Renal angiotensin II receptors and protein kinase C in diabetic rats: effects of insulin and ACE inhibition

Farhad Amiri and Raul Garcia

Laboratory of Experimental Hypertension and Vasoactive Peptides, Clinical Research Institute of Montreal, Université de Montréal, Montreal, Quebec, Canada H2W 1R7


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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. PKCalpha , PKCdelta , PKCepsilon , 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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 (alpha , beta I, beta II, and gamma ) are Ca2+- and phospholipid-dependent through their C2 domain; the novel PKC isoforms (delta , epsilon , eta  and theta ) lack this region and are, accordingly, Ca2+ independent; the third class, consisting of atypical PKC isoforms (zeta , iota  and lambda ); 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.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 10-5 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 10-12 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 alpha ; 1:2,000 for gamma ; 1:1,000 for delta , iota , µ, 1:500 for beta , epsilon , theta , lambda , zeta ) 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-(alpha -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.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.

                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Physiological characteristics of different groups of rats

Figure 1 presents the representative competition binding curves of preglomerular vascular membranes from low-insulin-treated STZ-diabetic rats using the specific AT1-receptor antagonist losartan and the specific AT2-receptor antagonist PD-123319. Preglomerular vessels and glomeruli from STZ-diabetic and control animals revealed only the AT1 receptor because no displacement was observed with PD-123319.


View larger version (18K):
[in this window]
[in a new window]
 
Fig. 1.   Representative competition binding curves of preglomerular vessels from low-insulin-treated streptozotocin (STZ)-diabetic rats using nonselective ANG II-receptor antagonist [Sar1, Ile8]-ANG II, AT1-receptor antagonist losartan, and AT2-receptor antagonist PD-123319. B and Bo, binding in respective presence and absence of competitor.

Figure 2 demonstrates the Bmax values of AT1 receptors on preglomerular vessels and glomeruli, respectively. The density of AT1 receptors on preglomerular vessels of STZ rats treated with low insulin was significantly higher (P < 0.05) than in the other groups (Fig. 2). Even though the Bmax values of preglomerular vascular receptors in low-insulin-treated STZ animals showed significant differences, no significant difference in Kd was observed in these or any other groups with values, ranging from 1.6 ± 0.9 to 2.3 ± 1.1 nM for preglomerular vessels, and from 1.1 ± 0.3 to 1.4 ± 0.3 nM for glomeruli. Moreover, glomerular Bmax was also significantly different in STZ-treated rats. In low-insulin-treated animals, AT1 receptor density was significantly lower (P < 0.05) compared with the control rats (Fig. 2) but was significantly higher (P < 0.05) in both high-insulin and captopril groups (Fig. 2).


View larger version (35K):
[in this window]
[in a new window]
 
Fig. 2.   ANG II receptor density (Bmax) of preglomerular vessels and glomeruli from low-dose insulin-, high-dose insulin-, low-dose insulin+captopril-treated, and control rats using nonspecific ANG II-receptor antagonist [Sar1,Ile8]-ANG II. Similar results were obtained with losartan. Values are means ± SE; n = 4 binding experiments performed in duplicate. # P < 0.05 vs. control animals. * P < 0.05 vs. control and low insulin-treated animals.

PKC isoform analysis. All PKC isoforms were present in vascular preglomerular fractions (Fig. 3). In preglomerular vessels, PKCalpha and PKCgamma were activated significantly (P < 0.05) in rats treated with either high-dose insulin compared with all other groups (Fig. 4), whereas PKCbeta 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 (delta , epsilon , and theta ), PKCepsilon and PKCtheta were significantly activated (P < 0.05) in high-insulin STZ-diabetic rats (Fig. 5), whereas PKCepsilon 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 (lambda  and zeta ) were unchanged in any of the experimental groups compared with the control rats (Fig. 6). However, PKCiota 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).


View larger version (31K):
[in this window]
[in a new window]
 
Fig. 3.   Representative immunoblots of protein kinase C isoforms in preglomerular vessels of low-dose-insulin-treated STZ-diabetic rats and brain of normal Sprague-Dawley rats. M.W., molecular weight.



View larger version (27K):
[in this window]
[in a new window]
 
Fig. 4.   Fold activation of classic PKC isoforms (alpha , beta  and gamma ; A, B, and C, respectively). PKC isoforms from control animals were considered to be 1.0-fold activated. Each graph contains both renal tissues studied, namely, renal arterioles and glomeruli. Open bars, control group; solid bars, low-insulin-treated STZ-rats; hatched bars, high-insulin-treated STZ-rats; crosshatched bars, captopril- and low-insulin-treated STZ rats. Values are means ± SE; n = 4 Western blot analyses. Area under peak of PKC isoform scanned (both cytosolic and membrane fractions) was determined, and membrane-to-cytosol ratio was used to calculate fold translocation (or activation).* P < 0.05 vs. control group.


                              
View this table:
[in this window]
[in a new window]
 
Table 2.   Total quantity of PKC isoforms in the different experimental groups



View larger version (28K):
[in this window]
[in a new window]
 
Fig. 5.   Fold activation of novel preglomerular vascular PKC isoforms (delta , epsilon , and theta ; A, B, and C, respectively). PKC isoforms from control animals were considered to be 1.0-fold activated. Each graph contains both renal tissues studied, namely, renal arterioles and glomeruli. Bars are defined as in Fig. 4. Values are means ± SE; n = 4 Western blot analyses. Area under peak of PKC isoform scanned (both cytosolic and membrane fractions) was determined, and membrane-to-cytosol ratio was used to calculate fold translocation (or activation). * P < 0.05 vs. control group. # P < 0.001 vs. control group.



View larger version (36K):
[in this window]
[in a new window]
 
Fig. 6.   Fold activation of atypical PKC isoforms (lambda , iota , zeta ) and PKCµ isoform (A, B, C, and D, respectively). PKC isoforms from control animals were considered to be 1.0-fold activated. Each graph contains both renal tissues studied, namely, renal arterioles and glomeruli. Bars are defined as in Fig. 4. Values are means ± SE; n = 4 Western blot analyses. Area under peak of PKC isoform scanned (both cytosolic and membrane fractions) was determined, and membrane-to-cytosol ratio was used to calculate fold translocation (or activation). * P < 0.05 vs. control group. # P < 0.001 vs. control group.

With respect to glomerular PKC isoforms, it was found that the classic PKC isoforms, PKCbeta and PKCgamma , were activated in animals prevented from developing glomerulosclerosis, i.e., the high insulin- and captopril-treated groups (Fig. 4). With respect to the quantity of classic PKC isoforms, PKCbeta was increased significantly in low-insulin-treated rats, whereas PKCalpha was increased in high-insulin-treated animals (Table 2). On the other hand, there were no differences in the quantity of PKCgamma among the different groups compared with the control rats (Table 2). As far as novel PKC isoforms (delta , epsilon , and theta ) were concerned, only PKCtheta was significantly activated (P < 0.05) in animals where the development of glomerulosclerosis was prevented (i.e., in high-insulin- and captopril-treated rats) (Fig. 5), whereas no differences were observed for PKCdelta and PKCepsilon (Fig. 5). However, no significant differences were found in the quantity of any of the novel PKC isoforms compared with the control group (Table 2). Moreover, no significant differences were seen in quantity of the atypical (lambda , iota , and zeta ) and µ isoforms in any of the groups with the exception of an increase of PKCiota in the captopril-treated group and PKCzeta in the high-insulin-treated group (Table 2). With respect to atypical PKC isoform activation (including PKCµ), PKCzeta was the only isoform activated (Fig. 6).

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).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 PKCalpha 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, PKCalpha 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.


    ACKNOWLEDGEMENTS

The authors thank Suzanne Diebold for excellent technical assistance and Ovid Da Silva for editorial input.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Allen, TJ, Cao Z, Youssef S, Hulthen UL, and Cooper ME. Role of angiotensin II and bradykinin in experimental diabetic nephropathy. Functional and structural studies. Diabetes 46: 1612-1618, 1997[Abstract].

2.   Amiri, F, and Garcia R. Differential regulation of renal glomerular and preglomerular vascular angiotensin II receptors. Am J Physiol Endocrinol Metab 270: E810-E815, 1996[Abstract/Free Full Text].

3.   Amiri, F, and Garcia R. Renal angiotensin II receptor regulation in two-kidney, one clip hypertensive rats. Effect of ACE inhibition. Hypertension 30: 337-344, 1997[Abstract/Free Full Text].

4.   Amiri, F, and Garcia R. Regulation of angiotensin II receptors and PKC isoforms by glucose in rat mesangial cells. Am J Physiol Renal Physiol 276: F691-F699, 1999[Abstract/Free Full Text].

5.   Anderson, S, Meyer TW, Rennke HG, and Brenner BM. Control of glomerular hypertension limits glomerular injury in rats with reduced renal mass. J Clin Invest 76: 612-619, 1985[ISI][Medline].

6.   Arima, S, Ito S, Omata K, Takeuchi K, and Abe K. High glucose augments angiotensin II action by inhibiting NO synthesis in in vitro microperfused rabbit afferent arterioles. Kidney Int 48: 683-689, 1995[ISI][Medline].

7.   Awazu, M, Parker RE, Harvie BR, Ichikawa I, and Kon V. Down-regulation of endothelin-1 receptors by protein kinase C in streptozotocin diabetic rats. J Cardiovasc Pharmacol 17, Suppl7: S500-S502, 1991[ISI][Medline].

8.   Babazono, T, Kapor-Drezgic J, Dlugosz JA, and Whiteside C. Altered expression and subcellular localization of diacylglycerol-sensitive protein kinase C isoforms in diabetic rat glomerular cells. Diabetes 47: 668-676, 1998[Abstract].

9.   Bottari, SP, De Jasper M, Stockings UM, and Levens NR. Angiotensin II receptor subtypes. Characterization, signaling mechanism and possible physiological implications. Front Neuroendocrinol 14: 123-171, 1993[ISI][Medline].

10.   Carney, SL, Wong NLM, and Dirks JH. Acute effects of streptozotocin diabetes on rat renal function. J Lab Clin Med 93: 950-961, 1979[ISI][Medline].

11.   Cheng, H-F, Burns KD, and Harris RC. Reduced proximal tubule angiotensin II receptor expression in streptozotocin-induced diabetes mellitus. Kidney Int 46: 1603-1610, 1994[ISI][Medline].

12.   Chiu, AT, Dunscomb J, Kosierowski J, Burton CRA, Santomenna LD, Corjay MH, and Benfield P. The ligand binding signatures of the rat AT1A, AT1B and the human AT1 receptors are essentially identical. Biochem Biophys Res Commun 197: 440-449, 1993[ISI][Medline].

13.   Chiu, AT, Herblin WF, McCall DE, Ardeckly RJ, Carini DJ, Duncia JV, Pease LJ, Wong PC, Wexler RR, Johnson AL, and Timmermans PBMWM Identification of angiotensin II receptor subtypes. Biochem Biophys Res Commun 165: 196-203, 1989[ISI][Medline].

14.   Craven, PA, Davidson CM, and DeRubertis FR. Increase in diacylglycerol mass in isolated glomeruli by glucose from de novo synthesis of glycerolipids. Diabetes 39: 667-674, 1990[Abstract].

15.   Craven, PA, and DeRubertis FR. Protein kinase C is activated in glomeruli from streptozotocin diabetic rats: possible mediation by glucose. J Clin Invest 83: 1667-1675, 1989[ISI][Medline].

16.   De Gasparo, M, and Levens NR. Pharmacology of angiotensin II receptors in the kidney. Kidney Int 46: 1486-1491, 1994[ISI][Medline].

17.   De León, H, and Garcia R. Angiotensin II receptor subtypes in rat renal preglomerular vessels. Receptor 2: 253-260, 1992[ISI][Medline].

18.   Eadington, DW, Swainson CP, Frier BM, and Semple PF. Renal responses to angiotensin II infusion in early type 1 (insulin-dependent) diabetes. Diabetic Med 8: 524-531, 1991[ISI][Medline].

19.   Freener, EP, and King GL. Vascular dysfunction in diabetes mellitus. Lancet 350, Suppl 1: 9-13, 1997.

20.   Gauquelin, G, and Garcia R. Characterization of glomerular angiotensin II receptor subtypes. Receptor 2: 207-212, 1992[ISI][Medline].

21.   Griendling, KK, Delafontaine P, Rittenhouse SE, Gimbrone MAJ, and Alexander RW. Correlation of receptor sequestration with sustained diacylglycerol accumulation in angiotensin II-stimulated cultured vascular smooth muscle cells. J Biol Chem 262: 14555-14562, 1987[Abstract/Free Full Text].

22.   Griendling, KK, Rittenhouse SE, Brock TA, Ekstein LS, Gimbrone MAJ, and Alexander RW. Sustained diacylglycerol formation from inositol phospholipids in angiotensin II-stimulated vascular smooth muscle cells. J Biol Chem 261: 5901-5906, 1986[Abstract/Free Full Text].

23.   Gutkowska, J, Boucher R, and Genest J. Dosage radioimmunologique de l'activité rénine plasmatique. Union Méd Can 106: 446-450, 1977[ISI][Medline].

24.   Healy, DP, Ye M-Q, and Troyanovskaya M. Localization of angiotensin II type 1 receptor subtype mRNA in rat kidney. Am J Physiol Renal Fluid Electrolyte Physiol 268: F220-F226, 1995[Abstract/Free Full Text].

25.   Herr, RR, Eble TE, Bergy ME, and Jahnke HK. Isolation and characterization of streptozotocin. Antibiotics Annu 7: 236-240, 1960.

26.   Hill, MA, and Larkins RG. Altered microvascular reactivity in streptozotocin-induced diabetes in rats. Am J Physiol Heart Circ Physiol 257: H1438-H1445, 1989[Abstract/Free Full Text].

27.   Inoguchi, T, Battan R, Handler E, Sportsman JR, Health W, and King GL. Preferential elevation of protein kinase C isoform beta II and diacylglycerol levels in the aorta and heart of diabetic rats: differential reversibility to glycemic control by islet cell transplantation. Proc Natl Acad Sci USA 89: 11059-11063, 1992[Abstract].

28.   Iwai, N, and Inagami T. Identification of two subtypes in the rat type 1 angiotensin II receptor. FEBS Lett 298: 257-260, 1992[ISI][Medline].

29.   Kalinyak, JE, Sechi LA, Griffin CA, Don BR, Tavangar K, Kraemer FB, Hoffman AR, and Schambelan M. The renin-angiotensin system in streptozotocin-induced diabetes mellitus in the rat. J Am Soc Nephrol 4: 1337-1345, 1993[Abstract].

30.   Kambayashi, Y, Bardhan S, Takahashi K, Tsuzuki S, Inui H, Hamakubo T, and Inagami T. Molecular cloning of a novel angiotensin II receptor isoform in phosphotyrosine phosphatase inhibition. J Biol Chem 268: 24543-24546, 1993[Abstract/Free Full Text].

31.   Kikkawa, R, Kitamura E, Fujiwara Y, Haneda M, and Shigeta Y. Biphasic alteration of renin-angiotensin-aldosterone system in streptozotocin-diabetic rats. Renal Physiol 9: 187-192, 1986[ISI][Medline].

32.   Liu, F, and Roth RA. Insulin-stimulated tyrosine phosphorylation of protein kinase Calpha : evidence for direct interaction of the insulin receptor and protein kianse C in cells. Biochem Biophys Res Commun 200: 1570-1577, 1994[ISI][Medline].

33.   Liu, J-P. Protein kinase C and its substrates. Mol Cell Endocrinol 116: 1-29, 1996[ISI][Medline].

34.   Maeda, S, Kikkawa R, Haneda M, Togawa M, Koya D, Horide N, Kajiwara N, Uzu T, and Shigeta Y. Reduced activity of renal angiotensin-converting enzyme in streptozotocin-induced diabetic rats. J Diabetes Complications 5: 225-229, 1991.

35.   Mogensen, CE. Glomerular filtration rate and renal plasma flow in short-term and long-term diabetes mellitus. Scand J Clin Lab Invest 28: 91-100, 1971[ISI][Medline].

36.   Morris, AD, Boyle DIR, McMahon AD, Greene SA, MacDonald TM, and Newton RW. Adherence to insulin treatment, glycemic control, and ketoacidosis in insulin-dependent diabetes mellitus. Lancet 350: 1505-1510, 1997[ISI][Medline].

37.   Munson, P, and Rodbard D. LIGAND: a versatile computerized approach for characterization of ligand-binding systems. Anal Biochem 107: 220-239, 1980[ISI][Medline].

38.   Nakajima, M, Hutchinson HG, Fujinaga M, Hayashida W, Morshita R, Zhang L, Horiuchi M, Pratt RE, and Dzau VJ. The angiotensin II type 2 (AT2) receptor antagonizes the growth effects of the AT1 receptor: gain-of-function study using gene transfer. Proc Natl Acad Sci USA 92: 10663-10667, 1995[Abstract].

39.   Nishizuka, Y. The molecular heterogeneity of protein kinase C and its implications for cellular regulation. Nature 334: 661-665, 1988[ISI][Medline].

40.   Pfeilschifter, J. Protein kinase C from rat renal mesangial cells: its role in homologous desensitization of angiotensin II-induced polyphosphoinositide hydrolysis. Biochim Biophys Acta 969: 263-270, 1988[ISI][Medline].

41.   Reineck, HJ, and Kreisberg JI. Renal vascular response to angiotensin II in rats with streptozotocin-induced diabetes mellitus (Abstract). Kidney Int 23: 247, 1983[ISI].

42.   Spector, T. Refinement of the Coomassie blue method of protein quantification. Anal Biochem 86: 142-146, 1978[ISI][Medline].

43.   Tang, EY, Parker PJ, Beattie J, and Houslay MD. Diabetes induces selective alterations in the expression of protein kinase C isoforms in hepatocytes. FEBS Lett 326: 117-123, 1993[ISI][Medline].

44.   Timmermans, PBMWM, Wong PC, Chiu AT, and Herblin WF. Nonpeptide angiotensin II receptor antagonists. Trends Pharmacol Sci 12: 55-62, 1991[ISI][Medline].

45.   Wilkes, BM. Reduced glomerular angiotensin II receptor density in diabetes mellitus in the rat: Time course and mechanism. Endocrinology 120: 1291-1298, 1987[Abstract].

46.   Williams, B, Tsai P, and Schrier RW. Glucose-induced downregulation of angiotensin II and arginine vasopressin receptors in cultured rat aortic vascular smooth muscle cells. Role of protein kinase C. J Clin Invest 90: 1992-1999, 1992[ISI][Medline].

47.   Yamada, T, Horiuchi M, and Dzau VJ. Angiotensin II type 2 receptor mediates programmed cell death. Proc Natl Acad Sci USA 93: 156-160, 1996[Abstract/Free Full Text].

48.   Zatz, R, Dunn BR, Meyer TW, Anderson S, Rennke HG, and Brenner BM. Prevention of diabetic glomerulopathy by pharmacological amelioration of glomerular capillary hypertension. J Clin Invest 77: 1925-1930, 1986[ISI][Medline].


Am J Physiol Renal Fluid Electrolyte Physiol 278(4):F603-F612
0363-6127/00 $5.00 Copyright © 2000 the American Physiological Society