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Aldosterone Stimulates Reactive Oxygen Species Production through Activation of NADPH Oxidase in Rat Mesangial Cells

Kayoko Miyata^*, Matlubur Rahman^,, Takatomi Shokoji, Yukiko Nagai, Guo-Xing Zhang, Guang-Ping Sun, Shoji Kimura, Tokihito Yukimura^||, Hideyasu Kiyomoto, Masakazu Kohno, Youichi Abe and Akira Nishiyama

^* Radioisotope Research Center, Second Department of Internal Medicine, Department of Pharmacology and Research Equipment Center, Kagawa Medical University, Kagawa, Japan; and ^|| Department of Pharmacology, Osaka City University Graduate School of Medicine, Osaka, Japan

Address correspondence to: Dr. Akira Nishiyama, Department of Pharmacology, Kagawa Medical University, 1750-1 Ikenobe, Miki-cho, Kita-gun, Kagawa 761-0793, Japan. Phone: +81-87-898-5111 ext. 2502; Fax: +81-87-891-2126; E-mail: akira{at}kms.ac.jp

Received for publication April 12, 2005. Accepted for publication July 28, 2005.

	Abstract

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It has recently been shown that glomerular mesangial injury is associated with increases in renal cortical reactive oxygen species (ROS) levels in rats treated chronically with aldosterone and salt. This study was conducted to determine the mechanisms responsible for aldosterone-induced ROS production in cultured rat mesangial cells (RMC). Oxidative fluorescent dihydroethidium was used to evaluate intracellular production of superoxide anion (O₂^–) in intact cells. The lucigenin-derived chemiluminescence assay was used to determine NADPH oxidase activity. The staining of dihydroethidium was increased in a dose-dependent manner by aldosterone (1 to 100 nmol/L) with a peak at 3 h in RMC. Aldosterone (100 nmol/L for 3 h) also significantly increased NADPH oxidase activity from 232 ± 18 to 346 ± 30 cpm/5 x 10⁴ cells. Immunoblotting data showed that aldosterone (100 nmol/L for 3 h) increased p47phox and p67phox protein levels in the membrane fraction by approximately 2.1- and 2.3-fold, respectively. On the other hand, mRNA expression of NADPH oxidase membrane components, p22phox, Nox-1, and Nox-4, were not altered by aldosterone (for 3 to 12 h) in RMC. Pre-incubation with the selective mineralocorticoid receptor (MR) antagonist, eplerenone (10 µmol/L), significantly attenuated aldosterone-induced O₂^– production, NADPH oxidase activation and membranous translocation of p47phox and p67phox. These results suggest that aldosterone-induced ROS generation is associated with NAPDH oxidase activation through MR-mediated membranous translocation of p47phox and p67phox in RMC. These cellular actions of aldosterone may play a role in the pathogenesis of glomerular mesangial injury.

	Introduction

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A growing body of evidence supports a role for aldosterone in the progression of renal injury (1–4). In rats, chronic administrations of aldosterone and salt led to severe proteinuria and glomerular injury (5,6). Similarly, exogenous infusion of aldosterone reversed the renoprotective effects of angiotensin-converting enzyme (ACE) inhibitors in remnant kidney hypertensive rats (7) and stroke-prone, spontaneously hypertensive rats (SHRSP) (8). In addition, treatment with the mineralocorticoid receptor (MR) antagonists ameliorated glomerular injury in SHRSP (9) and in rats treated with angiotensin II (AngII) and nitric oxide synthase inhibitor (10), cyclosporine A (11) or radiation (12), independent of BP reduction. In patients with chronic renal failure (13) and early diabetic nephropathy (14), addition of a nonselective MR antagonist, spironolactone, to ACE inhibitors did not exert hemodynamic effects, but markedly reduced urinary excretion rate of protein (U_proteinV). These observations suggest that aldosterone has direct deleterious effects on the kidney via activation of the MR. However, the mechanisms responsible for the aldosterone/MR-induced renal injury remain undetermined.

Recent studies have indicated the potential participation of reactive oxygen species (ROS) in the pathophysiology of aldosterone-induced cardiovascular tissue injury (5,15–22). In aldosterone/salt-treated hypertensive rats, vascular NADPH oxidase activity and ROS production were markedly increased (15,16). In these animals, treatment with an NADPH oxidase inhibitor, apocynin, prevented BP elevation and cardiovascular hypertrophy (17). It was also shown that treatment with a selective MR antagonist, eplerenone, improved endothelial dysfunction and reduced vascular superoxide anion (O₂^–) generation in diet-induced atherosclerosis (18). Similarly, eplerenone reduced aortic atherosclerotic lesions and O₂^– generation in peritoneal macrophages of apolipoprotein E-deficient mice (19,20). Mazak et al. (21) showed that aldosterone potentiates AngII-induced signaling in vascular smooth muscle cells, and that these effects of aldosterone were blocked by antioxidants. The authors also showed that spironolactone decreased NADPH oxidase-dependent ROS generation after AngII stimulation. Further studies by Callera et al. (22) showed that aldosterone increased NADPH oxidase activity in vascular smooth muscle cells, which were prevented by treatment with eplerenone.

Recently we demonstrated that in aldosterone/salt-treated rats, glomerular injury, characterized by mesangial matrix expansion and cell over-growth, is associated with increases in renal tissue ROS levels (5). We also observed that treatment with an antioxidant, tempol, or eplerenone normalized ROS levels and markedly attenuated glomerular injury in these animals (5). These results suggest that the glomerular mesangium is a target for injuries induced by aldosterone and the MR, and prompt us to perform further in vitro experiments to investigate possible mechanisms responsible for the aldosterone and MR-induced ROS generation. In this study, we hypothesized that aldosterone has a direct effect on ROS generation through MR-dependent activation of NADPH oxidase in glomerular mesangial cells. To test this hypothesis, we examined the effects of aldosterone and MR blockade on O₂^– generation and NADPH oxidase activity in cultured rat mesangial cells (RMC).

	Materials and Methods

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Reagents
Aldosterone was purchased from Across Organics (Geel, Belgium). Dihydroethidium was obtained from Molecular Probes Inc. (Eugene, OR). Diphenyleneiodonium, apocynin, lucigenin, NADPH and anti–-actin antibody were obtained from Sigma Chemical Co. (St. Louis, MO). Anti-p47phox and p67phox antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Eplerenone was provided by Pfizer Inc. (New York, NY).

Cell Culture
All experimental procedures were performed according to the guidelines for the care and use of animals established by Kagawa Medical University. RMC were isolated from male Sprague-Dawley rats and were maintained according to published methods (23,24). Control solutions always contained the appropriate amount of vehicle (ethanol for aldosterone and DMSO for eplerenone, apocynin, and diphenyleneiodonium, <1:1000 for each). After stimulation, protein or mRNA was extracted as described previously (23,24). In some RMC, membrane fraction was isolated as described previously (20,25). The protein concentration was determined using the Bradford or Lowry protein assay kit (Bio-Rad Laboratories, Hercules, CA).

Dihydroethidium Staining
The oxidative fluorescence dihydroethidium was used to evaluate intracellular O₂^– levels as described previously (25). Briefly, RMC were plated in a glass-bottom dish (Matsunami Glass Ind. Ltd., Kishiwada, Japan) and allowed to adhere for at least 18 h. At the appropriate time after stimulation, dihydroethidium (10 µmol/L) was added to the medium, and the incubation was continued for 15 min. Then, cells were washed with PBS and images were obtained with a laser scanning confocal microscope system (Bio-Rad Laboratories). The averages of fluorescence intensity values from 20 to 30 cells of 5 to 8 different examinations were calculated using image software from the National Institutes of Health (NIH).

NADPH Oxidase Activity
NADPH oxidase-dependent O₂^– production by intact RMC was measured by lucigenin-enhanced chemiluminescence (22,25). Briefly, RMC were detached from the culture dishes using 0.25% trypsin/EDTA (1 mmol/L), washed with PBS, and resuspended at 1 x 10⁶ cells/ml in Krebs-HEPES buffer. Fifty microliter of cell suspension (5 x 10⁴ cells) was transferred into glass scintillation vials containing 5 µmol/L lucigenin in Krebs-HEPES buffer (950 µl). The chemiluminescence value was recorded at 30-s intervals over 10 min (BLR-301, Aloka Co., Tokyo, Japan), and readings in the last 5 min were averaged. After measurement of background lucigenin chemiluminescence, NADPH (100 µmol/L) was added to the incubation medium as a substrate for O₂^– production.

Real-Time Reverse Transcriptase-PCR
mRNA expression levels of the NADPH membrane components, p22phox, Nox-1, and Nox-4, were analyzed by real-time PCR (5). All data were normalized by the expression of glyseraldehyde-3-phosphate dehydrogenase (GAPDH). The primers for p22phox, Nox-1, Nox-4, and GAPDH were synthesized as described previously (5).

Western Blotting Analysis
The protein expression of p47phox and p67phox was measured by Western blotting analysis as described previously (20,25). All values were normalized by setting the densitometry of control samples to 1.0. In samples from total lysates, blotting membranes were re-probed with an antibody against -actin to check for equal loading.

Statistical Analyses
The values are presented as means ± SEM. One-way ANOVA was used to determine significant differences among groups, after which the modified t test with the Bonferroni correction was used for comparison between individual groups. P < 0.05 was considered statistically significant.

	Results

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Effects of Aldosterone on O₂^– Production
To determine whether aldosterone stimulates ROS production in RMC, intracellular O₂^– levels were measured using dihydroethidium and fluorescence microscopy. Figure 1A shows the time course of aldosterone-induced increases in dihydroethidium staining (n = 5 to 6 for each). Aldosterone (100 nmol/L)-induced increases in dihydroethidium staining peaked at 3 h. Figure 1B shows the concentration-dependent effects of aldosterone (3 h) on the staining of dihydroethidium (n = 5 to 6 for each). Aldosterone-induced increases in dihydroethidium staining were maximal at 100 nmol/L (4.1 ± 0.9-fold). Representative results of dihydroethidium staining are shown in Figure 1C.

View larger version (21K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 1. Effect of aldosterone on dihydroethidium staining in rat mesangial cells (RMC). The oxidative fluorescence dihydroethidium was used to evaluate intracellular superoxide anion (O2–) levels. A, Time course of aldosterone-induced increases in dihydroethidium staining. Aldosterone (100 nmol/L)-induced increases in dihydroethidium staining peaked at 3 h. B, Concentration-dependent effects of aldosterone (3 h) on the staining of dihydroethidium. Aldosterone-induced increases in dihydroethidium staining were maximal at 100 nmol/L. The average of fluorescence intensity values was calculated from 20 to 30 cells of 5 to 8 different examinations. Data are expressed as the relative differences compared with control. *P < 0.05 versus control. C, Representative results of dihydroethidium are shown. Original magnification, x200.

View larger version (17K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 2. A, Effects of eplerenone, apocynin, and diphenyleneiodonium on aldosterone-induced increase in dihydroethidium staining. The average of fluorescence intensity values was calculated from each 20 to 30 cells of 5 to 8 different examinations. Eplerenone (10 µmol/L), apocynin (300 µmol/L), and diphenyleneiodonium (10 µmol/L) significantly attenuated aldosterone (100 nmol/L for 3 h)-induced increases in dihydroethidium staining. Data are expressed as the relative differences compared with control. *P < 0.05 versus control. B, Representative results of dihydroethidium are shown. Original magnification, x1600.

View larger version (22K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 3. Effects of aldosterone, eplerenone, apocynin, and diphenyleneiodonium on NADPH oxidase activity in RMC. NADPH oxidase-dependent O2– production by intact RMC was measured by lucigenin-enhanced chemiluminescence. Incubation with aldosterone (100 nmol/L) for 3 h significantly enhanced NADPH-dependent O2– production. The aldosterone-induced enhancement of NADPH-dependent O2– production was markedly attenuated by eplerenone (10 µmol/L), apocynin (300 µmol/L), or diphenyleneiodonium (10 µmol/L) (n = 4 to 7 for each).

View larger version (17K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 4. Effects of aldosterone on p22phox, Nox-1, and Nox-4 mRNA expression in RMC. mRNA expression levels of p22phox, Nox-1, and Nox-4 were analyzed by real-time PCR. All data were normalized by the expression of GAPDH, and the ratio to control is shown. Treatment with aldosterone (100 nmol/L) for 3 to 12 h did not alter mRNA expression of p22phox, Nox-1, and Nox-4 expression in RMC (n = 6 for each).

View larger version (33K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 5. Effects of aldosterone on membranous translocation of p47phox and p67phox proteins in RMC. A, Aldosterone (100 nmol/L)-induced increases in p47phox and p67phox protein levels in the membrane fraction peaked at 3 h. On the other hand, protein levels of p47phox and p67phox in total lysates were not changed by aldosterone treatment. B, Incubation with aldosterone (100 nmol/L) for 3 h increased p47phox and p67phox protein levels in the membrane fraction. Aldosterone-induced membranous translocation of p47phox and p67phox was attenuated by treatment with eplerenone (10 µmol/L), or apocynin (300 µmol/L) (n = 6 to 7 for each). *P < 0.05 versus control.

	Discussion

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NADPH oxidase is one of the major sources of O₂^– in a variety of cells (28,29). Our study showed that aldosterone-induced O₂^– production was accompanied by increases in NADPH oxidase activity in RMC. Furthermore, O₂^– production as well as NADPH oxidase activation induced by aldosterone were virtually abolished by pretreatment with the NADPH oxidase inhibitors, apocynin or diphenyleneiodonium. Keidar et al. (20) showed that increased NADPH oxidase activity was observed in macrophages isolated from aldosterone-treated apolipoprotein E-deficient mice. Similarly, increases in vascular NADPH oxidase activity were observed in rats treated with aldosterone and salt, and this increase was prevented by concurrent administration of apocynin (17). These in vivo observations as well as those in the in vitro experiments support the hypothesis that aldosterone stimulates ROS generation via the NADPH oxidase-dependent pathway. Our study also showed that eplerenone markedly attenuated aldosterone-induced increases in NADPH oxidase activity and ROS generation in RMC. These results are consistent with those observed in previous animal studies (15–18).

NADPH oxidase is composed of membrane-associated components (gp91phox, Nox-1, Nox-4, and p22phox) and cytosolic regulatory subunits (p40phox, p47phox, p67phox, and Rac) (28). Activation of NADPH oxidase requires the translocation of the cytosolic components to the cell membrane (28,29). Recent studies have shown that macrophages from aldosterone-treated apolipoprotein E-deficient mice exhibited higher membranous translocation of the cytosolic p47phox compared with those derived from placebo-treated mice (20). Our study showed that aldosterone directly induces the translocation of p47phox and p67phox to the RMC membrane. Of note, the effects of aldosterone on O₂^– production, NADPH oxidase activation and membranous translocation of p47phox and p67phox were shown within a similar time span. Thus, these data are consistent with the concept that membranous translocation of p47phox and p67phox is involved, at least in part, in the overall increased NADPH oxidase activity resulting in O₂^– production in RMC. The finding that aldosterone-induced translocation of p47phox and p67phox were markedly attenuated by treatment with eplerenone indicates the potential contribution of the MR to these effects of aldosterone.

Weber and co-workers (30,31) showed that immunohistochemical staining for gp91phox and 3-nitrotyrosine (a marker of nitrosative stress) were significantly increased in the heart of aldosterone/salt-treated uninephrectomized rats. In these animals, increased mRNA expression of p22phox was also observed in the aortic tissues (17). Similarly, we previously showed that in kidneys of aldosterone/salt-treated rats, elevated renal tissue ROS levels were associated with increased mRNA expression of p22phox, Nox-4, and gp91phox (5). Therefore, we anticipated that these components would be increased by aldosterone in RMC. However, we observed that aldosterone did not affect mRNA expression of p22phox, Nox-1, and Nox-4 in RMC. In this study, apparent mRNA expression of gp91phox was not detected in RMC (data not shown). At present, we can not explain the discrepancy between previous in vivo and current in vitro data, but it may be due to differences in the experimental conditions or cell types. Alternatively, the exposure time of aldosterone alone would not be enough for the overexpression of NADPH oxidase membrane components in RMC. Further in vitro studies in different cells and time courses will be needed to determine the effects of aldosterone on the expression of NADPH oxidase membrane components.

AngII also induces ROS generation through the NADPH oxidase-dependent pathway (28,29). Recent studies have indicated that MR interacts with AngII-induced NADPH oxidase activation and ROS production. Schiffrin et al. (15) showed that increased vascular NADPH oxidase activity and ROS production in AngII-induced hypertensive rats were markedly attenuated by treatment with spironolactone. Schiffrin et al. also showed that aldosterone-induced increases in vascular NADPH oxidase activity and ROS production were attenuated by AT1 receptor antagonist (16). Further in vitro studies demonstrated that AngII-induced ROS generation is attenuated by spironolactone in vascular smooth muscle cells (21). Although the mechanisms by which MR interacts with AngII-dependent NADPH oxidase activation are not clear, it is possible that the beneficial effects of aldosterone/MR blockade on AngII-induced glomerular injury reported in previous studies (10,32,33) are mediated, at least partially, through inhibition of its ROS generation.

In our study, we examined the effects of aldosterone at concentrations of 0.1 to 100 nmol/L in cultured RMC. The concentrations of aldosterone used here were determined on the basis of results from previous in vitro studies (21,22,24,26). However, some of these aldosterone concentrations would be higher than rat plasma levels (<1 nmo/L) (15,16), and, therefore, physiologic impacts on these data regarding the roles of aldosterone in NADPH oxidase activation and ROS generation are not clear. In addition, we cannot rule out the possibility that some of the effects of aldosterone observed in our study might be mediated via the glucocorticoid receptors. Nevertheless, our results show that the highly selective MR antagonist, eplerenone (34), significantly attenuated aldosterone-induced ROS production, NADPH oxidase activation and membranous translocation of p47phox and p67phox. Thus, it seems likely that MR could play a role, at least partially, in these effects of aldosterone. It seems also important to note that although this study has focused on the effects of aldosterone, glucocorticoids are also able to activate MR under certain pathophysiologic conditions (35). Further studies will be required to investigate the roles of glucocorticoids in mediating MR-dependent ROS generation.

In summary, this study presented evidence that aldosterone directly induces ROS generation through activation of NADPH oxidase in RMC. Aldosterone-induced NADPH oxidase activation may be, at least in part, due to membranous translocation of p47phox and p67phox. Our data also indicate the contribution of the MR to these effects of aldosterone. These findings might provide novel insights into the mechanisms responsible for aldosterone-induced ROS generation during the progression of renal injury. In addition, it can be speculated that some BP-independent renoprotective effects of MR antagonists reported in recent clinical studies (13,14,36 to 38) are mediated through their direct antioxidative actions on renal cells. Further in vitro studies will be performed to determine the specific roles of ROS in mediating aldosterone-dependent renal cell damage.

	Acknowledgments

 
This work was supported by grants-in-aid for scientific research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (15790136), by grants from the Salt Sciences Research Grant (05C2), Pfizer Inc., Japan Research Foundation for Clinical Pharmacology, the Naito Foundation and the Kao Foundation for Arts and Sciences (to A.N.). Part of this work was presented at the 58th Annual Fall Conference and Scientific Sessions of the Council for High Blood Pressure Research in association with the Council on the Kidney in Cardiovascular Disease, in Chicago, IL, October 9 to 12, 2004.

	Footnotes

 
Published online ahead of print. Publication date available at www.jasn.org.

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