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

Abstract

Aim: To investigate whether NO over-production in rat mesangial cells cultured in high glucose (HG) is related to activation of the TGF-β1/PI3K/Akt pathway. Methods: Rat mesangial cells line (HBZY-1) was exposed to HG (24.44 mmol/L) or H 2 O 2 (10 μmol/L) for 16 h. NO release was quantified using the Griess assay. The TGF-β1 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-β1 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, H 2 O 2 , or TGF-β1 (5 ng/mL) significantly increased the NO level that was substantially inhibited by co-treatment with the NADPH oxidase inhibitor diphenylene iodonium (DPI), TGF-β1 inhibitor SB431542, or PI3K inhibitor LY294002. Both HG and H 2 O 2 significantly increased the protein and mRNA levels of TGF-β1 in the cells, and HG-induced increases of TGF-β1 protein and mRNA were blocked by co-treatment with DPI. Furthermore, the treatment with HG or H 2 O 2 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 H 2 O 2 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-β1/PI3K/Akt pathway.

Danshen injection ameliorates STZ-induced diabetic nephropathy in association with suppression of oxidative stress, pro-inflammatory factors and fibrosis

“…Since high glucose-induce oxidative stress and inflammation could both lead to increased ECM production, such as collagen, via TGF-β1 pathway [37,38]. TGF-β1, a key fibrogenic cytokine, mediates glomerulosclerosis and tubulointerstitial fibrosis in DN by enhancing ECM synthesis.…”

NADPH Oxidase-Dependent Production of Reactive Oxygen Species Induces Endoplasmatic Reticulum Stress in Neutrophil-Like HL60 Cells

“…Interestingly, ATF4, GADD34 and CHOP mRNA levels were increased by PMA treatment, although this was not inhibited by DPI, suggesting an NADPH oxidase (and ER stress) independent effect. Indeed, in some cases DPI potentiated the effect of PMA, which might relate to other cellular effects of DPI, such as inhibition of O 2 o and H 2 O 2 production by mitochondria [ 61 ], induction of DNA damage [ 62 ] and increase in mRNA and protein levels related to cell death [ 63 ].…”

Reduced β2GPI Inhibiting Glomerular Mesangial Cells VEGF-NO Axis Uncoupling Induced by High Glucose

“…In this present study, high glucose could significantly increase the expressions of VEGF and VEGFR-2 and upregulated its downstream Akt phosphorylation in glomerular mesangial cells, which is consistent with previous study. The phosphorylated Akt could increase the production of NO [ 21 ]. However, NO is significantly reduced in high glucose treated glomerular mesangial cells, indicating the fact that high-glucose treatment caused glomerular mesangial cells VEGF-NO axis uncoupled, which coincides with previous research [ 20 , 22 ].…”