Effects Of Spironolactone On Aldosterone Mediated Oxidative Stress And Transforming Growth Factor β-1 Expression In Cultured Rat Glomerular Mesangial Cells

Objective To observe the effects of spironolactone on aldosterone mediated oxidative stress and transforming growth factorβ-1 (TGF-β1) expression in glomerular mesangial cells and explore its renoprotective mechanism.Methods Rat glomerular mesangial cells were cultured in the medium with nomal glucose (5.6 mmol/l) and then treated with medium containing 10-5mol/L (group ALD1), 10-7mol/L (group ALD2) and 10-9mol/L (group ALD3) aldosterone, respectively. Another, different concentrations of spironolactone (group SPI1 10-7 mol/L, group SPI2 10-8 mol/L and group SPI3 10-9 mol/L) were added into group ALD2, respectively. Cells and supernatants were collected after 48h separately. The levels of intracellular reactive oxygen species (ROS) were measured by flow cytometry. The expressions of TGF-β1, CYP11B2, MR and 11β-HSD2 mRNA were detected by semi-quantitative RT-PCR, while the levels of TGF-β1 protein in supernatants were measured by ELISA.Results (1) CYP11B2, MR and 11β-HSD2 mRNA were detected in cultured rat glomerular mesangial cells. (2) Compared with group NC, the levels of ROS and the expression of TGF-β1 mRNA and protein were all significantly increased in group ALD (P<0.05 or P<0.01), moreover, a dose-dependent manner was detected among group ALD1, group ALD2 and group ALD3 (P<0.05 or P<0.01). (3) Compared with group ALD2 (17.33±0.49), the levels of ROS in group SPI1 (7.34±0.89), SPI2 (9.25±0.65) and SPI3 (10.9±0.97) were all decreased significantly (P<0.05). Concomitantly, the expressions of TGF-β1 mRNA and protein were also significantly inhibited by spironolactone. (4) The changes in ROS were positively correlated with the change in TGFβ-1 mRNA and protein, respectively (r=0.866, P<0.01; r=0.823, P<0.01, respectively.).Conclusions CYP11B2, MR and 11β-HSD2 mRNA were detected in cultured glomerular mesangial cells by RT-PCR. Spironolactone can competitively binding with MR, then suppress aldosterone mediated oxidative stress and TGF-β1 expression in a dose-dependent manner in rat glomerular mesangial cells, which may partly contribute to its reno-protection.