption. Final proof of this, however, must depend on electron microscopy.
Similarly, our observations with controlled
pH staining showed differences that are,
at present, difficult to interpret. The increased cytoplasmic basophilia of tubules
from cold-stored bats, regardless of their
state of dehydration, may represent concentration of globulins or similar serum
protein resulting in increased binding of
basic dyes. Increases in the levels of serum protein are known to occur in hibernating hamsters (South and Jeffay, ’58).
Female bats (Myotis Zucifugus) from a
summer colony were adapted to laboratory
conditions for several weeks following
which they were divided into groups and
treated as follows: (1) active bats exposed
to normal summer laboratory temperature
and humidity; (11) active bats exposed to
normal summer laboratory temperature
but conditions of low humidity; (111) coldstored bats; (IV) cold-stored bats rapidly
exposed to low humidity. Criteria for deep
hibernation included the presence of typical reflexes upon awakening and histological examination of the thyroid gland.
Frozen sections of formalin-fixed kidneys, stained with the method of Wachstein
and Meisel for ATPase activity, showed
localization of enzymic activity in the
basal cell membranes of the proximal convolutions. With active bats, there was
decreased infolding of basal membranes
of the tubular epithelium and apparent
decreased ATPase activity. Proximal convoluted tubules from cold-stored bats
showed an increase both in the degree of
infolding and enzymic activity of the basal
inf oldings.
The periodic acid Schiff reaction revealed irregular staining of the brush border of the proximal convolutions from active bats. Cold-stored animals showed intense, regular staining of the brush border.
Acid phosphatase activity was present
in proximal convoluted tubules from all
the bats studied, enzymic activity being
restricted to intracellular “phagosomes.”
These were more numerous in cells from
kidneys of active animals. There was
virtual absence of phagosomes in kidneys
from cold-stored bats. Following staining
with acid and basic dyes at controlled pHs,
the cytoplasm of proximal tubules from
active bats showed the least basophilia.
Cells from kidneys of cold-stored animals
showed the most basophilia.
Physiological studies on alterations in
the water balance of normal, hypothermic
and hibernating mammals serve to suggest that the morphological observations
presented in this study reflect differences
in kidney function between active and
hibernating bats.
Adler, L. 1920 Schiilddriise und Warmeregulation. Untersuchungen an Winterschlser.
Arch. Exp. Path. Pharm., 86: 159-244.
Deane, H. W., and C. P. Lyman 1954 Body
temperature, thyroid and adrenal cortex of
hamsters during cold exposure and hibernation, with comparisons to rats. Endocrin., 55:
Eisentraut, M. 1934 Der Winterschlaf der
Fledermouse mit besonderer Beruchsichtigung
der Warmeregulation. 2. Morph. Okol. Tiere.,
29: 231-267.
Gomori, G. 1952 Microscopic Histochemistry.
U. of Chicago Press.
Hall, V. 1957 The protoplasmic basis of
glomerular ultrafiltration. Am. Heart J., 54:
Hock, R. 1951 The metabolic rates and body
temperature of bats. Biol. Bull., 101: 289-299.
Hong, K. S. 1958 Renal function during hypothermia and hibernation. Am. J. Physiol.,
188: 137-150.
Kallen, F. C. 1960 Vascular changes related
to hibernation in the vespertilionid bat Myotis
Zucifugus. Bull. Mus. Comp. Zool. Harvard
coi., 124: 373-386.
Lyman, C. P., L. P. Weiss, R. C. O’Brien and
A. A. Barbeau 1957 The effect of hibernation
on the replacement of blood in the golden
hamster. J. Exp. Zool., 136: 471-486.
Miller, F. 1962 Acid phosphatase localization
in renal protein absorption droplets. Proc. 5th
Int. Cong. Electron Microscopy, vol. 2, p. Q-2.
Novikoff, A. B., and B. Masek 1958 Survival
of lactic dehydrogenase and DPNH-diaphorase
activities after formol-calcium fixation. J.
Histochem. Cytochem., 6: 217.
Novikoff, A. B. 1960 The rat kidney: Cytochemical and electron microscopic studies, in
“The Biology of Pyelonephritis” (ed. by E. L.
Quinn and E. H. Kass), Little Brown and Co.,
Riedesel, M. 1960 The internal environment
during hibernation. Bull. Mus. Comp. Zool.
Harvard Col., 124: 421-435.
Rhodin, J. 1958 Anatomy of kidney tubules.
Intern. Rev. Cytol., 7: 485-534.
Rosenbaum, R. M. 1962 Intracellular localization of enzymes, in “Handbuch der Histochemie” (ed. by K. Neumann and W. Grauman),Bd. VII/3.
Ruska, H., D. Moore and J. Weinstock 1957
The base of the proximal convoluted tubule
cells of rat kidney. J. Biophys. Biochem. Cytol.,
3: 249-254.
South, F., and H. Jeffay 1958 Alterations in
serum proteins of hibernating hamsters. Proc.
SOC.Exp. Biol. Med., 98: 885-887.
Spater, H. W., A. B. Novikoff and B. Masek
1958 Adenosine triphosphatase activity in
the cell membranes of kidney tubule cells. J.
Biophys. Biochem. Cytol., 4: 765-770.
Wachstein, M., and E. Meisel 1957 Histochemistry of hepatic phosphatases at a physiologic
pH. Am. J. Clin. Path., 37: 13-23.
Central thyroid follicles from a summer bat stored at 4°C for three
weeks and deemed in a state of deep hibernation. The follicular epithelium is low, and flattened nuclei are evident. Hematoxylin and
eosin. 130 x .
2 Central thyroid follicles from an active bat. The follicular epithelium is cuboidal and nuclei are rounded. Hematoxylin and eosin.
130 X .
3 Proximal convoluted tubules from a kidney of a bat stored a t 4°C
and subjected to low humidity for one hour. The section, fixed i n
alcohol-formol-acetic acid, was stained with the periodic acid Schiff
method. Note the relatively intense and uninterrupted staining of
the brush border. X 130, green filter.
4 Proximal convoluted tubules from a kidney of an active bat exposed
to low humidity for one hour. Note irregular staining of the brush
border. PAS with hematoxylin counterstain. 130 X, green filter.
Arnold Melman and Robert M. Rosenbaum
ATPase activity i n proximal convoluted tubules from a bat stored in
the cold at 4°C for three weeks. The animal was subjected to low
humidity for three hours at room temperature. Strong ATPase activity delineates membranes of the intercellular basal lamellae, many
of which extend from the basement membrane ( b m ) nearly to the
luminal surface (1) of the cell. Nuclei ( n ) are unstained. In this
and the following three figures, a false-positive deposition of reaction product appears in the brush border, the basement membrane
and, especially, within the tubular lumen. Cold-form01 calcium fixation, method of Wachstein and Meisel ('57). 250 X .
ATPase activity i n proximal convoluted tubules of a n active bat exposed to low humidity for four hours. Compare with figure 5. Staining of the membranes shows that there is a decrease in the degree
of invagination and the intercellular compartments between basal
lamellae appear swollen. 250 X.
Proximal convoluted tubule from a cold-stored bat treated as in figure
5. Strong ATPase activity is present in the basal membranes (arrows)
which extend for variable distances toward the lumen (1). Cold calcium-formalin fixation, method of Wachstein and Meisel. 480 x.
Frozen section of a kidney from a n active bat treated as in figure
6. There is less staining for ATPase activity and the degree of infolding seen i n figure 7 is not present. Arrows indicate what appear to
be swollen interlameller spaces some of which extend nearly to the
lumen (1). Cold calcium-formalin fixation, method of Wachstein
and Meisel. 480 x.
Arnold Melman and Robert M. Rosenbaum
Cold calcium-formalin fixed frozen section of kidney from a n active
bat stained following a 40 minute incubation i n the lead nitrateglycerophosphate method of Gomori at pH 5.2. Large numbers of
droplets within the proximal convoluted tubule stain for acid phosphatase activity. 230 x.
Cold calcium-formalin fixed frozen section of kidney from a bat coldstored for three weeks and subjected to decreased humidity for
three hours. Stained as in figure 9. Acid phosphatase activity is
present in only a few droplets within the proximal convoluted tubule
(arrows). 230 X.
Cold calcium-formalin fixed frozen section of a kidney from a n active bat stained for DPNH diaphorase activity with the method of
Novikoff and Masek ('58). Staining of mitochondria is evident. No
difference between the localization of enzymic activity could be
detected in any of the animals studied. Nuclei show no activity.
450 X.
Arnold Melman and Robert M. Rosenbaum
Без категории
Размер файла
900 Кб
differences, storey, kidney, batse, histochemical, lucifugus, correlates, activ, activity, cold, function, summer, myotis
Пожаловаться на содержимое документа