Over-production of nitric oxide by oxidative stress-induced activation of the TGF-β1/PI3K/Akt pathway in mesangial cells cultured in high glucose

Aim: To investigate whether NO over-production in rat mesangial cells cultured in high glucose (HG) is related to activation of the TGF-beta 1/PI3K/Akt pathway. Methods: Rat mesangial cells line (HBZY-1) was exposed to HG (24.44 mmol/L) or H2O2 (10 mu mol/L) for 16 h. NO release was quantified using the Griess assay. The TGF-beta 1 level was measured using ELISA. The protein expression of p-Akt, t-Akt, Bim, and iNOS was examined by Western blotting. The mRNA levels of TGF-beta 1 and Bim were measured using RT-PCR. The cell proliferation rate was estimated using a BrdU incorporation assay. Results: Treatment of the cells with HG, H2O2, or TGF-beta 1 (5 ng/mL) significantly increased the NO level that was substantially inhibited by co-treatment with the NADPH oxidase inhibitor diphenylene iodonium (DPI), TGF-beta 1 inhibitor SB431542, or PI3K inhibitor LY294002. Both HG and H2O2 significantly increased the protein and mRNA levels of TGF-beta 1 in the cells, and HG-induced increases of TGF-beta 1 protein and mRNA were blocked by co-treatment with DPI. Furthermore, the treatment with HG or H2O2 significantly increased the expression of phosphorylated Akt and iNOS and cell proliferation rate, which was blocked by co-treatment with DPI, SB431542, or LY294002. Moreover, the treatment with HG or H2O2 significantly inhibited Bim protein and mRNA expression, which was reversed by co-treatment with DPI, SB431542, or LY294002. Conclusion: The results demonstrate that high glucose causes oxidative stress and NO over-production in rat mesangial cells in vitro via decreasing Bim and increasing iNOS, which are at least partially mediated by the TGF-beta 1/PI3K/Akt pathway.