Renal type I inositol 1,4,5-trisphosphate receptor is reduced
in streptozotocin-induced diabetic rats and mice
Kumar
Sharma1,
Lewei
Wang1,
Yanqing
Zhu1,
Aurora
DeGuzman1,
Gao-Yuan
Cao2,
Richard B.
Lynn2, and
Suresh K.
Joseph3
1 Nephrology Division and
2 Gastroenterology and Hepatology
Division of the Department of Medicine and
3 Department of Anatomy,
Pathology, and Cell Biology, Thomas Jefferson University School of
Medicine, Philadelphia, Pennsylvania 19107
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ABSTRACT |
The mechanisms
underlying glomerular hypertrophy and hyperfiltration in diabetes
remain unclear. We have previously demonstrated that the cytokine
transforming growth factor-
1 (TGF-
1) is increased in early
diabetic kidney disease and TGF-
1 inhibits the expression of the
inositol 1,4,5-trisphosphate
(IP3)-gated calcium channel, the
type I IP3 receptor
(IP3R), in mesangial cells. To
test the hypothesis that reduced type I
IP3R may be important in diabetic kidney disease, we evaluated type I
IP3R expression in the kidney of
streptozotocin-induced diabetic rats and mice. Two-week-old diabetic
rats have decreased renal type I
IP3R protein and mRNA levels.
Immunostaining of normal rat kidney demonstrated presence of type I
IP3R in glomerular and vascular
smooth muscle cells, whereas diabetic rats had reduced staining in both
compartments. Reduction of type I
IP3R also occurred in parallel
with renal hypertrophy, increased creatinine clearance, and increased
renal TGF-
1 expression in the diabetic rats. Two-week-old diabetic mice also had reduced renal type I
IP3R protein and mRNA expression in association with renal hypertrophy and increased TGF-
1 mRNA expression. These findings demonstrate that there is reduced type I
IP3R in glomerular and vascular
smooth muscle cells in the diabetic kidney, which may contribute to the
altered renal vasoregulation and renal hypertrophy of diabetes.
inositol 1,4,5-trisphosphate receptor; diabetic renal hypertrophy; transforming growth factor-
; hyperfiltration
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INTRODUCTION |
DIABETES CHARACTERISTICALLY induces glomerular
hyperfiltration in experimental animals and in patients.
Hyperfiltration likely occurs as a result of enhanced glomerular blood
flow due to afferent arteriolar vasodilatation, increased filtration
surface area from mesangial cell relaxation, and enhanced glomerular
capillary pressure due to relative efferent arteriolar vasoconstriction
(10). Despite intensive investigation, it remains unclear how diabetes
induces afferent arteriolar and mesangial cell relaxation (20). The glomerulus from diabetic animals exhibits decreased contraction to
vasoconstrictors (12) that is likely due to reduced responsiveness of
the mesangial cell to various vasoconstrictors, such as ANG II,
endothelin, vasopressin, and norepinephrine (9, 16, 26). Although
reduction of surface receptors may play a role (3), it remains possible
that diabetes induces effects on common intracellular signaling
pathways that may alter response to a variety of vasoconstricting agonists.
It is widely accepted that vasoconstrictors such as ANG II, endothelin,
norepinephrine, and vasopressin all raise free intracellular calcium
([Ca2+]i)
to promote contraction of the cell (4). The observation that mesangial
cells from diabetic rat glomeruli, as well as human and rat mesangial
cells cultured in high glucose, exhibit an impaired [Ca2+]i
increase in response to these vasoconstrictors (9, 16) is
consistent with the argument that an intracellular alteration may be
responsible. It is interesting that apart from high glucose, exposure
of several cell types to transforming growth factor-
(TGF-
) also
impairs agonist-induced
[Ca2+]i.
Pretreatment of vascular smooth muscle cells with TGF-
1 (35) inhibits ANG II-induced
[Ca2+]i
release, and in our study with mesangial cells, pretreatment with
TGF-
1 inhibited platelet-derived growth factor (PDGF)-induced [Ca2+]i
(2). A common pathway of raising
[Ca2+]i
with PDGF and ANG II is the generation of inositol 1,4,5-trisphosphate (IP3) from phosphatidylinositol
4,5-bisphosphate. The increased intracellular levels of
IP3 bind to
IP3 receptors
(IP3Rs) in the endoplasmic
reticulum to release stored
[Ca2+]i
into the cytoplasmic space (4). Of the various
IP3Rs identified (types I, II, and
III), the types I and III IP3Rs
appear to be the predominant isoforms expressed in glomerular mesangial
and vascular smooth muscle cells (17, 32). Recently, we found that
TGF-
1 inhibits the expression of the type I
IP3R in rat and mouse mesangial
cells (24). It is clear that diabetes leads to overexpression of
TGF-
1 in the kidney of streptozotocin (STZ)-induced diabetes
mellitus in the rat and mouse (18, 21, 23, 25, 31). Therefore, it is
possible that, in these models of diabetic kidney disease that are
associated with overexpression of TGF-
, there would be consequent
downregulation of the type I IP3R.
Reduction of IP3R expression may
contribute to impaired
[Ca2+]i
mobilization to vasoconstrictors and diabetes-related alterations in
mesangial cell contraction. In the present study, we demonstrate that
expression of the type I IP3R is
downregulated in two models of diabetic kidney disease and associated
with enhanced renal TGF-
1 expression.
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MATERIALS AND METHODS |
Animals. Sprague-Dawley rats weighing
220-260 g were made diabetic by a single intraperitoneal injection
of STZ (65 mg/kg body wt; Sigma, St. Louis, MO) in 10 mmol/l sodium
citrate, pH 5.5. Controls were injected with buffer alone. The levels
of blood glucose were determined 2 days after injection, and rats with blood glucose >16 mmol/l were used as diabetic rats. The diabetic rats were divided into two groups of six animals each. One group received daily subcutaneous insulin of 2.0 U of NPH
insulin (Eli Lilly, Indianapolis, IN) to maintain
hyperglycemia but avoid ketosis. The other diabetic group received
daily high-dose insulin (7-10 U) to maintain relative euglycemia
(<15 mmol/l). Diabetic and nondiabetic rats were given standard rat
diet and water ad libitum. After 14 days of diabetes, rats were placed
in metabolic cages for 24-h urine collection for creatinine clearance,
as measured by a colorimetric assay (Sigma). At the end of the
experiment, blood was collected, the right kidney was weighed, and the
kidney cortex was excised and stored at
70°C. After removal
of the right kidney, the left kidney was perfused with PBS and
formaldehyde, removed, snap-frozen in liquid nitrogen, and stored at
70°C.
C57Bl mice were fed a standard pellet laboratory diet and provided with
water ad libitum. Diabetes was induced in 7- to 8-wk-old mice
(17-21 g) by two consecutive daily intraperitoneal injections of
STZ (200 mg/kg). On the same day that glucosuria was noted, 0.5 U NPH
insulin was administered to allow for hyperglycemia (>20 mmol/l) and
to prevent ketonuria. Following 2 wk of diabetes, mice were killed, and
kidneys were weighed and snap-frozen in liquid nitrogen and stored at
70°C for Western and Northern analysis (see below).
Immunohistochemistry. The left kidney
of the rats in each group was perfused with PBS and formaldehyde in
vivo prior to extraction and postfixed in PBS-formaldehyde for 1 h
prior to storage at
20°C. Five-micron sections from kidney
blocks were obtained by microtome, placed on
poly-L-lysine-plated glass
slides, and incubated in 0.3%
H2O2
in methanol for 30 min to block endogenous peroxidase prior to
immunostaining. The primary antibody against the type I
IP3R that was used for
immunostaining was obtained from Affinity Bioreagents (Golden, CO) and
used at a dilution of 1:500. Sections were incubated with primary
antibody in Tris-buffered saline (TBS) for 16 h at 4°C. The
sections were washed in TBS and incubated with a goat biotin-conjugated
anti-rabbit IgG serum (Vector Laboratories, Burlingame, CA). The avidin
biotin-peroxidase complex technique using diaminobenzidine (Vectastain
Elite ABC kit, Vector) was then employed. Negative controls were
performed with the absence of primary antibody and revealed no
background staining. Slides were viewed on a Laborlux 5 microscope, and
photographs were taken of representative sections. Semiquantitative
assessment of immunostaining intensity was scored on a scale of
0-3 from 15 glomeruli, separately from their
accompanying arteriolar structures, in each section. The scale was
based on a value of 0 being completely negative and a value of 3 being
maximally positive.
Immunoblot analysis of type I
IP3R. Kidney
cortical tissue from rat and mouse origin were homogenized in lysis
buffer containing 50 mM Tris · HCl (pH 7.2), 150 mM
NaCl, 1% (wt/vol) Triton X-100, 1 mM EDTA, 1 mM phenylmethylsulfonyl
fluoride, and 5 µg/ml each of aprotonin and leupeptin. Protein
concentration of samples were quantitated (Bio-Rad, Hercules, CA), and
equal amounts of protein were run on a 7% SDS-PAGE gel, transferred to
nitrocellulose, and immunoblotted with an antibody raised to the COOH
terminus of the type I IP3R from
brain (13). The primary antibody was then removed, and the membrane was
incubated with horseradish peroxidase-conjugated secondary antibody.
Immunoreactive bands were detected using enhanced chemiluminescence
(Amersham, Arlington Heights, IL). Densitometric analysis of scanned
images was performed on a Macintosh 7600/132 computer using the public
domain NIH Image program. Measurements in control samples were assigned
a relative value of 100%.
Northern analysis. Total RNA was
isolated from rat and mouse kidney using acid guanidinium
thiocyanate-phenol-chloroform (7). Poly(A)+ mRNA was
isolated from total RNA by oligo(dT) affinity column (Promega, Madison,
WI). Three micrograms of poly(A)+
mRNA was loaded onto a 1.2% agarose gel containing 2.2 M formaldehyde, electrophoresed, and transferred onto nylon membrane by capillary blotting, then ultraviolet cross-linked. Prehybridization was performed
for 1 h at 65°C with a buffer containing 10% dextran sulfate, 1 M
NaCl, and 1% SDS. DNA probes were labeled with 10 µCi
[32P]dCTP (3,000 Ci/mmol; Du Pont-New England Nuclear, Boston, MA) via the random primer
method (DNA labeling kit; Boehringer-Mannheim, Indianapolis, IN) and
added to prehybridization fluid. The hybridization was performed at
65°C overnight after the addition of labeled DNA probe. The filters
were washed twice at 65°C for 30 min in a solution containing 1 mM
EDTA, 40 mM
Na2PO4,
pH 7.2, and 5% SDS and then in a solution of 1 mM EDTA, 40 mM
Na2PO4,
pH 7.2, and 1% SDS. The filters were exposed at
80°C for
1-3 days to Kodak XAR film.
The probes for the type I IP3R and
TGF-
1 were synthesized by the PCR using specific
oligonucleotide primers, based on the published cDNA sequence and
murine kidney cDNA as template as previously described (24, 25). A cDNA
probe for rat kidney type I IP3R
was similarly prepared by PCR using rat kidney cDNA as template.
Nucleotide sequencing of the PCR products confirmed the identity of the
probes. To standardize for loading, membranes were stripped and
reprobed with a
-actin cDNA probe (kindly provided by Dr. Pamela A
Norton). Densitometry was performed as described above, and mRNA levels
were calculated relative to those of
-actin.
Statistical analyses. Results are
expressed as means ± SE. One-way ANOVA was used to test for
differences between two groups and analyzed by Student's unpaired
t-test. Bonferroni's correction was
applied for comparisons between three groups. The variability within
the groups was random. P < 0.05 was
considered significant.
 |
RESULTS |
Metabolic parameters and kidney hypertrophy in the
STZ-induced diabetic rat. Table
1 shows the body weight, kidney weight, blood glucose, and creatinine clearance measured in normal rats and
diabetic rats at the end of the experimental period. Diabetic rats
treated with a low dose of insulin (2 U of NPH) had significant hyperglycemia (23.3 ± 1.4 mmol/l), whereas diabetic rats treated with high-dose insulin (7-10 U) had mild hyperglycemia (13.2 ± 1.9 mmol/l) that was not significantly different from control values
(8.9 ± 0.7 mmol/l). Body mass of hyperglycemic diabetic rats was
significantly lower compared with control. However, absolute kidney
weight was significantly increased by 28% and creatinine clearance was
increased by 82% in hyperglycemic diabetic rats compared with normal
rats. All of the above parameters were attenuated in diabetic rats
treated with high-dose insulin and not significantly different compared
with control rats.
Distribution of type I
IP3R in normal and diabetic
kidney.
The expression and distribution of type I
IP3R protein in the kidney was
evaluated by immunoperoxidase staining using a specific rabbit
anti-type I IP3R antibody.
Immunohistochemistry from the normal rat kidney demonstrates presence
of type I IP3R primarily in
glomerular cells and in arteriolar smooth muscle cells (Fig. 1A).
Type I IP3R staining in cortical
tubular cells was less prominent than in glomerular and vascular cells.
In contrast to the normal rat kidney, the diabetic rat kidney
demonstrates reduced immunostaining for the type I
IP3R in glomerular cells and
vascular smooth muscle cells (Fig.
1B). The diabetic rats treated with
high-dose insulin had a pattern of immunostaining of the type I
IP3R similar to that of the
control group (Fig.
1C). The results of glomerular and
arteriolar immunostaining intensity scores for type I
IP3R of all the animals in each of
the three groups are shown in Fig. 2.

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Fig. 1.
Type I inositol 1,4,5-trisphosphate
(IP3) receptor
(IP3R) is reduced in diabetic
glomeruli and vascular smooth muscle cells.
A: normal rat kidney (control); note
distinct staining for type I IP3R
in glomerulus and vascular arterioles (see arrow).
B: diabetic rat kidney; much reduced
staining for type I IP3R is
present in glomerular cells and vascular smooth muscle cells (see
arrow). C: kidney from
diabetic rat treated with high-dose insulin; note similar intensity of
immunostaining in glomerular and arteriolar cells compared with control
(A).
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Fig. 2.
Semiquantification of glomerular and arteriolar immunostaining for type
I IP3R. Intensity of
immunostaining for type I IP3R was
scored on a scale of 0 (negative staining) to 3 (maximal staining) on
kidney sections from all rats in each of the three groups
(n = 6 per group). N, normal rats; D,
diabetic rats treated with low-dose insulin; D + I,
diabetic rats treated with high-dose insulin; Glom, glomeruli; Vessels,
glomerular arteriolar vessels. Data shown are means ± SE of
staining scores from each of the 3 groups
(n = 6 per group).
* P < 0.05 vs. control.
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Type I IP3R
protein in kidney cortex of normal and diabetic rats.
Figure 3 shows a representative immunoblot
of rat cerebellum and rat kidney cortex protein with an antibody raised
against the COOH-terminal end of the rat type I
IP3R (13). A prominent ~240-kDa
band was observed in rat kidney cortex from normal and diabetic rats
that corresponds to the known size of the type I IP3R from rat cerebellum (Fig.
3A). Densitometric analysis of all
samples in each of the three groups revealed that diabetic rats with
hyperglycemia have significantly decreased type I
IP3R expression compared with
control rats (29 ± 12% of control values, P = 0.04; Fig.
3B). Decreased expression of type I
IP3R in diabetic kidney cortex was
largely prevented with high-dose insulin treatment and tight control of
blood glucose (81 ± 35% of control values, P = not significant).

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Fig. 3.
Reduction of type I IP3R protein
expression in diabetic rat kidney. A:
samples (50 µg protein/lane) of kidney cortical protein from 2 separate normal rats (lanes 2 and
3), hyperglycemic diabetic rats
treated with low-dose insulin (lanes 4 and 5), and diabetic rats treated
with high-dose insulin (lanes 6 and
7) were resolved on 7% SDS-PAGE,
transferred to nitrocellulose, and probed with type I
IP3R antibody. The ~240-kDa band
migrated to the same position on the gel as the type I
IP3R from rat cerebellum
(lane 1).
B: densitometric quantitation of
immunoreactive protein expressed relative to control. Data are means ± SE of band intensities from each of the 3 groups
(n = 6 per group).
* P < 0.05 vs. control.
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Northern analysis of type I
IP3R and TGF-
1
expression in diabetic rat kidneys.
A representative hybridization of
poly(A)+ mRNA of rat kidney cortex
from the normal, diabetic, and diabetic group treated with high-dose
insulin probed for the type I IP3R
is shown in Fig. 4. The diabetic
rat kidney has decreased expression of type I
IP3R mRNA compared with the normal
rat kidney. In addition, reduction of type I
IP3R mRNA in the diabetic kidneys
is associated with enhanced expression of TGF-
1 mRNA. Densitometric
analysis of pooled samples in the diabetic group demonstrates a
reduction of type I IP3R mRNA
(standardized for
-actin) of 27 ± 5% of control values
(P = 0.01) (Fig.
4B), whereas TGF-
1 mRNA is
increased by 196 ± 26% of control value
(P = 0.03) (Fig.
4C). Treatment of diabetic rats with
high-dose insulin prevented changes in type I
IP3R and TGF-
1 mRNA expression
compared with control.

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Fig. 4.
Reduced expression of type I IP3R
mRNA occurs in association with enhanced transforming growth
factor- 1 (TGF- 1) mRNA in diabetic rat kidney.
A,
top: representative autoradiograph of
a Northern blot of poly(A)+ mRNA
isolated from kidney cortex from normal (N), diabetic (D), and diabetic
rat treated with high-dose insulin (D + I) probed with rat
type I IP3R. The same blot was
probed with TGF- 1 cDNA (A,
middle) and was finally probed with
-actin cDNA (A,
bottom) as a control for loading.
B: densitometric quantitation of renal
type I IP3R mRNA/ -actin mRNA
from each of the 3 groups. C:
densitometric quantitation of renal TGF- 1 mRNA/ -actin mRNA from
each of the 3 groups. Data are means ± SE of band intensities of
pooled data from all samples in each of the 3 groups
(n = 6 per group).
* P < 0.05 vs. normal group.
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Metabolic parameters and kidney hypertrophy in the
STZ-induced diabetic mouse. Table
2 shows the body weight, kidney weight, and
blood glucose from normal and STZ-induced diabetic mice after 2 wk of
diabetes. Diabetic mice demonstrated significant hyperglycemia and
kidney hypertrophy despite a tendency toward reduced body weight
compared with the normal group.
Renal type I IP3R
protein and mRNA expression in diabetic mice.
Type I IP3R expression in the
kidneys from normal and diabetic mice was analyzed by immunoblot and
Northern analysis. A representative immunoblot analysis of kidney
protein with antibody to the type I
IP3R demonstrated decreased type I
IP3R protein in the diabetic kidney compared with normal mouse kidney (Fig.
5). Pooled analysis of all samples revealed
that renal expression of type I
IP3R protein in the diabetic mice
was reduced to 26 ± 6% of control values (P = 0.01) (Fig.
5B). This is a reduction of type I
IP3R protein similar
to that noted in the kidneys from hyperglycemic diabetic rats.

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Fig. 5.
Reduction of type I IP3R protein
expression in diabetic mouse kidney.
A: representative immunoblot analysis
of samples (50 µg protein/lane) from normal (NL) mouse kidney and
diabetic mouse kidney were resolved on 7% SDS-PAGE, transferred to
nitrocellulose, and probed with type I
IP3R antibody.
B: quantitation of immunoreactive
protein expressed in diabetic kidney (D) relative to the control (N).
Data are means ± SE of band intensities from all samples in each of
the groups (n = 5 per group).
* P < 0.05 vs. normal
group.
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Hybridization of poly(A)+ mRNA
from mouse kidneys with the type I
IP3R cDNA probe revealed a band
for type I IP3R mRNA of the same
size as that noted from mouse cerebellum (Fig.
6). The diabetic mouse kidney has less
expression of type I IP3R mRNA
compared with the normal mouse kidney. A representative hybridization
of poly(A)+ mRNA from mouse
kidneys from the normal and diabetic groups with the type I
IP3R, TGF-
1 and
-actin cDNA
probes is shown in Fig. 7. Similar to the
diabetic rats, the kidneys from diabetic mice demonstrate reduction of
type I IP3R mRNA in association
with enhanced expression of TGF-
1 mRNA. Densitometric analysis of pooled samples demonstrates a reduction of type I
IP3R mRNA (standardized for
-actin) of 51.6 ± 8.6% of control values
(P = 0.006) (Fig. 7B), whereas TGF-
1 mRNA is
increased by 194 ± 12% of control value
(P = 0.05) (Fig.
7C).

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Fig. 6.
Type I IP3R mRNA expression in
mouse cerebellum and normal and diabetic mouse kidney.
Top: autoradiograph of a Northern blot
of poly(A)+ mRNA isolated from
mouse cerebellum, normal mouse kidney, and a diabetic mouse kidney
probed with cDNA encoding murine type I
IP3R. The single band noted in
mouse kidney corresponds to an identically located band from mouse
cerebellum. Bottom: same blot was
probed with -actin cDNA as a control for loading.
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Fig. 7.
Reduced expression of type I IP3R
mRNA occurs in association with enhanced TGF- 1 mRNA in diabetic
mouse kidney. A,
top: representative autoradiograph of
a Northern blot of poly(A)+ mRNA
isolated from normal (NL) mouse kidneys and diabetic mouse kidneys
probed with murine type I IP3R.
The same blot was probed with TGF- 1 cDNA
(middle) and was finally probed with
-actin cDNA (A,
bottom) as a control for loading.
B: densitometric quantitation of type
I IP3R mRNA/ -actin mRNA
expressed in diabetic mouse kidney (D) relative to the control (N).
C: densitometric quantitation of
TGF- 1 mRNA/ -actin mRNA expressed in diabetic mouse kidney (D)
relative to control (N). Data are means ± SE of band intensities of
pooled data from all mouse samples in both groups
(n = 5 per group).
* P < 0.05 vs. normal group.
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DISCUSSION |
The present results indicate that renal expression of type I
IP3R is reduced in two animal
models of early STZ-induced diabetes. Immunostaining of diabetic rat
kidneys demonstrates that the reduced expression of type I
IP3R is prominent in arteriolar
smooth muscle cells and glomerular cells. In STZ-induced diabetic rats,
there is an association of reduced renal type I
IP3R expression with diabetic
renal hypertrophy and diabetic renal hyperfiltration, as measured by
creatinine clearance. In both models of STZ-induced diabetic rats and
mice, reduction of type I IP3R
mRNA expression is associated with increased renal TGF-
1 mRNA
expression suggesting a possible relationship between these processes.
The IP3R family has previously
been evaluated in mouse and rat kidney. Northern analysis of various
mouse tissues revealed that mouse kidney had the strongest expression
of type I IP3R mRNA outside the
brain (8). In situ hybridization of mouse kidney demonstrated type I
IP3R expression primarily in
vascular structures (8). Immunostaining of mouse kidney with an
antibody raised against the IP3R
from cerebellum showed expression of
IP3R primarily in glomerular
mesangial cells (19) and light immunostaining in tubular epithelial
cells (6). Immunostaining of normal rat kidneys with monoclonal
antibodies specific for each of the
IP3R isoforms revealed expression
of only the type I and type III
IP3R isoforms in glomerular
mesangial cells and glomerular arteriolar structures (17).
Microdissection of rat nephron segments and subsequent RT-PCR with
primers specific for the type I and II IP3R demonstrated that glomeruli
only express type I IP3R, whereas distal tubular cells express both types I and II
IP3R (32). RT-PCR analysis of
Madin-Darby canine kidney cells, a model of polarized tubular
epithelial cells, revealed expression of all three types of
IP3R (6) with the type III isoform
appearing to be the most predominant. On the basis of the above
results, it appears that the type I
IP3R is present predominantly in
glomerular mesangial cells and glomerular arterioles. Our recent study
in murine and rat mesangial cells in culture demonstrated expression of
type I IP3R protein and mRNA (24).
Our present demonstration of immunostaining for type I
IP3R in rat kidney glomerular
cells and smooth muscle cells corresponds to similar findings recently reported in rat kidney (17). Expression of type I
IP3R mRNA in rodent kidneys
confirms prior findings by Furuichi et al. (8).
Regulation of IP3R expression is
likely to have functional consequences. Reduction of type I
IP3R protein expression has
previously been shown to result in decreased
IP3 sensitivity and
[Ca2+]i
release in response to IP3
mobilizing agonists in neuroblastoma cells and rat liver epithelial
cells (5, 29, 30). In a rat model of dehydration expression of type II
IP3R mRNA, but not type I
IP3R mRNA, in collecting ducts was
found to be downregulated (32). The authors speculated that type II
IP3R regulation may be important
in maintaining body fluid homeostasis (32). In the present study, we
demonstrate that type I IP3R
protein is reduced in glomerular cells and vascular smooth muscle cells
of the diabetic rat in association with the development of renal hypertrophy and increased creatinine clearance. It is possible that
reduced type I IP3R expression in
vascular smooth muscle cells and glomerular mesangial cells may play an
important role in mediating diabetic glomerular hyperfiltration.
Reduced type I IP3R would lead to
decreased IP3 sensitivity and
consequently an impaired rise in
[Ca2+]i
in response to a variety of vasoconstrictors, such as ANG II, norepinephrine, vasopressin, and endothelin. Impaired rise in [Ca2+]i
would prevent maximal vasoconstrictive response to the above agents,
thus leading to vascular dilatation and enhanced glomerular blood flow.
Enhanced glomerular blood flow, due to afferent arteriolar dilatation,
is likely the most important determinant in increasing glomerular
capillary pressure and promoting glomerular hyperfiltration (10).
Although downregulation of the type I
IP3R may play a crucial role in
mediating the vasodilatory effect of diabetes at the glomerulus, other
isoforms of the IP3R may
compensate, and overall calcium mobilization may not be altered. In
this context, regulation of the type III
IP3R isoform and possibly other
intracellular calcium channels, such as the ryanodine receptor, will
need to be evaluated. Although this remains an important consideration, it is also possible that discrete intracellular calcium channels control discrete pools of calcium and thus may regulate different functions in response to intracellular calcium-mobilizing agonists. In
this regard, during the process of apoptosis in lymphocytes, there is
upregulation of the type III IP3R
in the membrane fraction, whereas type I
IP3R expression in the cytosolic
compartment is not affected (15).
The role of
[Ca2+]i
mobilization in explaining the basis for reduced contraction of
mesangial cells to vasoconstrictors remains controversial. Studies by
Whiteside and colleagues (11, 26) have not demonstrated
reduced
[Ca2+]i
mobilization in mesangial cells grown in high glucose, although several
other studies have demonstrated this phenomenon (9, 16). The basis for
these discrepant results may be due to technical issues in calcium
measurements and the passage number of mesangial cells used. It has
also been demonstrated that actin filaments may play a role in the
altered contractile function of mesangial cells grown in high glucose
(33). Additionally, the involvement of the aldose reductase pathway and
the protein kinase C (PKC) pathway have been implicated in disturbing
actin assembly (34). Interestingly, PKC may also be involved in the
inhibition of calcium mobilization by high glucose in rat mesangial
cells (16) and rat aortic vascular smooth muscle cells (27, 28).
Receptor downregulation to angiotensin II, arginine vasopressin, and
endothelin has also been described in experimental models of diabetes
and may also play a role in decreased responsiveness to
vasoconstrictors (3, 28). Thus several pathways may contribute to
altered vascular responsiveness of mesangial and smooth muscle cells
grown in high glucose or in the diabetic milieu.
How might the diabetic state lead to reduced type I
IP3R expression in the kidney? Two
recent studies from our group have demonstrated that both ANG II and
TGF-
are capable of reducing IP3R expression. ANG II reduces
both type I and III IP3R
expression in rat liver epithelial cells, possibly via enhanced
ubiquitination and proteasomal degradation (5). TGF-
1
leads to reduced expression of both the mRNA and the protein expression
of the type I IP3R in mesangial
cells (24). Numerous studies have demonstrated that both the renin-ANG
II system (1, 14, 22) and the TGF-
system are stimulated in the
glomeruli of diabetic kidneys (18, 21, 23) at a very early stage of
diabetes. Our present study demonstrates an association with reduced
type I IP3R and overexpression of
mRNA for TGF-
1 in diabetic rat and mouse kidneys. Whether reduced
expression of the type I IP3R in
the diabetic kidney is due to upregulation of the TGF-
system or the
renin-ANG II system remains to be determined.
In summary, we have demonstrated that renal hypertrophy, renal
hyperfiltration and enhanced renal expression of TGF-
1 is associated
with reduced expression of the type I
IP3R in diabetic rodents. Reduced
type I IP3R expression may play an
important role in altered responsiveness of vascular structures in the
pathogenesis of diabetic renal hyperfiltration and subsequent diabetic nephropathy.
 |
ACKNOWLEDGEMENTS |
We are grateful to Dr. Peter McCue (Department of Anatomy,
Pathology, and Cell Biology) for expert advice on the
immunohistochemistry studies and to Stephen Dunn (Division of
Nephrology, Department of Medicine) for expert advice on statistical analysis.
 |
FOOTNOTES |
This work was supported in part by National Institute of Diabetes and
Digestive and Kidney Diseases Grant KO8-DK-02308 (to K. Sharma), an
American Diabetes Association Research Award (to K. Sharma), and
National Institutes of Health Grant R01-AA-10971 (to S. K. Joseph).
Address for reprint requests: K. Sharma, Division of Nephrology, Dept.
of Medicine, Thomas Jefferson Univ., 1020 Locust St., JAH suite 353, Philadelphia, PA 19107.
Received 19 November 1997; accepted in final form 17
September 1998.
 |
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