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Published ahead of print on August 31, 2005
J Am Soc Nephrol 16: 2906-2912, 2005
© 2005 American Society of Nephrology
doi: 10.1681/ASN.2005040390
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Cell Biology

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

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

* Radioisotope Research Center, {dagger} Second Department of Internal Medicine, {ddagger} 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.

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.




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