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(Hypertension. 2008;51:507.)
© 2008 American Heart Association, Inc.

Original Articles Part 2

Osteopontin in Rat Renal Fibroblasts

Functional Properties and Transcriptional Regulation by Aldosterone

Jun Irita; Takafumi Okura; Mie Kurata; Ken-ichi Miyoshi; Tomikazu Fukuoka; Jitsuo Higaki

From the Department of Integrated Medicine and Informatics, Ehime University Graduate School of Medicine, Shitsukawa, Japan.

Correspondence to Takafumi Okura, MD, PhD, Department of Integrated Medicine and Informatics, Ehime University Graduate School of Medicine, Shitsukawa, Toon City, Ehime, 791-0295, Japan. E-mail okura{at}m.ehime-u.ac.jp

Osteopontin (OPN), a proinflammatory cytokine, plays an important role in renal fibrosis. We reported that plasma OPN levels were higher in patients with primary aldosteronism than with essential hypertension. However, the regulatory mechanism of OPN by aldosterone remains unclear. Here, we report the transcriptional regulation of OPN expression by aldosterone and the functional effects of aldosterone-mediated OPN transcription in renal fibroblasts. Aldosterone induced OPN expression in a dose-dependent manner with significant responses at 10 nmol/L (1.6±0.2-fold of controls, P<0.05, n=5) and elicited maximal effects at 10 µmol/L (3.5±0.4-fold of controls, P<0.01, n=5). Aldosterone increased OPN expression in a time-dependent manner with a maximal effect after 48 hours (2.7±0.3-fold of controls, P<0.01, n=5). This effect was abolished by the mineralocorticoid receptor (MR) antagonist spironolactone. Luciferase promoter deletion assays identified a novel cis regulatory element (–2153 to –1758) in the OPN promoter that is responsive to aldosterone. This element contains an activator protein-1 (AP-1) and nuclear factor kappa B (NFB) site. Electrophoretic mobility shift assays, supershift assays, and chromatin immunoprecipitation assays identified both AP-1 and NFB as the DNA binding proteins induced by aldosterone with spironolactone inhibiting aldosterone-induced AP-1 or NFB activity. OPN-siRNA inhibited completely the induction of cell proliferation, type I, III, and IV collagen synthesis by aldosterone. These results indicate that aldosterone induced MR-mediated OPN expression through AP-1 and NFB activation and suggest that aldosterone plays an important role in renal fibrosis through the induction of OPN.

Key Words: osteopontin • aldosterone • activator protein-1 • nuclear factor kappa B • mineralocorticoid receptor • renal fibrosis

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