Laboratory of Experimental Hypertension and Vasoactive Peptides, Clinical Research Institute of Montreal, Université de Montréal, Montreal, Quebec, Canada H2W 1R7
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ABSTRACT |
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It has been shown
that glomerular ANG II receptors are downregulated and protein kinase C
(PKC) activity is enhanced in diabetes mellitus. Therefore, we
investigated glomerular and preglomerular vascular ANG II receptors and
PKC isoform regulation in streptozotocin (STZ)-diabetic rats treated
with insulin and/or captopril. Diabetic rats were prepared by injecting
STZ (60 mg/kg). Those that developed diabetes after 48 h were treated
with low or high doses of insulin, or with a low dose of insulin as
well as captopril, and killed 14 days later. Their glomeruli and
preglomerular vessels were purified, competitive binding studies were
performed by using the ANG II antagonists losartan and PD-123319, and
PKC analysis was carried out by Western blotting. Competitive binding
studies showed that the AT1 receptor was the only ANG II
receptor detected on both glomeruli and preglomerular vessels of all
groups. Preglomerular vascular AT1 receptor density
(Bmax) was significantly upregulated in low insulin-treated
STZ rats, whereas glomerular AT1 Bmax was downregulated. Furthermore, both the captopril- and high
insulin-treated groups had less glomerulosclerosis and vascular damage
than the low insulin-treated group. PKC, PKC
, PKC
, and PKCµ
isoforms found in preglomerular vessels were upregulated by captopril
and high insulin doses, respectively, whereas no such regulation
occurred in glomeruli. We conclude that in STZ-diabetic rats ANG II
receptors and PKC isoforms on preglomerular vessels and glomeruli are
differentially regulated by treatment with insulin and/or captopril.
angiotensin-converting enzyme; receptors; streptozotocin; insulin
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INTRODUCTION |
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SEVERAL LINES OF EVIDENCE indicate that the renin-angiotensin system (RAS) plays an important role in the renal complications seen in patients with type 1 (insulin-dependent) diabetes mellitus (DM) as well as animals in the early stages of experimental DM, with above-normal increases in the glomerular filtration rate (GFR) (10, 35). Because ANG II is an important modulator of glomerular filtration and function, it is believed that glomerular structural injury can be prevented effectively, despite pronounced hyperglycemia, as long as glomerular pressure and flow are maintained within normal limits by the administration of angiotensin-converting enzyme (ACE) inhibitors (5), suggesting a possible role of ANG II in the development of glomerular injury, a hallmark of DM. Moreover, ANG II mediates its effects through high-affinity membrane-bound receptors, namely the ANG II type 1 (AT1) and type 2 receptor (AT2), which have been classified recently with the aid of specific nonpeptide antagonists (13, 44). All the known cardiovascular and mitogenic effects of ANG II have been attributed to AT1, which has a high affinity for the selective nonpeptide antagonist losartan. On the other hand, the AT2 receptor, which has a high affinity for the selective nonpeptide antagonist 1-(4-amino-3-methylphenyl) methyl-5-diphenyl-acetyl-4,5,6,7- tetrahydro-1H-imidazole (4,5-c) pyridine-6-carboxylic acid (PD-123319), has been suggested to act as a biological antagonist of the AT1 receptor because it has been shown to exert antiproliferative effects as well as mediating programmed cell death (38, 47). In addition to this first classification, in rodents, AT1 can be divided into two subtypes, namely AT1a and AT1b (13, 28), both of which are present in the rat kidney but cannot be distinguished pharmacologically (12). ANG II receptor distribution is not uniform in all somatic tissues. Some organs, such as the liver, lung, and kidneys, have a nearly homogenous population of AT1 receptors, whereas others, such as the pancreas and human uterus, almost uniquely contain the AT2 subtype (9, 17, 20). A mixture of both receptor subtypes characterizes certain tissues, such as the adrenals and heart (9). The signaling pathways coupled to AT1, a G-protein-coupled receptor, are diverse. Among these is phosphoinositide hydrolysis (22), the products of which, inositol 1,4,5-triphosphate (IP3) and diacylglycerol (DAG), have the ability to increase intracellular calcium and activate protein kinase C (PKC), respectively. Both DAG and PKC are activated in a variety of tissues in experimental diabetes (15, 27), and hyperglycemia can stimulate PKC through de novo synthesis of DAG (14). Furthermore, PKC is important in many vascular functions found to be abnormal in diabetes, including cell growth, permeability, contractility, and the synthesis of extracellular matrix proteins (19).
Molecular cloning analysis has shown that the PKC family comprises at
least 12 isoenzymes, all having closely related structures but
differing in their individual properties. They have been divided into
four classes: the conventional or classical PKC isoforms (,
I,
II, and
) are Ca2+- and
phospholipid-dependent through their C2 domain; the novel PKC isoforms
(
,
,
and
) lack this region and are, accordingly, Ca2+ independent; the third class, consisting of atypical
PKC isoforms (
,
and
); and the fourth group, which embodies
PKCµ are both Ca2+ and DAG independent (33). Different
tissue and cellular distributions have been noted for the PKC subtypes,
suggesting specific roles for each of them in cell regulation (39).
It has been shown previously that both renal plasma flow and the GFR are blunted in diabetic rats receiving ANG II infusions (41), a finding that can result from either changes in ANG II receptors or alterations in postreceptor actions of the hormone or intracellular signaling pathways. Furthermore, both glomerular and proximal tubular ANG II receptor densities are reduced in diabetic rats (11, 45). These observations could account for the diminished ANG II actions and hyperfiltration seen in DM.
In light of our previous findings of differential regulation of renal ANG II receptors located on preglomerular vessels and glomeruli (2, 3), we wanted to test the hypothesis that this differential regulation is due to the differential expression and/or activation of PKC isoforms. Thus the purpose of this study was to characterize glomerular and preglomerular vascular ANG II receptors and PKC isoforms in streptozotocin (STZ)-induced diabetic rats after treatment with low or high doses of insulin, or a low dose of insulin with captopril, an ACE inhibitor.
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MATERIALS AND METHODS |
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Animals. All studies performed respected the standards of the Canadian Council on Animal Care for the use of laboratory animals.
STZ-induced diabetes. Male Sprague-Dawley rats (225-250 g; Charles River Laboratories, St.-Constant, PQ) were rendered diabetic by a single intravenous STZ injection (60 mg/kg) made up in fresh 0.1 M citrate buffer, pH 4.5. Age-matched control rats received buffer only. The diabetic state was confirmed 48 h later by measurement of tail blood glucose (BG) level, using the Accu-chek glucometer. Rats with blood glucose concentration exceeding 15 mM were considered diabetic, divided randomly into three experimental groups (n = 40 animals/group) and treated for 14 days: the low group received 2 U Humulin U insulin daily subcutaneously (sc) to prevent ketoacidosis (36), whereas the high group was given 25 U insulin daily sc; the captopril group was treated with 2 U insulin daily sc in addition to captopril (75-85 mg/kg body wt) in their drinking water. All animals were fed standard Purina rat chow (Ralston- Purina, Richmond, IN), had free access to tap water ad libitium, and were kept on a 12:12-h light-dark cycle. Moreover, BG was measured on days 7 and 14, and systolic blood pressure (BP) was measured by the tail-cuff method under light ether anesthesia.
Biochemical methods.
Trunk blood was collected in ice-chilled tubes containing
105 M EDTA for the measurement of plasma
renin activity (PRA). Blood samples were immediately
centrifuged at 1,000 g for 10 min at 4°C, and PRA was
assessed by radioimmunoassay of ANG I generation (23).
Histological preparation. Decapsulated kidney halves were fixed for 24 h in Bouin's solution and embedded in paraffin. Sections (5 µm) were cut and stained with hematoxylin, lithium carbonate, and eosin. Readings of each kidney section were done in a "single-blinded manner", i.e., without knowledge of the experimental group from which they were taken. Each kidney section was classified as normal, or presenting vascular lesions or glomerulosclerosis.
Isolation of preglomerular vessels. Once the animals were killed, their kidneys were decapsulated, excised, and placed in ice-cold 0.9% NaCl solution, dissected longitudinally, and the medulla and papilla were discarded. Kidney halves were pressed against a 0.3-mm stainless steel grid. Interlobar arteries and their subsequent attached branches, arcuate, and interlobular arteries as well as afferent arterioles were retained on the grid surface, whereas glomeruli and tubules passed through and were kept at 4°C (17).
Preparation of vascular membranes. Preglomerular vessels were recovered immediately after isolation and minced into small pieces to facilitate the detachment of tubular and connective tissue. The isolated vessels were then placed on a 75-µm nylon mesh and washed with ice-cold 0.9% NaCl solution to eliminate adhering tubules and connective tissues. The purity of the preparations was assessed by light microscopy and estimated to be ~95%. The microvessels were then homogenized twice in fresh ice-cold 0.25 M sucrose solution with a Polytron (setting 7 × 30 s) and centrifuged at 1,000 g for 10 min at 4°C. The supernatant was kept on ice, and the process was repeated. Both supernatants were combined, filtered through a 20-µm nylon mesh, and centrifuged at 100,000 g for 30 min at 4°C. The pellet was resuspended in 50 mM Tris · HCl buffer, pH 7.4. Aliquots were taken for binding assays. Protein concentration was assessed by a modification of Bradford's method as described by Spector (42). Vascular membranes were used immediately thereafter for radioligand-binding studies.
Vascular membrane binding assay.
Optimal conditions for binding dependency on incubation time,
temperature, and protein concentration were ascertained as described previously (17). In competition experiments, 30-35 pM
125I-[Sar1, Ile8]-ANG
II were incubated with increasing concentrations of unlabeled displacing compounds, from 1012 to
10
6 M for both [Sar1,
Ile8]-ANG II, and PD-123319, and from
10
11 to
10
5 M for losartan. The assay buffer
contained 50 mM Tris · HCl (pH 7.4), 1 µM
aprotinin, 0.1% bacitracin, 5 mM MgCl2, 0.5 mM
phenylmethylsulfonyl fluoride (PMSF), 0.4 µM phosphoramidon, and
0.5% BSA. Incubations were undertaken with 40 µg of membrane protein
at 22°C for 90 min in a final volume of 250 µl. The reaction was
stopped by dilution with 3.5 ml ice-cold Tris · HCl
and rapid filtration through Whatman GF/C filters by using a cell
harvester (Brandel, Gaithersburg, MD). The filters were then rinsed
three times with 3 ml Tris · HCl, allowed to dry, and
counted in a LKB gamma counter (Turku, Finland) with 65% efficiency.
Nonspecific binding was determined by the amount of tracer bound in the
presence of 1 µM of unlabeled [Sar1,
Ile8]-ANG II, and specific binding was defined as
total minus nonspecific binding. All radioligand-receptor binding
assays were conducted in duplicate, and at least four separate binding
experiments were performed for each group.
Preparation of glomerular membranes. Glomeruli were isolated, as described previously (20), by filtration with ice-cold 0.9% NaCl solution through a 200-, 150-, 120-, and 50-µm nylon mesh. Those retained on the sieve were collected, washed by centrifugation (4°C, 2,000 g), suspended in 50 mM Tris · HCl (pH 7.4), and snap-frozen with liquid nitrogen for assay the following day. We found no significant differences in either ANG II receptor density (Bmax) or affinity (Kd) values when freshly prepared glomerular membranes were compared with snap-frozen preparations (data not presented). The purity of the glomerular suspensions was assessed by light microscopy and estimated to be ~95% at the end of each preparation. The next day, the glomerular suspensions were defrosted at room temperature, homogenized for 1 min in a Polytron (setting 7), centrifuged at 40,000 g for 20 min, and resuspended in 50 mM Tris · HCl (pH 7.4). Protein concentration was assessed by a modification of Bradford's method (42).
Glomerular membrane binding assay. Optimal conditions for binding dependency on incubation time, temperature, and the protein concentration of glomerular membrane preparations were determined previously (20). Thus the radioligand-receptor binding assay of glomerular membranes was performed similarly to that of vascular membranes, except that 35 µg of glomerular protein were assayed in a final volume of 1 ml binding buffer. As with vascular membrane radioligand-receptor binding assays, all these assays were conducted in duplicate and at least four separate experiments were undertaken for each group.
Cell fraction separation.
Preglomerular vascular and glomerular homogenates were prepared as
mentioned above with the exception that glomeruli were not snap-frozen
and were placed in a 50 mM Tris · HCl lysis buffer containing 1 µM pepstatin A, 1 µM leupeptin, 0.1 mM PMSF, 5 mM EGTA
(pH 8.6), and 2 mM EDTA (pH 6.5) with final pH 7.4. One-half of the
homogenates were used for the total fraction and thus were incubated
under constant shaking for 30 min at 4°C in lysis buffer containing
1% Triton X-100. Subsequently, the total fraction was centrifuged at
145,000 g for 30 min at 4°C, and the supernatant was
retained. The remaining portion of the homogenates was used for
cytosolic and particulate fractions. It was initially centrifuged at
145,000 g for 30 min at 4°C, thereby isolating the
cytosolic fraction in the supernatant. Then, the particulate fraction
was incubated under constant shaking for 30 min at 4°C in the lysis buffer containing 1% Triton X-100 and recentrifuged at 145,000 g for 30 min at 4°C. Protein concentration for each of the
fractions was assessed by a modification of Bradford's method (42),
and with each fraction being aliquoted and stored at 40°C
until Western blot analysis was performed.
PKC isoform analysis.
PKC isoforms in preglomerular vascular and glomerular fractions were
detected by using PKC isoform specific monoclonal antibodies. Solubilized proteins, mixed with Laemmli's sample buffer, were resolved by 10% SDS-PAGE, transferred to a nitrocellulose membrane, and blocked by 90-min incubation at room temperature (22°C) in PBS-T (PBS with 0.1% Tween-20, pH 7.4) plus 2.5% skimmed milk powder
and 1.0% polyvinylpyrrolidone. Affinity-purified anti-PKC isoform
antibodies were diluted (1:5,000 for ; 1:2,000 for
; 1:1,000 for
,
, µ, 1:500 for
,
,
,
,
) in PBS-T containing 0.3% BSA. After 90 min of incubation at room temperature, the nitrocellulose membranes were washed five times for 10 min each with
PBS-T and incubated with goat anti-mouse IgG horseradish peroxidase
conjugate (1:10,000). After extensive washing, bound antibody was
visualized on Kodak XRP-1 film, using the Pierce Supersignal substrate
chemiluminescence detection kit. Specificity of the bands was assessed
by molecular weight markers. The intensity of the bands was quantified
by Alpha Ease (Alpha Innotech, San Leandro, CA). The area under the
peak of the PKC isoform scanned (both cytosolic and membrane fractions)
was determined, and the membrane-to-cytosol ratio was used to calculate
fold activation (or translocation). This method to determine PKC
activation was employed instead of PKC activity assays such as
phosphorylation of histone or of endogenous substrates because these
assays are not isoform specific.
Chemicals.
All materials were of the highest reagent grade available. Bacitracin,
PMSF, N-(-rhamnopyranosyloxyhydroxyphosphinyl)-Leu-Trp (phosphoramidon), captopril, STZ, Tween-20, Triton X-100, pepstatin A,
EGTA, and BSA were all purchased from Sigma Chemical (St. Louis, MO);
aprotinin was obtained from Miles Laboratories (Rexdale, ONT), and
sucrose and EDTA from J. T. Baker (Toronto, ONT). Goat anti-mouse IgG
horseradish peroxidase conjugate and molecular weight markers were
procured from Biorad (Hercules, CA). Accu-chek was purchased from
Boehringer Mannheim (Laval, PQ), and Humulin U insulin from Eli Lilly
(Toronto, ONT). [Sar1, Ile8]-ANG II
was bought from Bachem California (Torrence, CA) and leupeptin from
Bachem Bioscience (King of Prussia, CA).
2-N-butyl-4-chloro-5-hydroxymethyl-1-(2-(H-tetrazole-5-yl)biphenyl-4-yl-methyl) imidazole, potassium salt (losartan potassium), and PD-123319 were
synthesized at EI DuPont Nemours (Wilmington, DE). Losartan potassium
and PD-123319 were generous gifts from the DuPont Merck Pharmaceutical
(Wilmington, DE) and Parke-Davis (Ann Arbor, MI), respectively. Eosin,
hematoxylin, and lithium carbonate were all purchased from Fisher
Scientific (Ottawa, ONT), and PKC isoform-specific monoclonal
antibodies were acquired from Transduction Laboratories (Lexington,
KY). The Pierce Supersignal substrate chemiluminescence detection kit
was obtained from Pierce (Rockford, IL).
Statistical analysis. Binding data were analyzed by processing the raw data with the EBDA program. Bmax and Kd of binding sites were then determined with the LIGAND program (37). Statistical analysis was carried out with the SigmaStat program (Jandel Scientific, San Rafael, CA), using one-way ANOVA followed by the Student-Newman-Keuls t-test to determine significance. The values presented are means ± SE. P < 0.05 values are considered to be significant.
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RESULTS |
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ANG II receptor characterization studies.
Table 1 enumerates several physiological
characteristics of STZ rats treated with either low or high doses of
insulin, or with both a low dose of insulin and captopril. STZ-diabetic
rats had significantly elevated (P < 0.001) initial and final
BG concentrations compared with control animals. In addition,
high-insulin-treated diabetic rats had significantly lower (P < 0.05) final BG than the other STZ-diabetic animals. BP was
significantly lower (P < 0.05) in the captopril-treated
group, whereas PRA was significantly increased (P < 0.05) in
the same animals compared with all other experimental groups.
Low-insulin- and captopril-treated groups had significantly lower body
weight (P < 0.0001) compared with control animals. However,
body weight loss was corrected by a high-insulin dose. No significant
differences (P > 0.05) were found in hematocrit values among
the different experimental groups compared with the control group.
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PKC isoform analysis.
All PKC isoforms were present in vascular preglomerular fractions (Fig.
3). In preglomerular vessels, PKC and
PKC
were activated significantly (P < 0.05) in rats
treated with either high-dose insulin compared with all other groups
(Fig. 4), whereas PKC
was not activated
(Fig. 4), even though it was present in a significantly large quantity
compared with the control rats (P < 0.05) (Table 2). With respect to novel preglomerular
vascular PKC isoforms (
,
, and
), PKC
and PKC
were
significantly activated (P < 0.05) in high-insulin
STZ-diabetic rats (Fig. 5),
whereas PKC
was the only novel isoform activated in
captopril-treated diabetic rats (Fig. 5). This increase in PKC-specific
isoform activation was accompanied by a significant elevation
(P < 0.05) of the total PKC amount found in preglomerular
vascular membranes of the respective groups (Table 2). In addition,
atypical PKC isoforms (
and
) were unchanged in any of the
experimental groups compared with the control rats (Fig.
6). However, PKC
expression
was enhanced by hyperglycemia (Table 2), whereas it was significantly
activated only in captopril-treated diabetic rats (Fig. 6). On the
other hand, the quantity of PKCµ was significantly augmented in all experimental groups compared with the control rats (Table 2), whereas
its activation was increased in high-insulin- and captopril-treated animals (Fig. 6).
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Histological evaluations. The most significant changes were focal glomerulosclerosis and glomerular structural abnormalities, with vascular lesions of renal arteries in the form of adventia deterioration in low-insulin-treated kidneys. All other experimental groups, high insulin- and captopril-treated as well as control rats, presented no vascular lesions or glomerulosclerosis (results not shown).
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DISCUSSION |
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In the present study, we have demonstrated that ANG II receptors on preglomerular vessels and glomeruli are differentially regulated in STZ-diabetic rats after either insulin or/and captopril treatment. In addition, we have characterized the different PKC isoforms present in both renal glomeruli and preglomerular vessels and their regulation after treatment with either different doses of insulin and/or captopril.
As described previously (25), STZ has the ability to induce experimental insulin-dependent DM, causing pronounced hyperglycemia that can be corrected by daily insulin injections. As a physiological indicator of DM induction, BG was significantly increased in STZ-diabetic rats compared with control animals but was partially corrected, as observed by others (11, 31), by daily treatment with a high dose of insulin. In agreement with earlier studies, BP, hematocrit, and PRA were unchanged in STZ-DM rats treated with insulin (1, 29, 31). However, as reported previously, BP was significantly reduced in STZ-diabetic animals treated with the ACE inhibitor captopril (1, 48). As in earlier studies, diabetic rats treated with a low-insulin dose either alone or with captopril had significant body weight loss (31, 48). This loss in body weight was corrected by high-dose insulin treatment.
Competitive binding studies with the specific AT1-antagonist losartan and the specific AT2-antagonist PD-123319 revealed that AT1 was the only ANG II receptor detected on preglomerular vessels and glomeruli in the kidneys of all groups, whereas the AT2 receptor was not detected. These results are in total agreement with earlier investigations (16, 30), which have clearly demonstrated the absence of renal AT2 receptors and mRNA, respectively. Furthermore, no significant differences in either Bmax or Kd values were observed when freshly prepared glomerular membranes were compared with snap-frozen preparations (data not shown). These binding studies also revealed no significant differences in Kd values among the various experimental groups.
When the density of vascular AT1 receptors from control and STZ-induced rats was considered, significant upregulation was seen in rats treated with only low-insulin dose. Because we have previously shown that preglomerular vascular ANG II receptors are regulated by the renal RAS (2, 3), a plausible explanation for vascular receptor upregulation is decreased activity of the renal RAS in STZ-diabetic rats, which has been clearly documented by Maeda et al. (34). This suggests that the renal RAS plays an important role in the upregulation of AT1 receptors located on preglomerular vessels of diabetic animals. The increase in preglomerular vascular ANG II Bmax can also explain the heightened microvascular resistance vessel responsiveness to ANG II in diabetic rats as reported by Hill and Larkins (26).
On the other hand, the density of glomerular ANG II receptors was downregulated in low-insulin-treated STZ-diabetic rats. These results are in total agreement with previous investigation (45), which have clearly shown glomerular ANG II receptor downregulation in diabetic rats. Furthermore, Wilkes has demonstrated in an elegant study that the induction of glomerular ANG II downregulation is not due to STZ because it can also be reproduced with alloxan, another diabetogenic agent (45). Not only did treatment with either high-dose insulin or a combination of a low-dose insulin with captopril prevent the glomerular ANG II Bmax downregulation observed in low-insulin-treated STZ-diabetic rats but it also caused significant Bmax upregulation compared with control animals. Moreover, it has been clearly demonstrated that insulin treatment prevents renal ANG II receptor downregulation (11).
To ascertain whether PKC isoforms were modified under diabetic conditions, we determined the different PKC isoforms present with their respective activation in both preglomerular vessels and glomeruli. We observed that, in addition to the differential regulation of renal ANG II receptors on preglomerular vessels and glomeruli, there was differential activation of the various PKC isoforms in both renal structures.
As mentioned above, in diabetic animals, hyperglycemia has the ability to activate glomerular PKC isoforms through de novo synthesis of DAG (14). However, to date, no reports have been published that ascertain the effects of hyperglycemia on PKC isoform activation in preglomerular vessels, which have been shown by Arima et al. (6) to be a crucial vascular segment in the control of glomerular hemodynamics.
With respect to the differential activation between glomerular and
preglomerular vascular PKC isoforms, we generally observed that when
PKC isoforms are activated in one of the two tissues, they are not in
the other. This differential activation of PKC isoforms is not unique
to our study, because several other investigators (27, 43) have
reported it previously. However, it must be noted that our results on
the absence of glomerular PKC activation do not agree with those of
Babazono et al. (8).
Furthermore, we report for the first time that in vivo treatments that prevent renal vascular or glomerular damage in STZ-diabetic rats, such as an elevated dose of insulin or low-dose insulin in combination with an ACE inhibitor, can also activate the majority of PKC isoforms found in both renal preglomerular vessels and glomeruli. These results are in agreement with previously published work by Liu and Roth (32), who have shown that insulin has the ability to activate PKC isoforms in vitro.
Interestingly, it has been shown that in cultured rat mesangial cells,
PKC downregulation potentiated the ANG II-induced IP3
formation (40). In addition, Griendling et al. (21) have demonstrated
that ANG II receptor internalization in vascular smooth muscle cells is
directly related to ANG II-induced DAG accumulation secondary to PKC
activation and accompanied by an increase in IP3.
Furthermore, DAG accumulation is inhibited when ANG II receptor
internalization is blocked, suggesting that DAG stimulation is a
necessary step to ANG II receptor internalization and regulation. These
results as well as our data suggest a potential role of PKC activation
in ANG II receptor regulation. It is also noteworthy that similar
receptor regulation by PKC has been observed for other vasoconstrictors
such as endothelin 1 and arginine vasopressin (7, 46).
In conclusion, we have shown that in STZ-diabetic rats, renal ANG II receptors on preglomerular vessels and glomeruli are regulated differentially and after treatment with either insulin and/or an ACE inhibitor. We have found that preglomerular ANG II receptors are upregulated in STZ-diabetic rats, whereas glomerular ANG II receptors are downregulated. However, when STZ-diabetic rats are prevented from developing glomerular structural and vascular damage, ANG II receptors on preglomerular vessels are no longer upregulated whereas glomerular ANG II receptors are significantly upregulated. In addition to these changes in receptor densities, we observed that PKC isoforms are also differentially regulated in both of renal structures. Treatments that prevent vascular and glomerular damage cause an increase in the activation of most PKC isoforms found in these structures.
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ACKNOWLEDGEMENTS |
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The authors thank Suzanne Diebold for excellent technical assistance and Ovid Da Silva for editorial input.
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FOOTNOTES |
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This study was supported by a grant from the Medical Research Council of Canada (MT-11558) and the Kidney Foundation of Canada.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: F. Amiri, Vascular Biology Center, Medical College of Georgia, Augusta, GA 30912-2500 (E-mail: Famiri{at}mail.mcg.edu).
Received 20 May 1999; accepted in final form 24 November 1999.
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