close

Вход

Забыли?

вход по аккаунту

?

The effect of alloxan and alloxan-induced diabetes on the kidney.

код для вставкиСкачать
THE ANATOMICAL RECORD 208:33-47 (1984)
The Effect of Alloxan, and Alloxan-Induced Diabetes
on the Kidney
ANDREW P. EVAN, STEPHEN A. MONG, BRET A. CONNORS,
GEORGE R. ARONOFF, AND FRIEDRICH C. LUFT
Departments of Anatomy and Medicine, Indiana University School of
Medicine, Indianapolis, IN 46223
ABSTRACT
Alloxan is known to induce diabetic renal changes as well as
causing nephrotoxic alterations. However, no ultrastructural study has been
performed to differentiate diabetic verses toxic affects of alloxan to the tubule
and/or glomerulus. Therefore the present study used the “protected” kidney
model to prevent one kidney from being exposed to the alloxan while allowing
the other to receive the drug immediately. In all experimental animals the
right renal hilum was gently occluded for 5 minutes and then released. This
was performed prior to the injection of alloxan. Subsequently, the left renal
hilum was occluded at the time of, and for 5 minutes after, alloxan adminstration (40 m g k g i.v.). The experimental rats were divided into three groups:
untreated diabetics, diabetics treated with protamine-zinc-insulin, and alloxantreated rats that failed to become diabetic. Three groups of controls were
included: one group received a n equal volume of saline diluent as the experimental rats but no clamping of either renal hilum; another group received the
saline and had the left renal hilum occluded for 5 minutes; and a third group
had both the right and left renal hila occluded. All animals were followed and
sacrificed after 9 weeks. Endogenous creatinine clearance did not change
among groups. Alloxan-treated nondiabetic rats displayed marked interstitial
nephritis in unprotected kidneys, while protected kidneys were normal. The
diabetic state resulted in mesangial proliferation and focal glomerular basement membrane thickening as well as glomerular capillary endothelial abnormalities and visceral epithelial foot-process fusion. The endothelial changes
consisted of focal areas showing a reduction in the size of endothelial fenestrae.
All glomerular changes were ameliorated by insulin treatment. We conclude:
1)alloxan per se is distinctly nephrotoxic; and 2) the glomerular endothelium
and epithelium are involved early in the course of experimental diabetes.
Diabetes mellitus is responsible for wellrecognized renal functional and ultrastructural changes in man and experimental animals (Mogensen, 1976; Osterby, 1974). In
animal models, diabetes is generally induced
by the administration of agents that are toxic
to pancreatic islet cells. Both alloxan and
streptozotocin are useful for this purpose;
however, both of these materials are also capable of causing renal damage apart from
that created by the diabetic state (Rerup,
1970). Light microscopic studies by Orskov
et al. (1965) and Vargas et al. (1970) have
shown both tubular and interstitial changes
0 1984 ALAN R. LISS, INC.
in the nonprotected kidney of a n alloxaninjected rat that would appear to be the result of direct alloxan damage to that kidney.
In particular, Vargas et al. (1970) described
cystic tubular dilations of the nephron leading to a spongy appearance of the cortex
which was related to alloxan toxicity.
More recent studies have employed both
transmission and scanning electron microscopy to follow changes in the kidney during
alloxan-induced diabetes (Hagg, 1974; Hagg
Received May 24, 1983;accepted September 2,1983.
34
A.P. EVAN ET AL.
and Winbland, 1975; Evan and Luft, 1980;
Bell et al., 1980). However, no study has differentiated the fine structural changes that
may result from alloxan toxicity versus diabetes. In the present experiments, we have
extended our ultrastructual observations of
alloxan diabetic rats to animals in which one
kidney was protected by means of a postalloxan-injection, renal-hilar-clamping technique.
METHODS
General
Male Sprague-Dawleyrats, initially weighing 200-225 gm, were used in the present
study. For the induction of diabetes, the animals were first anesthetized with pentabarbital.
All rats were allowed free access to tap
water and a standard rat diet (Purina). Following assignment to their groups, the rats
were placed in metabolic cages for 24-hour
urine collections. Blood was also obtained at
this time from the tail vein. These collections
were repeated at weeks 1 through 8, following which the rats were sacrificed for morphological studies. No more than three
deaths occurred prior to sacrifice in any
group. Only animals which were studied at
every time point were included in the data
analysis.
Alloxan- Treated Animals
diabetic. The last group was also diabetic,
but these animals were given protamine-zinc
insulin (Eli Lilly & Company) as a daily subcutaneous injection (1-5 units). The amount
of the insulin dose was adjusted for each animal so that their plasma and urine glucose
concentrations were maintained at control
levels.
Control Animals
Three groups of control animals were used
with 20 rats per division. The first group of
rats were age-matched controls that received
1ml of 0.9% saline solution by tail-vein injection but did not have either renal hilum
clamped. Another group had only the left
renal hilum occluded for 5 minutes in addition to receiving 1 ml of the saline solution.
The third group had the right renal hilum
occluded for 5 minutes followed by clamping
of the left hilum for 5 minutes while receiving 1ml of the saline solution.
An occlusion time of 5 minutes was chosen
for several reasons. First, it has been shown
that the concentration of alloxan rises rapidly in the plasma, kidney, and pancreatic
islets during the first 5 minutes after a bolus
injection. The concentrations in the plasma
and kidney then falls quickly over the next 2
hours but not in the pancreas (Janes and
Winnick, 1952; Hammarstrom and Ullberg,
1966; Bilic and Felber, 1969). Therefore the
5-minute interval would prevent the clamped
kidney from accumulating the drug while
allowing the other kidney t o excrete the compound. The clamped-protected kidney would
be exposed to alloxan after the time of clamp
removal; however, no toxicity to that organ
was observed, according to our morphological
results. Second, alloxan causes most of its
tissue necrosis, particularly in the pancreas,
within 5 minutes of injection (Hughes et al.,
1944). Third, Orskov et al., (1965) and we
(unpublished observations) have noted that
if the occlusion time was kept to 5 minutes
or less, there was minimal postoperative
mortality and a high percentage of animals
developed insulin-dependent diabetes. Last,
several authors have previously shown that
a 5-minute or shorter occlusion time was sufficient to protect the kidney from alloxaninduced nephrotoxicity (Orskov et al., 1965;
Arteta, 1952).
After anesthesia, both renal hila were exposed via a midline abdominal incision. First,
the right renal hilum was gently occluded for
5 minutes via a spring-clamp. The occlusion
was released and then applied t o the left
renal hilum as alloxan monohydrate (Sigma)
at 40 mgkg in 0.9% saline solution was rapidly injected as a bolus into the vena cava.
After 5 minutes the clamp was removed and
the incision closed in two layers. The 40-mgl
kg dose and bolus injection protocol was used
because of its effectiveness in inducing diabetes in the rat compared to giving several
smaller doses over time which creates a subdiabetic state. Eighty animals received alloxan, and 4 days later the presence of
diabetes was verified by determining the
nonfasting serum glucose level. Only those
rats with a glucose concentration of at least
250 mgldl were considered diabetic. At this
time, three separate groups were formed. The
Fixation Procedures
first group contained animals that had reAll
kidneys
were preserved by in vivo perceived alloxan but developed neither glycosuria nor hyperglycemia and therefore were fusion. Following pentabarbital anesthesia,
termed nondiabetic alloxan-treated. The sec- the abdominal cavity was opened and a polyond group was made up of animals that were ethylene catheter was introduced into the
ALLOXAN-INDUCED DIABETES
abdominal aorta below the renal arteries.
Using 100 mm Hg pressure, 30 ml of 0.9%
NaCl was perfused through the catheter followed by 150 ml 2.5% glutaraldehyde in
0.075 M cacodylateEIC1 buffer, pH 7.4. Following fixation, portions of the outer and inner cortex were removed and further fixed in
the original fixative for a n additional 48
hours. At this time, all specimens were assigned coded numbers and examined without
knowledge of the regimens. One-millimeter
cubes of tissue were routinely prepared for
transmission electron microscopy and viewed
with a Philips EM-400 electron microscope.
Larger pieces were washed in the buffer for
90 minutes, dehydrated through a series of
graded alcohols to 100%ethanol, fractured in
liquid nitrogen, transferred to a Samdri-critical point dryer and dried with liquid COz.
Tissues were attached to a n aluminum stub
and placed in a sputter coater (Hummer V)
and coated with gold-palladium. Specimens
were examined on a n AMR-1000A scanning
electron microscope. Sections were also obtained for light microscopy and were stained
by hematoxylin and eosin or by the periodic
acid-Schiff (PAS) reaction.
35
In addition to the semiquantitative assessments, we also performed quantitative measurements to determine basement-membrane
thickness. This determination was accomplished using a computer digitizing system
and Quantigraph program (Novus Instruments, Carmel, IN). Thickness of the glomerular basement membrane was determined
from transmission electron micrographs
printed at a fixed magnification of x 30,000.
Approximately ten micrographs were obtained from each of five randomly selected
glomeruli per kidney. Ten measurements
were made along a 10-mm length of normal
or thickened glomerular basement membrane. Only those lengths of basement membrane were used which appeared perpendicular to the capillary wall. All measurements were obtained on a line orthogonal to
the edge of the glomerular basement membrane starting at the endothelial surface. The
line that was measured extended from the
endothelial to the epithelial side.
Chemical Analysis
At weekly intervals, animals were placed
in metabolic cages in order to obtain a 24hour urine collection. A blood sample was
Tissue Analysis
also obtained from the tail vein. Both the
In order to obtain a statistical comparison blood and urinary glucose levels were deterof some of the changes noted in the kidneys mined on a Beckman Glucose I1 analyzer
of the different experimental groups as seen (Beckman Instruments, Inc., Creve Coeur,
by light, transmission, and scanning electron MO). The creatinine levels were measured
microscopy, we employed a semiquantitative with a Beckman Creatinine I1 analyzer. The
histologic analysis (Bohman et al., 1979; Pir- data were analyzed by means of repeated
ani et al., 1964). The severity and distribu- analysis of variance and Student’s t-test as
tion of each specific lesion was scored by the indicated. The 95% limits of probability were
following scale: 0 = absence of the lesion, 1 accepted as significant.
= lesion represented in less than 10% of the
nephrons and with minimal change, 2 = leRESULTS
sion represented in up to 50% of the nephThe plasma glucose concentrations appear
ions and with moderate change, 3 = lesion
represented in 50 to 90% of the nephrons in Figure 1. Untreated diabetes resulted in
with moderate change, and 4 = lesion repre- marked hyperglycemia throughout the pesented in over 50%of the nephrons and with riod of observation. The range of plasma glusevere change. The lesions observed by light cose levels during the last week was 280-628
microscopy included tubular atrophy, tubu- mg/dl. Insulin treatment brought blood sugar
lar dilation, interstitial nephritis, glomeru- values into the normal range except during
lar sclerosis, and distal tubular vacuolation. week 4. The control group in which both
Scanning and transmission electron micros- renal hila were clamped had values of 90copy were used to examine the visceral epi- 150 mg/dl throughout the experiment. Those
thelium to detect podocyte loss or fusion, the animals that received alloxan but had
glomerular endothelium for alterations of the plasma glucose concentrations similar to that
fenestral diameter and mesangial cells for a of the controls, formed a separate group, the
change in number. A mean diameter of indi- nondiabetic alloxan-treated animals. The
vidual endothelial fenestra was calculated age-matched controls, and controls with right
after determining both a maximum and min- hilar clamping only, had similar values as
imum caliper diameter off of micrographs those control animals in which both renal
hila were occluded.
printed a t a final magnification of 30,000.
36
A.P. EVAN ET AL.
500 -.
400---
300-
-
-I
0
\
-E"
-
u
g 200-
->
CI
0
0
-
E
h
100 -.
0
1
2
3
4
5
6
7
6
Weeks
0 Diabetes Untreated
0 Diabetes Treated
A Alloxan
No Diabetes
0 Control w/Both Renal Hila Clamped
-
Fig. 1. Plasma glucose concentrations of diabetic, nondiabetic alloxan-treated,
insulin-treated diabetic, and control rats.
Table 1outlines the effects on body weight,
urine volume, creatinine clearance and glucose excretion. Only data from week 0 and
the week of sacrifice are shown. Increases in
body weight were observed in every group (P
< .001); however, the increase was least in
the diabetes untreated group (P < .05). The
diabetic state resulted in polyuria, which was
ameliorated by insulin treatment (P < .001).
No significant interactions between treatment and creatinine clearance were observed. Urine glucose excretion was in the
range of 7,500 mglday in untreated diabetic
animals. Treatment brought glycosuria to
within the range observed in all control rats.
Figures 2-7 are light photomicrographic
sections from the protected and nonprotected
kidneys of nondiabetic animals killed 8
weeks following alloxan treatment. The entire protected kidney appears normal (Figs.
2, 4) while the unprotected kidney shows
areas of normal tubules adjacent to extensively injured tubules (Figs. 3, 5-7; Tables 2,
3). Damage to the nephron was noted by tubular atrophy, dilation, and glomerular sclerosis (Figs. 3, 5-7). A few tubules were filled
37
ALLOXAN-INDUCED DIABETES
TABLE 1. Changes in body weight, urine volume, creatinine clearance, and glucose excretion at week 0 compared to
week 8 in all experimental and control groups (mean f SEM)
Group
Diabetes
Untreated
Insulin-treated
Nondiahetic
Alloxan-treated
Control,
Both renal hila clamped
Left hilum clamped
Age-matched
(n)
Week 0
Variable
Weight (gm)
Urine volume
(m1124 hr)
Creatinine clearance
(ml/min)
Urine glucose
(mg/24 hr)
(17) Weight (gm)
Urine volume
(m1/24 hr)
Creatinine clearance
(mlimin)
Urine glucose
(mg/24 hr)
(13) Weight (gm)
Urine volume
(mli24 hr)
Creatinine clearance
(mlimin)
Urine glucose
(mgi24 hr)
(20) Weight (gm)
Urine volume
(m1124 hr)
Creatinine clearance
(mlimin)
Urine glucose
(mgi24 hr)
(20) Weight (gm)
Urine volume
(m1124 hr)
Creatinine clearance
(mlimin)
Urine glucose
(mg/24 hr)
(10) Weight (gm)
Urine volume
(mli24 hr)
Creatinine clearance
(mlimin)
Urine glucose
(mgi22 hr)
with cast material (Fig. 5). An interstitial
nephritis characterized by a cellular infiltration (mainly lymphocytes) and fibrosis was
associated with the tubular injury and extends from the cortex to the medulla of the
kidney. Figure 7 shows the same area by
both light microscopy and transmission electron microscopy of an unprotected kidney obtained from an animal that received alloxan
but did not develop diabetes. A proximal tubule recognized by apical microvilli shows
considerable evidence of atrophy, noted by its
reduced size. The individual cells present an
Week 8
222
I
8
308
f
22
110
f
12
128
+
8
0.535 f
8,720
218
103
0.10
f 1,340
7,495
8
12
421
22
f
i
0.554 f
0.2
f 1,600
8,800
0.481 t
0.04
t 1,160
*
0.577
18
3
0.09
86
f
29
230
k
9
472
f
15
10
f
1
13
f
2
0.547
0.12
0.513 f
0.05
74
I
17
68
k
9
217
5
3
498
i
14
t
5
12
f
1
7
-
0.528 f
0.10
0.581 t
+
9
82
k
f
3
423
f
f
1
9
i
1.67 f
0.2
107
202
7
2.79 5
0.06
20
6
1
0.3
8
f
1
12
f
1
208
9
f
4
5
430
6
I
8
1
i
1.58 f
0.3
6
1
+
5
2.01 t
10
f
0.3
2
apparent reduction in the number of organelles and interdigitating processes. The interstitium contains numerous cells not
normally found there as well as an increased
amount of collagen material. Scanning electron microscopy reveals a loss of microvilli in
approximately 10% of the injured proximal
tubules from the unprotected kidney when
compared t o the protected of the same nondiabetic animal (Figs. 8,9). The renal corpuscles from the protected kidney are normal by
both transmission and scanning electron microscopy except for pericapsular fibrosis. The
Fig. 2. Light micrograph showing the cortex and medulla of the left (protected) kidney from an animal that
did not develop diabetes, sacrificed 8 weeks after receiving alloxan. No glomerular (G) or tubular alterations
were noted. x 10.
Fig. 3. Light micrograph showing a similar region of
the right kidney from the same animal a s in Figure 2.
Areas of tubular atrophy and interstitial nephritis extend from the cortex to the outer medulla (arrows). Some
tubules are dilated (D), and contain cast material within
their lumens. x 10.
Fig. 4. Higher-magnification light micrograph froin
the same kidney as in Figure 2. No obvious changes are
noted in the renal corpuscle, tubular segments, or interstitium. x25.
Fig. 5. Higher-magnification light micrograph from
the same kidney as in Figure 3. Normal proximal tubules (P)are seen adjacent to atrophic tubular segments
(arrow). A tubule filled with cast material is seen. ~ 2 5 .
Fig. 6. A plastic thick section from the same kidney
as in Figure 3. Normal proximal tubules are seen next
to atrophic tubular segments (arrow). The injured tubules are surrounded by an interstitial nephritis which is
characterized by a cellular infiltrate and fibrosis. Some
of the injured proximal tubules show almost a complete
loss of their brush border (double arrow). x80.
40
A.P. EVAN ET AL.
Fig. 7. A light microscopic and transmission electron
micrograph (TEM) from an unprotected (right) kidney of
an animal that did not develop diabetes, sacrificed 8
weeks after receiving alloxan. A low-magnification light
microscopic inset shows the same tubules seen in the
TEM. Two atrophic segments are seen near a cortical
collecting tubule (CT). One of the injured tubules can be
identified as a proximal tubule (double arrow) due to the
presence of apical microvilli. This tubule is considerably
smaller than the more normal appearing proximal tubules (PI. The other atrophic segment (arrow) is difficult
to classify. TEM, X 3,800; LM, ~ 7 5 .
41
ALLOXAN-INDUCED DIABETES
TABLE 2. Semiquantitative changes in renal architecture: Mean pathological score
Control
Chance
Tubular atrophy
Tubular dilation
Interstitial
neDhritis
Glomerular
sclerosis
Distal tubular
vacuolation
Foot process loss
Fenestral diameter
reduction
Mesangial cell
proliferation
Nondiabetic
Diabetic
Treated diabetic
Rt
Lf
Rt
Lf
Rt
Lf
Rt
Lf
0.00
0.00
0.00
0.00
3.25
o.oo*
O.OO*
3.50
2.50
0.00
2.00
o.oo*
3.00
2.00
0.00';
0.00
0.00
3.75
3.50
0.00'
3.50
o.oo*
0.00
0.00
3.00
o.oo*
o.oo*
3.25
0.50*
3.00
0.25*
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
3.25
2.25
3.00
2.00
1.00
0.00
0.50
0.00
0.00
0.00
0.00
0.00
1.25
1.50
0.00
0.25
0.00
0.00
0.00
0.00
2.50
2.00
0.00
0.00
o.oo*
"N represents the number of animals used. The control group includes all three control groups: age-matched, right hilum clamp
Ynly, both right and left hila clamp.
Significant difference right vs. left or left vs. right (P< .05). Five sections from each kidney were examined.
TABLE 3. Basement membrane thickness (BMT) for
right (Rt) and left (LD kidneys BTM (nm)
Group
Controlb
Nondiabetic
Diabetic
Treated diabetic
Rt
Lt
Na
193 2 3'
195 k 5
190 i 5
235 k 3*
195 4
10
188 k 4
241 + 6*
200
+7
+
10
10
10
sN represents the number of animals used.
bIncludes only those animals in which both renal hila were
clamped.
'Values expressed as the mean f SEM.
"P < .05 for difference between control and diabetic animals.
mesangial cells, filtration barrier, and glomerular vasculature exhibit no changes. The
protected kidneys from the alloxan nondiabetic group resembles that of the control
group (Tables 2, 3; Figs. 2, 4).The data from
all three control groups were combined in
that no structural changes were found in any
of the kidneys. The 5-minute clamping period did not appear to induce chronic injury
to the tubules or glomeruli.
Diabetic untreated animals also exhibited
severe interstitial nephritis in the unprotected kidney after 8 weeks (Table 2). However, in protected kidneys, interstitial nephritis was not a feature. The cytoplasm of
tubular epithelium from some distal tubules
and collecting ducts from the protected kidney exhibited large clear vacuoles (PAS-negative) by light microscopy which by
transmission electron microscopy had a flocculent appearance. These electron microscopic changes are shown in Figure 10. In
addition the protected kidney from the untreated diabetic animals possessed glomeruli
that exhibited a n increased number of mesangial cells and focal areas of glomerular
basement membrane thickening and podocyte fusion (Fig. 11).
The four panels of Figure 12 display the
glomerular endothelial surface from control
and experimental groups as viewed by scanning electron microscopy. Panel A is from a
control animal in which both the right and
left hila were clamped. The en$othelial fenestrae are approximately 750 A in diameter. Occasional cytoplasmic ridges are observed. Panel B shows the glomerular endothelium from the protected kidney of a n animal after eight weeks of untreated diabetes.
There are areas of the capillary where the
endothelial fenestrae sbow a reduction in size
to as small as 350 A in diameter. These
changes are focal in that a capillary loop may
possess both normal and abnormal cells (Table 3). The semiquantitative data reveals the
relative increase or absence of injured endothelial cells. Small spherical structures,
which appear to be platelets, can be seen
attached to the luminal surface of the endothelial cells. Further injury of the endothelium is seen in panel C, where denuded areas
are found along the capillary wall. These cells
are from the same animal illustrated in panel
B. Panel D is from a n insulin-treated animal,
and all endothelial cells resemble those of
the control animals (Table 3).
Figure 13 shows the visceral epithelial surface of a n untreated diabetic animal. Panel
A shows a control animal and reveals numerous slender interdigitating foot processes.
However, the untreated diabetic animals
possess focal areas of clearly blunted and
42
A.P. EVAN ET AL.
Fig. 8. Scanning electron micrograph of a proximal
tubule from a protected (left) kidney of an animal that
did not develop diabetes, 8 weeks after receiving alloxan.
The apical surface presents an elaborate arrangement of
microvilli. ~ 3 , 0 0 0 .
Fig. 9. A proximal tubule from the unprotected (right)
kidney from the same animal as in Figure 8. These cells
show an extensive loss of microvilli (arrow). X3,OOO.
fused foot processes (B). Such changes are not
observed in animals of the nondiabetic group
or in those which received insulin treatment
(Table 3).
Tables 2 and 3 summarize by both semiquantitative and quantitative analysis the
changes in renal morphology just described.
By light microscopy it was noted that only
the right, or unprotected, kidney from all
animals receiving alloxan showed changes
consisting of tubular atrophy and dilation,
interstitial nephritis and glomerular sclerosis. These data plus the fact that the left,
or protected, kidney from the nondiaetic rats
showed no alteration clearly points out the
nephrotoxic effects that alloxan has by itself
on the kidney.
The diabetic changes are obvious when the
protected kidney from the nondiabetic animal is compared to that of the diabetic rat.
The protected kidney from the diabetic animals showed glomerular changes which in-
cluded a loss of the epithelial foot processes,
reduction in the diameter of the endothelial
fenestrae (Table 21, increase in basement
membrane thickness and proliferation of
mesangial cells (Table 3), as well as vacuolation of the distal portion of the nephron.
Fig. 10. Cortical collecting tubule from the protected
kidney of an untreated diabetic animal sacrificed 8 weeks
after the alloxan injection. Most cells of this tubule are
abnormal in that they display a lucent cytoplasm with
many organelles (arrows) positioned at the cell periphery. ~ 2 , 0 0 0 .
Fig. 11. Transmission electron micrograph of a portion of a glomerulus from the same kidney as in Figure
10. Two capillary loops are seen in cross section. The
basement membrane of the capillary on the right is
generally uniform in width while the one on the left
shows several areas (arrows) of increased thickness. The
foot processes (P) of both loops are irregular in width.
x 12,000.
Fig. 12. A) Scanning electron micrograph of glomerular endothelium from the left kidney of a control animal
in which both renal hila were clamped for 5 minutes;
the animal was sacrificed 8 weeks later. The cells display
numerous large fenestrae (arrow) and occasional cytoplasmic ridges (C). x 18,000. B) Glomerular capillary
from the protected kidney of an untreated diabetic rat
sacrificed 8 weeks after receiving alloxan. There is a
reduction in size of the fenestrae (arrow). Spherical
structures resembling platelets are seen (double arrow).
x 18,000. C) Endothelium from the protected kidney of
an untreated diabetic rat (same as animal in Fig. 12B)
showing focal degeneration (arrow) and exposure of the
underlying basement membrane. X 18,000. D) Glomerular capillary from the protected kidney of a n insulintreated diabetic animal sacrificed 8 weeks after receiving alloxan. The endothelium is normal in appearance.
x 18,000.
ALLOXAN-INDUCED DIABETES
45
Fig. 13. A) Visceral epithelium from the right kidney
of a control animal (same as animal in Fig. 12A) in which
both renal hila were clamped. The cells exhibit long,
. Visslender, interdigitating foot processes. ~ 5 , 0 0 0 B)
ceral epithelium from the protected kidney of an untreated diabetic animal (same as Fig. 12B). There are
focal areas of blunting and fusion of the foot processes
(arrow). ~ 5 , 0 0 0 .
These changes are seen in both the right and
left kidneys of the diabetic animals. Further
evidence that these alterations are related to
the diabetes is that the changes are reversible with insulin treatment (Tables 2, 3). Glomerular volume measured in all groups
remained unchanged from the control values
(Table 3).
nephropathy, identified a number of potentially important glomerular endothelial and
epithelial abnormalities which have not been
previously appreciated. The experiments of
Orskov et al. (1965)raised the possibility that
the alterations we identified were related to
alloxan rather than diabetes. These investigators used the approach to protect the kidney employed in the present study and
observed tubular atrophy, dilation, and interstitial inflammation, which they attributed
to alloxan. Current sophisticated ultrastructural techniques were not used by Orskov et
al. (19651, and the possibility remained that
significant glomerular damage occurred related to alloxan that would not be detected
by light microscopy. Such has been the case
in the study of other nephrotoxins, including
the aminoglycoside antibiotics and heavy
metals (Luft and Evan, 1980; Avasthi et al.
1980). The distinction between the effects of
alloxan and diabetes per se is important, not
only with respect to interpreting morphol-
DISCUSSION
Chemically induced diabetic models have
commonly been employed to study the functional and structural changes associated with
diabetes mellitus as well as the effects of
treatment with either insulin or pancreatic
islet cell transplantation (Black et al., 1980;
Rasch, 1979; Mauer et al., 1974; Orloff et al.,
1975; Weil et al., 1975). Recently we examined the renal glomerulus in a series of sequential studies which utilized rats made
diabetic with alloxan (Evan and Luft, 1980).
The scanning electron microscope, a tool not
previously employed in the study of diabetic
46
A.P. EVAN ET AL
ogy, but also in evaluating the results of renal
functional studies including micropuncture
experiments (Michaels et al., 1981).
Our studies indicate that alloxan caused
considerable interstitial nephritis and tubular atrophy. These changes were observed
not only in rats that developed diabetes, but
also in rats which received alloxan but failed
to develop diabetes. Thus this latter group
was particularly useful in defining the effects of alloxan. In those areas that showed
severe interstitial changes, glomerular sclerosis was also prominent. However, these
glomeruli had not presented the same
changes that were associated with diabetes,
such as podocyte fusion, mesangial hyperplasia, and basement membrane thickening.
Therefore the nephrotoxic and diabetic
changes could be differentiated.
The diabetic state resulted in mesangial
hyperplasia and focal glomerular basementmembrane thickening after only 9 weeks of
untreated diabetes. These structural changes
have been implicated in the functional
changes observed in diabetes (Steffes et al.,
1975). Experiments in which glucose and insulin concentrations in diabetic rats were
controlled have shown decreases in mesangial matrix, immunoglobulin deposition and
glomerular cellularity as well as improvment in renal function (Mauer et al. 1974;
Orloff, 1975; Weil et al. 1975). In addition, a
recent study examining the effects of insulin
treatment demonstrated that good blood glucose control in rats perserved normal glomerular basement-membrane thickness (Rasch,
1979).
The glomerular capillary endothelial
changes, namely, a decrease in size of the
fenestrae, proliferation of microvilli, and appearance of irregular endothelial cell processes, can now be attributed solely to the
diabetic state. The endothelial structural aberrations may be partially responsible for abnormalities in glomerular function described
in recent micropuncture studies (Hostetter et
al., 1981). The pathogenesis of the endothelial changes is unknown. Similarly, little is
known of the mechanisms responsible for the
increase in amount of glomerular basementmembrane material. The latter has been attributed to abnormal deposition due to excessive synthesis of glomerular basementmembrane material as well as faulty disposal (Romen, 1980).The functional relationships between capillary endothelium and
glomerular basement membrane are imper-
fectly defined. Our results suggest that endothelial damage antedates glomerular
basement-membrane thickening. Whether or
not endothelial damage and altered glomerular basement-membrane metabolism are
separate events or are specifically related is
not known. Elucidation of a possible connection must await detailed studies of endothelial cell growth and metabolism.
Streptozotocin is another commonly used
drug to induce diabetes mellitus in laboratory animals. Several studies have shown
this compound to produce direct injury to
kidney, in particular, the proximal tubule
(Sadoff, 1970; Myerowitz et al., 1975; Levine
et al., 1980). Alternatively, Steffes et al.
(1980) and Rasch (1979) have presented indirect data which suggests that streptozotocin
may not have direct toxic effects on the kidney since the glomerular changes can be
either ameliorated or prevented by treatment by insulin or islet transplantation.
However, a protection study as performed in
our present report has not been done for
streptozotocin.
In summary, our experiments differentiate
the effects on renal tissue caused by alloxan
from those evoked by diabetes. Alloxan induces marked interstitial nephritis but does
not appear to affect glomerular structure.
Untreated diabetes caused hyperplasia of
mesangium, changes in glomerular basement membrane, visceral epithelial foot process fusion, and endothelial damage. These
features were ameliorated by treatment with
insulin.
ACKNOWLEDGMENTS
This study was supported by Diabetes
Grant PHS P60 AM 20542-03. The authors
wish to give special thanks to Ms. Toni D.
Moore and Mrs. Sandra S. Wilson for typing
the manuscript.
LITERATURE CITED
Arteta, J.L. (1952) Mechanism of protective action of
clamping renal pedicles of dogs with alloxan diabetes.
J. Endocrinol., 8.245-249.
Avasthi, P.S., A.P. Evan, and D. Hay (1980) Glomerular
endothelial cells in uranyl nitrate-induced acute renal
failure in rats. J. Clin. Invest., 65:121-127.
Bell, R.H., L. Fernandez-Cruz,-J.E.Brimm, H.A. Sayers,
S. Lee, and M.J. Orloff (1980) Prevention of whole
pancreas transplantation of glomerular basement
membrane thickening on alloxan diabetes. Surgery,
88:31-40.
Bilic, N., and J.P. Felber (1969) An improved fluorometric method for measurement of alloxan in biological
fluids. Anal. Biochem., 29:91-94.
ALLOXAN-INDUCED DIABETES
47
Black, H.E., I.Y. Rosenblum, and C.C. Capen (1980) Myerowitz, R.L., G.P. Sartiawo, and T. Cavallo (1975)
Nephrotoxic and cytoproliferative effects of streptozoChemically induced diabetes mellitus in the dog. Am.
tocin. Cancer, 38:1550-1555.
J. Pathol., 98:295-310.
Bohman, S.O., N. Deguichi, H.J.G. Gundersen, J. Hest- Orloff, M.J., S. Lee, A.C. Charters, D.E. Gramfort, L.G.
Storck, and D. Knox (1975) Long term studies of panbech, A.B. Maunsbach, and S. Olsen (1979)Evaluation
creatic transplantation. Ann. Surg., 282198-205.
of a procedure for systematic semiquantitative analysis of glomerular ultrastructure in human biopsies. Orskov, H., T. Steen-Olsen, LK. Nielsen, O.J. Rafaelsen,
and K. Lundbaek (1965) Kidney lesions in rats with
Lab. Invest., 40:433-444.
severe long-term, alloxan diabetes. Diabetologia,
Evan, A.P., and F.C. Luft (1980)Effect of alloxan-induced
1:172-179.
diabetes on the glomerular filtration barrier of the rat.
Osterby, R. (1974) Early means in the development of
Renal Physiol., 3:257-264.
diabetic glomerulopathy. A quantitative electron miHagg, E. (1974) Glomerular basement membrane thickcroscopic study. Acta Med. Scand., 574(Suppl):13-81.
ening in rats with long-term diabetes. A quantitative
electron microscopic study. Acta Pathol. Microbiol. Pirani, C.L., V.E. Pollack, and F.D. Schwartz (1964) The
reproducibility of semiquantitative analyses of renal
Scand. [A], 82:211-219.
histology. Nephron, 1:230-237.
Hagg, E., and B. Winblad (1975) A scanning electron
microscopic study of the glomerular epithelial cells in Rasch, R. (1979) Prevention of diabetic glomerulopathy
in streptozotocin diabetic rats by insulin treatment:
alloxan diabetic rats. Diabetologia, llt245-248.
Glomerular basement membrane thickness. DiabetoHammarstrom, L., and S. Ullberg (1966)Specific uptake
logia, 16:319-324.
of labeled alloxan in the pancreatic islets. Nature,
Rerup, C.C. (1970) Drugs producing diabetes through
212:708-709.
damage
ofthe insulin secreting cells. Pharmacol. Rev.,
Hostetter, T.H., J.L. Troy, and B.M. Brenner (1981) Glo22:485-518.
merular hernodynamics in experimental diabetes melRomen, W. (1980) Fur morphologie und Pathogenese der
litus. Kidney Int., 19r410-415.
diabetischen glomerulosklerose. Klin. Wochenschr.,
Hughes, H., L.L. Ware, and F.G. Young (1944) Diabeto58: 1013-1022.
genic action of alloxan. Lancet, 246:148.
Janes, R.G., and T. Winnick (1952) Distribution of CI4- Sadoff, L. (1970) Nephrotoxicity of streptozotocin NSC85998). Cancer Chemother. Rep., 54:457-459.
labeled alloxan in the tissues of the rat and its mode
Steffes, M.W., D.M. Brown, J.M. Basgen, and S.M. M a w
of elimination. Proc. SOC.EXD.Biol. Med.. 81.226-229.
(1980) Amelioration of mesangial volume and surface
Levine, B.S., M.C. Henry, ank E. Rosen (1980) Toxicoalterations following islet transplantation in diabetic
logic evaluation of streptozotocin (NSC85998) in mice,
rat. Diabetes, 29:509-515.
dogs and monkeys. Drug Chem. Toxicol, 3:201-212.
Luft, F.C., and A.P. Evan (1980) Glomerular filtration Steffes, M.W., D.E.R. Sutherland, S.M. Mauer, R.J.
Leonard, J.S. Najanau, and D.M. Brown (1975)Plasma
barrier in aminoglycoside-induced nephrotoxic acute
insulin and glucose levels in diabetic rats prior to and
renal failure. Renal Physiol., 3:265-271.
following islet cell transplantation. J. Lab. Clin. Med.,
Mauer, S.M., D.E.R., Sutherland, M.W. Steffes, R.J.
85:75-81.
Leonard, J.S. Najarian, A.F. Michael, and D.M. Brown
(1974) Pancreatic islet cell transplantation. Diabetes, Vargas, L., H.H.R. Friederici, and H.C. Maibenco (1970)
Cortical sponge kidneys induced in rats by alloxan.
23:748-753.
Diabetes, 19:34-44.
Michaels, L.D., M. Davidman, W.F. Keane (1981) Glomerular function in alloxan diabetes and the effects of Weil, R., M. Nozawa, M. Koss, C. Weber, K. Reemtsma,
and R.M. McIntosh (1975) Pancreatic transplantation
insulin. J. Lab. Clin. Med., 98t869-885.
in diabetic rats: Renal function, morphology, ultraMogensen, C.E. (1976)Renal function changes in diabestructure, and immunohistology. Surgery, 78t142-148.
tes. Diabetes, 25872-879.
Документ
Категория
Без категории
Просмотров
1
Размер файла
1 725 Кб
Теги
effect, induced, alloxan, diabetes, kidney
1/--страниц
Пожаловаться на содержимое документа