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
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ABSTRACT |
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)-
1 secretion, and anti-TGF-
antibody significantly blocked 25 mM glucose-induced downregulation of 125I-ANG II
binding. Furthermore, the 25 mM glucose-induced increase in TGF-
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-
signal cascade in primary cultured rabbit renal proximal
tubule cells.
angiotensin II receptor; protein kinase C; transforming growth
factor-
1
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INTRODUCTION |
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)-
1, an important cytokine in the development of diabetic
nephropathy, especially in PTCs in the diabetic condition (22,
35). Treatment with anti-TGF-
-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-
decreased
expression of the AT2 receptor and mRNA (29).
These reports suggest the possibility that PKC, oxidative stress, and
TGF-
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
-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-
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-
cascade in PTCs.
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MATERIALS AND METHODS |
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-
1 from porcine platelets and anti-TGF-
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
-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-
.
Active TGF-
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-
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.
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RESULTS |
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 -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.
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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.
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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.
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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.
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Involvement of TGF-
in high-glucose-induced LPO formation and
125I-ANG II binding.
Next, we examined the involvement of TGF-
in the
high-glucose-induced 125I-ANG II binding. As shown in Fig.
9A, TGF-
1 downregulated
125I-ANG II binding in a dose-dependent manner.
Furthermore, studies with a neutralizing antibody to TGF-
were
performed to examine the involvement of TGF-
in the
high-glucose-induced downregulation of 125I-ANG II binding.
Figure 9B depicts that anti-TGF-
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-
1 secretion
compared with 5 mM glucose (Fig.
10A). We also examined which
signal pathways are involved in high-glucose-induced TGF-
1
secretion. Figure 10B demonstrates that H-7, TPA
pretreatment, methoxyverapamil, NAC, and taurine significantly blocked
25 mM glucose-induced TGF-
1 secretion. These results suggest that
TGF-
1 may be involved in the high-glucose-induced downregulation of
125I-ANG II binding and high glucose stimulates TGF-
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-
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-
1 secretion, and 125I-ANG II binding.

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Fig. 9.
A: effect of transforming growth factor
(TGF)- 1 on 125I-ANG II binding. PTCs were treated
with different dosages of TGF- 1 (0.1-10 ng/ml) for 48 h.
B: effect of anti-TGF- antibody on high-glucose-induced
125I-ANG II binding. PTCs were treated with
anti-TGF- -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- 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- 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- 1 secretion, and
downregulation of 125I-ANG II binding
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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-
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- 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.
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DISCUSSION |
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-
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-
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-
system in diabetic animals
suggests that TGF-
is one of the common mediators of diabetic nephropathy. Sharma et al. (37) also reported that
anti-TGF-
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-
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-
1 may be responsible for
the high-glucose-induced inhibition of ANG II binding, because high
glucose increased TGF-
1 secretion, exogenous TGF-
1 treatment
downregulated 125I-ANG II binding, and anti-TGF-
antibody prevents high-glucose-induced downregulation of
125I-ANG II binding. Such stimulation of TGF-
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-
isoform inhibitor,
prevented glomerular TGF-
1 mRNA expression in diabetic rat
(25). Although we did not measure TGF-
mRNA expression,
high glucose may increase TGF-
1 mRNA expression, because actinomycin
D and cycloheximide attenuate high-glucose-induced downregulation of
125I-ANG II binding and secretion of TGF-
1. Hoffman et
al. (21) reported that the stimulation of TGF-
1
production is attributed to transcriptional activation involving a
region in the TGF-
1 promoter containing a glucose-response element.
Surprisingly, up until now, there were no studies about the involvement
of TGF-
1 in the downregulation of ANG II receptor binding. We first
demonstrated here that TGF-
1 is involved in the high-glucose-induced
downregulation of ANG II receptor binding, because anti-TGF-
antibody significantly prevented high-glucose-induced ANG II binding
and TGF-
1 also significantly downregulates ANG II binding. This
result is not consistent with the report of Lebrethon et al.
(28), who demonstrated that TGF-
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-
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-
immunohistochemical staining. The stimulatory
effect of oxidative stress and TGF-
1 secretion by high glucose are
also dependent on PKC activation, suggesting the role of a
PKC-oxidative stress-TGF-
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-
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-
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.
 |
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