The mechanism of angiotensin II binding downregulation by high glucose in primary renal proximal tubule cells

Soo Hyun Park and Ho Jae Han

Hormone Research Center, Department of Veterinary Physiology, College of Veterinary Medicine, Chonnam National University, Kwangju 500-757, Korea


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The renin-angiotensin system plays an important role in the development of diabetic nephropathy. However, the mechanism of ANG II receptor regulation in the renal proximal tubule in the diabetic condition has not been elucidated. Thus we investigated the signal pathways involved in high-glucose-induced downregulation of ANG II binding in primary cultured renal proximal tubule cells. Twenty-five millimolar glucose, but not mannitol and L-glucose, induced downregulation of the AT1 receptor (AT1R) because of a significant decline in maximal binding with no significant change in the affinity constant. Twenty-five millimolar glucose also decreased AT1R mRNA and protein levels. The 25 mM glucose-induced increase in the formation of lipid peroxides was prevented by antioxidants, protein kinase C (PKC) inhibitors, or L-type calcium channel blockers. These agents also blocked 25 mM glucose-induced downregulation of 125I-ANG II binding. In addition, 25 mM glucose increased transforming growth factor (TGF)-beta 1 secretion, and anti-TGF-beta antibody significantly blocked 25 mM glucose-induced downregulation of 125I-ANG II binding. Furthermore, the 25 mM glucose-induced increase in TGF-beta 1 secretion was inhibited by PKC inhibitors, L-type calcium channel blockers, or antioxidants. In conclusion, high glucose may induce downregulation of 125I-ANG II binding via a PKC-oxidative stress-TGF-beta signal cascade in primary cultured rabbit renal proximal tubule cells.

angiotensin II receptor; protein kinase C; transforming growth factor-beta 1


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE RENIN-ANGIOTENSIN SYSTEM (RAS) has been recently reported to be deeply associated with the development of diabetic nephropathy (27). All of the biological actions of ANG II, an active component of RAS, have been attributed to cell surface receptors. There are two major subtypes of ANG II receptors, referred to as angiotensin types 1 (AT1) and 2 (AT2). Recent studies have indicated that animals with untreated diabetes manifest a significant decrease in expression of AT1 receptor mRNA, which was associated with decreases in 125I-ANG II binding (6, 8, 23). However, Becker et al. (4) reported that AT1 receptor expression and ANG II binding were increased in the high-glucose condition in SV40 immortalized rabbit proximal tubule cells (PTCs). Thus it is unclear whether high glucose upregulates or downregulates ANG II receptors in the diabetic condition. Moreover, the factors responsible for ANG II receptor regulation in diabetic nephropathy also remain undefined.

It has been widely accepted that free radicals are involved in the pathogenesis of diabetic nephropathy by their severe cytotoxic effects, such as lipid peroxidation and protein denaturation in cell membrane, followed by the alteration of the membrane receptor, fluidity, and properties (17, 41). In addition, excessive glucose can be transported intracellularly and metabolized to change redox potential, increase sorbitol production via aldose reductase, or alter signal transduction pathways, such as the activation of diacylglycerol (DAG) and protein kinase C (PKC) levels (24, 26, 38). It has been known that activation of DAG and PKC pathways in diabetic animals contributes to development of diabetic nephropathy (2). Our groups and others have also reported that high glucose activates PKC in the rabbit proximal tubule and rat renal glomerulus (19, 25), suggesting the possible role of PKC in the regulation of ANG II receptor binding. Recently, many researchers have shown increases in the production and activity of transforming growth factor (TGF)-beta 1, an important cytokine in the development of diabetic nephropathy, especially in PTCs in the diabetic condition (22, 35). Treatment with anti-TGF-beta -neutralizing antibodies was shown to prevent glomerular hypertrophy in diabetic animals (37) and high-glucose-induced inhibition of PTC proliferation (36). In R3T3 cells, TGF-beta decreased expression of the AT2 receptor and mRNA (29). These reports suggest the possibility that PKC, oxidative stress, and TGF-beta 1 may be involved in the high-glucose-induced regulation of ANG II receptor binding. However, signal transduction pathways linked to high-glucose-induced ANG II receptor regulation have not been fully elucidated even in other tissues, including renal PTCs.

The primary rabbit kidney PTC culture system that was utilized in this report retains in vitro the differentiated phenotype typical of the renal proximal tubule, which includes a polarized morphology, apical membrane proteins (leucine aminopeptidase and gamma -glutamyl transpeptidase), distinctive proximal tubule transport systems (9, 40), as well as hormonal responses (34). Therefore, PTCs in hormonally defined, serum-free culture conditions would be a powerful tool for studying the effect of high glucose on the ANG II receptor (19, 20, 32). The binding method was used for the ANG II receptor assay because the binding assay reflects biological specificity of ANG II and is quantitatively reproducible. Therefore, on the physiological level, we investigated interaction between ANG II binding and oxidative stress, PKC, or TGF-beta in PTCs under normal- and high-glucose concentrations. We first demonstrated herein that high glucose decreased ANG II binding through a PKC-oxidative stress-TGF-beta cascade in PTCs.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Materials. DMEM and Ham's F-12 nutrient mixture (DMEM/F-12) and class IV collagenase were purchased from Life Technologies (GIBCO BRL, Grand Island, NY). D-Glucose, L-glucose, mannitol, taurine, ascorbic acid (vitamin C; Vc), or N-acetylcystein (NAC), methoxyverapamil, nifedipine, H-7, rabbit immunoglobulin G, actinomycin D, cycloheximide, 12-O-tetradecanoylphorbol-13-acetate (TPA), 1,2-dioctanoyl-sn-glycerol (DOG), and 1-oleoyl-2-acetyl-sn-glycerol (OAG) were obtained from Sigma (St. Louis, MO). 125I-[Sar1, Ile8]-ANG II and losartan (DuP-753) were purchased from DuPont-NEN (Boston, MA). PD-123319 was purchased from Parke-Davis. A PKC enzyme assay system (code RPN 77) was purchased from Amersham (Buckinghamshire, UK). TGF-beta 1 from porcine platelets and anti-TGF-beta antibody were purchased from R&D Systems (Minneapolis, MN). Antibody to AT1R was obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Isolation of rabbit renal proximal tubules and culture conditions. Rabbit renal PTCs in primary culture were prepared by a modification of the method of Chung et al. (9). The PTCs were grown in DMEM/F-12 (i.e., 5 mM glucose) supplemented with 15 mM HEPES buffer (pH 7.4), 20 mM sodium bicarbonate, and three growth supplements (5 µg/ml insulin, 5 µg/ml transferrin, and 5 × 10-8 M hydrocortisone). Kidneys were perfused via the renal artery, first with PBS, and subsequently with DMEM/F-12 containing 0.5% iron oxide (wt/vol) until the kidney turned gray black in color. Renal cortical slices were prepared by cutting of the renal cortex and then homogenization with four strokes of a sterile glass homogenizer. The homogenate was poured first through a 253- and then through a 83-µm mesh filter. Tubules and glomeruli on top of the 83-µm filter were transferred into sterile DMEM/F-12 medium containing a magnetic stirring bar. Glomeruli (containing iron oxide) were removed with a magnetic stirring bar. The remaining proximal tubules were briefly incubated in DMEM/F-12 containing 60 µg/ml collagenase (class IV) and 0.025% soybean trypsin inhibitor. The dissociated tubules were then washed by centrifugation, resuspended in DMEM/F-12 containing the three growth supplements, and transferred into tissue culture dishes. PTCs were maintained at 37°C, in a 5% CO2-humidified environment in DMEM/F-12 medium containing the three supplements. Medium was changed 1 day after plating and every 2 days thereafter. In this experiment, a 25 mM glucose condition was prepared by addition of 20 mM glucose to basal medium (5 mM glucose).

 125I-ANG II binding. The ANG II binding assays were performed as described by Becker and Harris (3). To summarize, after the incubation of PTCs with 25 mM glucose for 48 h, confluent monolayers of PTCs were washed twice with ice-cold PBS containing 0.1% albumin (PBS-A), and cells were incubated in PBS with 0.1% albumin, supplemented with 125I-[Sar1, Ile8]ANG II (0.1 nM) at 4°C for 4 h, followed by three washes with the same ice-cold PBS-A. After solubilization in 0.5 N NaOH (1 ml), 900 µl of each sample were transferred into a scintillation tube and counted in a gamma counter (Wallac Wizard 1470, Turku, Finland). The remainder of each sample was used for protein determination by the Bradford method (5). When 125I-ANG II binding was conducted at 4°C for further incubation time, there was no significant difference between two groups, suggesting that equilibrium binding has occurred by 4 h. Internalization studies were performed after the final binding study wash. A subset of cells was placed in PBS-A at 37°C for 20 min. PBS-A then was replaced with ice-cold acid buffer (pH 3, 50 mM acetic acid, 150 mM NaCl) for 5 min at 4°C. The acid wash was removed, and the cells were lysed with 0.05 M NaOH. An aliquot of lysate was counted in a gamma counter to determine cell-associated radioactivity, a measure of internalized 125I-ANG II. Acid wash radioactivity was also counted. The radioactive counts in each sample were then normalized with respect to protein.

AT1R analysis by RT-PCR. Total RNA was prepared from the cells using TRIzol according to the manufacturer's instructions (GIBCO BRL). mRNA was reverse transcribed with oligo (dT) to first-strand cDNA using Superscript II RT (Qiagen, Hilden, Germany). The sequences of PCR primers are as follows: rabbit AT1R (sense 5'-CAT CAT CTT TGT GGT GGG AA-3'; antisense 5'-GCC AGC CAG CAG CCA AAT AA-3') and beta -actin (sense 5'-AAC CGC GAG AAG ATG ACC CAG ATC ATG TTT-3'; antisense 5'-AGC AGC CGT GGC CAT CTC TTG CTC GAA GTC-3'). The PCR reactions were run for 35 cycles of 94°C (30 s), 57°C (30 s), and 72°C (30 s) using a MJ thermal cycler (Watertown, MA). After the amplification, the RT-PCR products were separated in 1.5% (wt/vol) agarose gels and stained with ethidium bromide.

Western blot analysis of the AT1 receptor. The effect of glucose on AT1 receptor protein was evaluated by using AT1 receptor antibodies. Briefly, PTCs were incubated with 5 or 25 mM glucose for 48 h. Cells were lysed in 100 µl of lysis buffer [62.5 mM Tris-HCl, pH 6.8 containing 2% SDS (wt/vol), 10% glycerol, 50 mM dithiothreitol, and 0.1% bromophenol blue (wt/vol)] and harvested in Eppendorf tubes. The cell lysates were sonicated for 2 s, heated at 95°C for 5 min, and then centrifuged at 12,000 g for 2 min at 4°C. Small aliquots (20 µl) of the supernatants were subjected to PAGE (10%) containing SDS-PAGE and then transferred onto a nitrocellulose membrane (Hybond C Extra, Amesharm Pharmacia Biotech). The nitrocellulose sheet was blocked with 5% nonfat dry milk in Tris-buffered saline. AT1R protein was detected with specific polyclonal antibodies for AT1R. The blots were developed using a peroxidase-conjugated secondary antibody, and proteins were visualized by the ECL system (ECL, Amersham Life Science).

Measurement of lipid peroxide. The levels of lipid peroxides (LPO) in the monolayer cells was determined by measuring malondialdehyde content according to the method of Ohkawa et al. (31). The cells were harvested and sonicated. One hundred microliters of sonicated cells were mixed with 8% SDS (100 µl), 0.8% 2-thiobarbituric acid (TBA; 200 µl), and 20% acetic acid (200 µl). The mixture was heated to 95°C for 60 min. After reaction time, this mixture was cooled in ice-cold water. To extract nonspecific red pigment, n-butanol-pyridine mixture (15:1 vol/vol, 1 ml) was added, and the mixture was shaken vigorously and then centrifuged at 8,000 g for 10 min. The upper organic layer was measured by spectrofluorometry at emission wavelength of 553 nm with an excitation wavelength of 515 nm. 1,1,3,3-Tetraethoxypropane was used as a standard, and the values of LPO for samples were expressed as nanomoles per milligram protein. In this study, 2 mM taurine was added to cell mixtures to prevent any initiation of membrane lipid peroxidation during the assay.

PKC assay. For PKC assay, PTCs grown in 35-mm plates were incubated with 25 mM glucose for 48 h and were washed by ice-cold buffer [10 mM Tris · HCl (pH 7.5), 0.25 M sucrose, 0.2 mM CaCl2, 1 mM phenylmethylsufonyl fluoride (PMSF), 10 µg/ml leupeptin and 10 mM benzamidine] and were separated into cytosolic and membrane fractions, using ultracentrifugation. Aliquots of cytosolic and membrane fractions were assayed for PKC activity by using the PKC enzyme assay system kit and expressed as picomoles per phosphate per minute per milligram protein.

Assay for TGF-beta . Active TGF-beta 1 concentration in 200 µl of media collected from PTCs was measured using an enzyme-linked immunosorbent assay (R&D Systems). The total protein content of lysed cells was measured, and TGF-beta 1 was expressed as nanograms per milligram protein.

Statistical analysis. Results were expressed as means ± SE. The difference between two mean values was analyzed by ANOVA. The difference was considered statistically significant when P < 0.05.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Effect of high glucose on 125I-ANG II binding. This study examined the relationship between glucose concentration and 125I-ANG II binding to PTCs. Confluent PTCs were incubated with different concentrations of glucose (5-50 mM) for 48 h before the binding of 125I-ANG II to PTCs was determined. As shown in Fig. 1, glucose significantly downregulated 125I-ANG II binding in a dose-dependent manner. Although 15 mM glucose significantly downregulated 125I-ANG II binding, we used 25 mM glucose for 48 h in the sequential experiments to manifest the effect of high glucose. High-glucose concentrations can result in altered medium osmolarity. To rule out an influence of altered osmolarity on 125I-ANG II binding, we also tested the effect of L-glucose or mannitol. 125I-ANG II binding in monolayers treated with mannitol or L-glucose (0-45 mM) for 48 h did not cause downregulation of 125I-ANG II binding (Fig. 1). In addition, we also investigated which ANG II receptor was downregulated by high glucose. Figure 2A depicted that, in the 125I-ANG II binding assay, losartan inhibits 125I-ANG II binding in a dose-dependent manner. However, PD-123319 even at a high dosage (10-5 M) has a mildly inhibitory effect on 125I-ANG II binding. Furthermore, the AT1R binding site is greatly downregulated by high glucose (Fig. 2B), and high-glucose-induced downregulation of 125I-ANG II was due to the decrease in both the membrane and internalized 125I-bound forms (Fig. 2C). In addition, high glucose decreased AT1 mRNA and the AT1R protein level (Fig. 3).


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Fig. 1.   Dose-dependent effect of glucose, L-glucose, and mannitol on specific binding of 125I-ANG II. When cultures were confluent, different dosage of glucose, L-glucose, or mannitol (0-45 mM) were added to proximal tubule cells (PTCs) for 48 h. Values are means ± SE of 3 independent experiments with triplicate dishes. *P < 0.05 vs. control.



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Fig. 2.   A: effect of losartan and PD-123319 on specific binding of 125I-ANG II binding. Cells were incubated with losartan or PD-123319 at different dosage (10-9, 10-7, 10-5 M) in the presence of 125I-ANG II (0.1 nmol/ml), and the level of 125I-ANG II binding was determined as described in MATERIALS AND METHODS. B: effect of losartan and PD-123319 on specific binding of 125I-ANG II in the presence of 25 mM glucose. HG, high glucose. C: effect of 25 mM glucose on specific 125I-ANG II membrane binding and internalization. Values are means ± SE of 3 (A) or 4 (B and C) independent experiments with triplicate dishes. *P < 0.05 vs. control. **P < 0.05 vs. 25 mM glucose alone.



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Fig. 3.   A: effect of 25 mM glucose on ANG II receptor type 1 (AT1R) mRNA using RT-PCR. PTCs were treated with 25 mM glucose for 48 h before RT-PCR analysis. Band represents 364 bp of AT1R. For standardization, corresponding beta -actin PCR products (354 bp) are shown. B: effect of 25 mM glucose on AT1R protein. Band represents ~50 kDa of AT1R. The example shown is a representative of 3 experiments.

The next study examined the effect of high glucose on the kinetic properties of specific 125I-ANG II binding. In presence of 5 mM glucose, binding data for specific 125I-ANG II binding to confluent PTCs demonstrated a single class of 125I-ANG II binding sites with a maximum number of binding sites of 119.11 ± 7.74 fmol/mg protein and an affinity constant of 4.79 ± 0.49 nM (Fig. 4B). In contrast, however, preexposing PTCs for 48 h to a 25 mM glucose medium markedly reduced 125I-ANG II binding by ~52% compared with 5 mM glucose medium, due to a significant decline in maximal binding (57.61 ± 1.83 fmol/mg protein) with no significant change in the affinity constant (4.68 ± 0.20 nM). The high-glucose-induced decrease in 125I-ANG II binding did not represent PTC toxicity or accelerated cell death because the lactate dehydrogenase assay was also similar after a 48-h exposure to all test media, as previously described (19).


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Fig. 4.   A: kinetic binding analysis of ANG II by high glucose. PTCs were treated with different dosages of [Sar1, Ile8]ANG II in the presence of 0.1 nM 125I-ANG II, and the level of 125I-ANG II binding was determined as described in MATERIALS AND METHODS. B: Scatchard plots of ANG II binding to PTCs under 5 or 25 mM glucose condition. The affinity constant (Kd) was 4.79 ± 0.49 and 4.68 ± 0.20 nM in the presence of 5 or 25 mM glucose, respectively. Values are means ± SE of 4 independent experiments with triplicate dishes. *P < 0.05 vs. control.

Role of oxidative stress in the high-glucose-induced downregulation of 125I-ANG II binding. To examine the involvement of oxidative stress in the high-glucose-induced downregulation of 125I-ANG II binding, we measured the effect of high glucose on LPO formation. When cultured cells were preincubated for 48 h with 25 mM glucose, 25 mM glucose significantly increased LPO formation to 0.76 nmol/mg protein compared with control (0.45 nmol/mg protein; P < 0.05). However, mannitol and L-glucose did not affect LPO formation (data not shown). Figure 5A showed that 25 mM glucose or H2O2 (10-7 M) significantly increased LPO formation (P < 0.05), although there was no synergistic effect. This 25 mM glucose-induced increase in LPO was effectively inhibited by addition of taurine (2 mM), ascorbic acid (1 mM), or NAC (10-5 M) although taurine, ascorbic acid, or NAC alone did not cause any significant effect on LPO formation produced by 5 mM glucose. Therefore, to examine the role of oxidative stress in the 25 mM glucose-induced decrease in 125I-ANG II binding, H2O2, taurine, ascorbic acid, and NAC were used. As shown in Fig. 5B, H2O2 significantly downregulated 125I-ANG II binding by 38% compared with control (P < 0.05). However, taurine, ascorbic acid, or NAC prevented the downregulation of 25 mM glucose on 125I-ANG II binding although they had no significant effect per se on 125I-ANG II binding.


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Fig. 5.   Effects of antioxidants and hydrogen peroxide on high-glucose-induced lipid peroxide (LPO) formation (A) and 125I-ANG II binding (B). PTCs were treated with taurine (2 mM), ascorbic acid (vitamin C; Vc, 1 mM), or N-acetylcystein (NAC; 10-5 M) before the treatment with 25 mM glucose or were incubated with 25 mM glucose alone or together with hydrogen peroxide (10-7 M) for 48 h. Values are means ± SE of 3 independent experiments with triplicate dishes. *P < 0.05 vs. control. **P < 0.05 vs. 25 mM glucose alone.

Effects of PKC on LPO formation and 125I-ANG II binding. To examine the effects of PKC on LPO formation and 125I-ANG II binding, confluent monolayers were treated with TPA, DOG, or OAG (100 ng/ml), artificial PKC activators, for 48 h. Figure 6 showed that TPA, DOG, or OAG alone increased LPO formation by 52, 48, and 46% and downregulated 125I-ANG II binding by 36, 49, and 36% compared with control, respectively. H-7 (10-7 M, a PKC inhibitor) blocked 25 mM glucose-induced stimulation of LPO formation. As evidence of this, downregulation of PKC activity by preincubation with TPA (500 ng/ml) for 24 h also prevented 25 mM glucose action on LPO formation (Fig. 7A). Therefore, we examined the relationship between oxidative stress and PKC in the 25 mM glucose-induced downregulation of 125I-ANG II binding. As shown in Fig. 7B, H-7 and TPA pretreatment significantly blocked 25 mM glucose-induced inhibition of 125I-ANG II binding. These results strongly suggest that high glucose increased LPO formation and downregulated 125I-ANG II binding via a PKC signal pathway. Indeed, 25 mM glucose for 48 h increased the PKC activity (Fig. 8). In addition, we examined the possibility that calcium may be involved in the high-glucose-induced increase in LPO formation and downregulation of 125I-ANG II binding. As shown in Fig. 7, nifedipine or methoxyverapamil, L-type Ca2+ channel blockers, prevented high-glucose-induced increase of LPO formation and inhibition of 125I-ANG II binding. This result suggests that extracellular calcium may be involved in the high-glucose-induced action of LPO formation and 125I-ANG II binding.


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Fig. 6.   Effects of 12-O-tetradecanoylphorbol-13-acetate (TPA), 1,2-dioctanoyl-sn-glycerol (DOG), or 1-oleoyl-2-acetyl-sn-glycerol (OAG) on LPO formation (A) and 125I-ANG II binding (B). PTCs were treated with TPA, DOG, or OAG (100 ng/ml, respectively) for 48 h. Then, an LPO formation assay and 125I-ANG II binding assay were conducted. Values are means ± SE of 3 independent experiments with triplicate dishes. *P < 0.05 vs. control.



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Fig. 7.   Effects of H-7, TPA pretreatment, nifedipine, and methoxyverapamil on high-glucose-induced LPO formation (A) and 125I-ANG II binding (B). PTCs were treated with H-7 (10-7 M) for 30 min or TPA pretreatment (pretreat; 500 ng/ml) for 24 h, nifedipine, or methoxyverapamil (10-6 M) for 30 min before the treatment with 25 mM glucose. Values are means ± SE of 3 independent experiments with triplicate dishes. *P < 0.05 vs. control. **P < 0.05 vs. 25 mM glucose alone.



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Fig. 8.   Effect of high glucose on protein kianse C (PKC) activity. PTCs were treated with 25 mM glucose for 48 h. Then, PTCs were scraped off into a tube after being washed with ice-cold PBS and were homogenized. With this sample, PKC activity was measured as described in MATERIALS AND METHODS. Values are means ± SE of 3 independent experiments with triplicate dishes. *P < 0.05 vs. control.

Involvement of TGF-beta in high-glucose-induced LPO formation and 125I-ANG II binding. Next, we examined the involvement of TGF-beta in the high-glucose-induced 125I-ANG II binding. As shown in Fig. 9A, TGF-beta 1 downregulated 125I-ANG II binding in a dose-dependent manner. Furthermore, studies with a neutralizing antibody to TGF-beta were performed to examine the involvement of TGF-beta in the high-glucose-induced downregulation of 125I-ANG II binding. Figure 9B depicts that anti-TGF-beta antibody (20 µg/ml) significantly blocked 25 mM glucose-induced downregulation of 125I-ANG II binding. In contrast, rabbit IgG (20 µg/ml) did not block the 25 mM glucose action on 125I-ANG II binding. Indeed, 25 mM glucose, TPA, DOG, OAG, or H2O2 significantly increased TGF-beta 1 secretion compared with 5 mM glucose (Fig. 10A). We also examined which signal pathways are involved in high-glucose-induced TGF-beta 1 secretion. Figure 10B demonstrates that H-7, TPA pretreatment, methoxyverapamil, NAC, and taurine significantly blocked 25 mM glucose-induced TGF-beta 1 secretion. These results suggest that TGF-beta 1 may be involved in the high-glucose-induced downregulation of 125I-ANG II binding and high glucose stimulates TGF-beta 1 secretion via a PKC-oxidative stress signal pathway. In addition, we investigated the relationship between PKC and extracellular Ca2+ influx. As shown in Table 1, TPA increased LPO formation and TGF-beta 1 secretion and downregulated ANG II binding. However, TPA did not exhibit these effects in the presence of nifedipine (10-6 M). These results suggest that PKC may cause the Ca2+ influx from the extracellular portion, which mediates LPO formation, TGF-beta 1 secretion, and 125I-ANG II binding.


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Fig. 9.   A: effect of transforming growth factor (TGF)-beta 1 on 125I-ANG II binding. PTCs were treated with different dosages of TGF-beta 1 (0.1-10 ng/ml) for 48 h. B: effect of anti-TGF-beta antibody on high-glucose-induced 125I-ANG II binding. PTCs were treated with anti-TGF-beta -antibody (20 µg/ml) or rabbit IgG (20 µg/ml) for 30 min before the treatment with 25 mM glucose. Values are means ± SE of 3 independent experiments with triplicate dishes. *P < 0.05 vs. control. **P < 0.05 vs. 25 mM glucose alone.



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Fig. 10.   A: effect of 25 mM glucose, mannitol, L-glucose, TPA, DOG, OAG, or H2O2 on TGF-beta 1 secretion. PTCs were treated with 25 mM glucose, mannitol, L-glucose, TPA (100 ng/ml), DOG (100 ng/ml), OAG (100 ng/ml), or H2O2 (10-7 M) for 48 h. B: effect of H-7, TPA pretreatment, methoxyverapamil, NAC, and taurine on high-glucose-induced TGF-beta 1 secretion. PTCs were treated with H-7 (10-7 M) for 30 min or TPA pretreatment (500 ng/ml) for 24 h, methoxyverapamil (10-6 M), NAC (10-5 M), or taurine (2 mM) for 30 min before treatment with 25 mM glucose. Values are means ± SE of 3 independent experiments with duplicate dishes. *P < 0.05 vs. control. **P < 0.05 vs. 25 mM glucose alone.


                              
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Table 1.   Effects of Ca2+ channel blocker on TPA-induced lipid peroxide formation, TGF-beta 1 secretion, and downregulation of 125I-ANG II binding

Furthermore, we examined whether RNA and/or protein synthesis is involved in the high-glucose-induced downregulation of 125I-ANG II binding. Actinomycin D (10-8 M) and cycloheximide (10-6 M) alone did not induce cell toxicity, as assayed by a trypan blue exclusion test (data not shown) and had no effect on 125I-ANG II binding. However, they blocked the action of 25 mM glucose on 125I-ANG II binding and TGF-beta 1 secretion, suggesting the role of transcription and translation (Fig. 11).


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Fig. 11.   Effects of actinomycin D and cycloheximide on high-glucose-induced TGF-beta 1 secretion (A) and 125I-ANG II binding downregulation (B). PTCs were treated with actinomycin D (10-7 M) or cycloheximide (10-5 M) for 1 h before treatment with 25 mM glucose. Values are means ± SE of 3 independent experiments with triplicate dishes. *P < 0.05 vs. control. **P < 0.05 vs. 25 mM glucose alone.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In the present study, we first demonstrated that 125I-ANG II binding was downregulated in a 25 mM glucose environment through PKC-oxidative stress-possibly TGF-beta 1 pathways in the primary cultured rabbit renal proximal tubule cells. The direction of change in AT1 receptor density may depend on the specific cell type and the capacity for that cell type to use glucose as an energy substrate (4, 6). 125I-ANG II binding by PTCs exposed to 25 mM glucose was significantly decreased after 48 h, compared with cells exposed to 5 mM glucose. This downregulation of 125I-ANG II binding was not reproduced with mannitol or L-glucose, suggesting that the action is specific for glucose specificity but not for hyperosmolarity. When receptor density (Bmax) from PTCs grown in 5 and 25 mM glucose concentration was compared, significant decrease was observed in 25 mM glucose. In contrast, the affinity constant (Kd) for ANG II binding was not altered in PTCs from different concentration of glucose. Competitive displacement studies using the ANG II antagonists losartan and PD-123319 demonstrated that the AT1 receptor was the major subtype localized in PTCs. This result is inconsistent with the report of Dulin et al. (12), in which it was demonstrated that rabbit proximal tubule has the AT2 receptor as its major receptor. However, Burns et al. (7) reported that specific binding of 125I-ANG II of rabbit proximal tubule was inhibited by the AT1 ANG II-receptor antagonist losartan, but not by the AT2 antagonist PD-123319. These differences may be due to cell culture condition, as reported in the previous report (20). In the present study, we demonstrated that high glucose predominantly downregulated AT1 receptor binding, consistent with the results regarding AT1R mRNA and AT1R protein expression. This result is similar to the reports of some investigators who demonstrated that downregulation of ANG II binding was associated with AT1R mRNA and protein in diabetic rat kidney (8, 30). Because it has been established by several investigators that ANG II receptor densities in renal proximal tubule cells are significantly reduced in diabetic rats (8, 30), our present results suggest that PTCs grown in 25 mM glucose mimic the in vivo situation of ANG II receptors in diabetic animals. This result is also consistent with observations in glomerular and aortic vascular smooth cells (1, 39). Recent studies showed that hyperglycemia may be toxic either by nonenzymatic reaction of glucose with proteins and subsequent formation of advanced glycosylation end products or by increased metabolism, which leads to increased oxidative stress and activation of PKC that results in increased production of cytokines (13, 16). Nonetheless, many questions remain unanswered regarding ANG II receptor regulation in diabetic nephropathy. Thus to elucidate the mechanisms involved in high-glucose-induced alterations of ANG II binding, we examined key modulators involved in 125I-ANG II receptor regulation in PTCs exposed to high glucose.

Several lines of evidence suggest that hyperglycemia may increase the generation of free radicals in many ways, such as glucose autoxidation, autoxidative glycosylation (glycoxidation), increased polyol pathway metabolism with subsequent "pseudohypoxia," and decreased natural antioxidant defenses (14). Generation of different kinds of reactive oxygen radicals can oxidize membrane lipids or proteins and inactive enzymes, which can impair cellular functions and lead to cell death (17, 41). However, direct evidence demonstrating the relationship between 125I-ANG II binding and LPO formation in the high-glucose condition is not yet known. In the present study, we clearly demonstrated that high glucose can increase the levels of LPO in PTCs as a result of glucose-induced oxidative stress and taurine; the endogenous antioxidant in the kidney can block high glucose-induced LPO formation and decrease in 125I-ANG II binding. In addition, other antioxidants (NAC and ascorbic acid) also blocked it. The effect of glucose and H2O2 on ANG II binding was not mediated by cellular toxicity, because glucose and H2O2 did not increase lactate dehydrogenase (19). Craven et al. (10) demonstrated that elevated glucose concentrations enhanced de novo synthesis of DAG in isolated glomeruli and concluded that this increase in DAG mass may contribute to glucose-induced activation of PKC. Williams et al. (39) reported that high-glucose-induced downregulation of the ANG II receptor and PKC inhibitor attenuated high-glucose-induced downregulation of ANG II binding in cultured rat aortic vascular smooth muscle cells, suggesting the role of PKC as its regulating factor. However, they did not elucidate the PKC-induced downstream signal pathway. In the present study, we demonstrate that the decrease in 125I-ANG II binding induced by a high glucose level is mimicked by TPA, DOG, OAG, and PKC activators and its effect is restored to normal by H-7, PKC inhibitor or TPA pretreatment, or PKC downregulation. These present results provide strong evidence that high-glucose-induced ANG II receptor downregulation is dependent on the capacity of glucose to activate PKC in PTCs. Thus it is possible that the PKC activation induced by high glucose could be the result of excessive oxidants, which are known to activate PKC. However, we showed that H-7 strongly suppressed LPO formation in PTC exposed to high glucose, suggesting the role of PKC in high glucose-induced increase of oxidative stress. Therefore, the possibility that extracellular glucose might directly mediate receptor downregulation is suggested by studies of PTCs, in which a high-glucose environment acutely activates PKC and reduces AT1 receptor abundance. It is also possible that PKC activation mediates AT1 receptor desensitization because intracellular accumulation of DAG, the stimulator of PKC, has been shown to stimulate ANG II receptor internalization in vascular smooth muscle cells (15). Our present study also revealed that extracellular Ca2+ influx as well as PKC activation was involved in high-glucose-induced downregulation of 125I-ANG II binding. In our previous report (33) and the present results, high glucose stimulated Ca2+ uptake via PKC activation, and the L-type Ca2+ channel blocker prevented high-glucose-induced downregulation of 125I-ANG II binding. TPA-induced LPO formation, secretion of TGF-beta 1, and downregulation of 125I-ANG II binding were also prevented by the L-type Ca2+ channel blocker, suggesting that PKC is an upstream regulator of Ca2+ influx in high-glucose-induced downregulation of 125I-ANG II binding. This result is correlated with the in vivo situation. In the PTCs of streptozotocin-induced diabetic rats, the elevation of intracellular Ca2+ concentration ([Ca2+]i) is associated with the downregulation of AT1 receptor mRNA and the Ca2+ channel blocker amilodipine prevents downregulation of mRNA of AT1 receptor (30). Thus our present results also suggest that the activation of the L-type Ca2+ channel via PKC may play an important role in increase of oxidative stress, causing downregulation of ANG II binding in PTCs under high-glucose conditions.

Increased activity of the renal TGF-beta system in diabetic animals suggests that TGF-beta is one of the common mediators of diabetic nephropathy. Sharma et al. (37) also reported that anti-TGF-beta antibody attenuated kidney hypertrophy and the enhanced extracellular matrix gene expression in streptozotocin-induced diabetic mice. Han et al. (18) demonstrated that treatment with anti-sense TGF-beta 1 oligodeoxynucleotide attenuates high-glucose-induced PTC hypertrophy in vitro and partially prevents the increase in kidney weight and extracellular matrix expression in diabetic mice. In our present study, TGF-beta 1 may be responsible for the high-glucose-induced inhibition of ANG II binding, because high glucose increased TGF-beta 1 secretion, exogenous TGF-beta 1 treatment downregulated 125I-ANG II binding, and anti-TGF-beta antibody prevents high-glucose-induced downregulation of 125I-ANG II binding. Such stimulation of TGF-beta 1 secretion may be mediated by PKC activation due to increased DAG and [Ca2+]i by high glucose. Consistent with our result, oral treatment with LY-333531, a PKC-beta isoform inhibitor, prevented glomerular TGF-beta 1 mRNA expression in diabetic rat (25). Although we did not measure TGF-beta mRNA expression, high glucose may increase TGF-beta 1 mRNA expression, because actinomycin D and cycloheximide attenuate high-glucose-induced downregulation of 125I-ANG II binding and secretion of TGF-beta 1. Hoffman et al. (21) reported that the stimulation of TGF-beta 1 production is attributed to transcriptional activation involving a region in the TGF-beta 1 promoter containing a glucose-response element. Surprisingly, up until now, there were no studies about the involvement of TGF-beta 1 in the downregulation of ANG II receptor binding. We first demonstrated here that TGF-beta 1 is involved in the high-glucose-induced downregulation of ANG II receptor binding, because anti-TGF-beta antibody significantly prevented high-glucose-induced ANG II binding and TGF-beta 1 also significantly downregulates ANG II binding. This result is not consistent with the report of Lebrethon et al. (28), who demonstrated that TGF-beta 1 enhanced AT1 mRNA and its binding site in adrenal cells. Although it remains to be determined, this may be due to differences in animal species and cell culture condition. In the present study, antioxidants blocked high-glucose-induced TGF-beta 1 secretion, suggesting the involvement of oxidative stress, consistent with a study of the diabetic rat (17). Craven et al. (11) also reported that treatment with vitamin C or E prevented the increase in glomerular TGF-beta immunohistochemical staining. The stimulatory effect of oxidative stress and TGF-beta 1 secretion by high glucose are also dependent on PKC activation, suggesting the role of a PKC-oxidative stress-TGF-beta 1 signal cascade in downregulation of ANG II binding. Thus the present findings support the hypothesis that the high-glucose milieu of diabetes increases PKC activity, which results in stimulation of TGF-beta 1 secretion, leading to decreased ANG II receptor binding. Further studies to identify other related factors, such as AT1 receptor transcription or recycling linked high glucose, will be required to understand the mechanisms of diabetic nephropathy. In conclusion, high glucose decreased 125I-ANG II binding through PKC-oxidative stress and possibly TGF-beta 1 in PTCs.


    ACKNOWLEDGEMENTS

The authors thank Drs. Tae Sung Kim and Bok Yun Kang of the College of Pharmacy, Chonnam National University, for technical support of RT-PCR (HRC G0301).


    FOOTNOTES

This work was supported by Ministry of Health and Welfare Grant 01-PJ1-PG3-20700-0010 (to H. J. Han).

Address for reprint requests and other correspondence: H. J. Han, Dept. of Veterinary Physiology, College of Veterinary Medicine, Chonnam National Univ., Kwangju 500-757, Korea (E-mail: hjhan{at}chonnam.ac.kr).

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. Section 1734 solely to indicate this fact.

First published October 23, 2001; 10.1152/ajprenal.00080.2001

Received 8 March 2001; accepted in final form 31 October 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Am J Physiol Renal Fluid Electrolyte Physiol 282(2):F228-F237
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