Protective Effects Of Reduced Glutathione On The Human Renal Tubular Epithelial Cells Cultured In High Glucose

Objective: To investigate the effect of high glucose on the production of the reactive oxygen species (ROS), nuclear factor-kappa B (NF-κB) and osteopontin (OPN) in the human renal tubular epithelial cells (HK-2), the relation with oxidative stress in diabetic nephropathy and the production of ROS, NF-κB and OPN. We also studied the effect of reduced glutathione on oxidative stress in diabetic nephropathy in vitro.Methods: HK-2 cells were cultured with different concentrations of GSH for 48 hours and the cell proliferation was examined by MTT colorimetry to determine the experimental dose of GSH. The cells cultured in vitro were divided into 5 groups: normal glucose group, high glucose group, high glucose with GSH of 1 mmol/L group, high glucose with GSH of 5 mmol/L group, and high glucose with GSH of 10 mmol/L group. After 24h, 48h and 72h, the activity of SOD and the contents of MDA in supernatant were detected by spectrophotometry, meanwhile the expression of NF-κBmRNA was determined by RT-PCR. And the contents of OPN in supernatant were detected by ELISA. Apoptotic changes were detected by DNA agarose gel electrophoresis after 6 hours. The differences of data among groups were compared by One-Way ANOVA Test.Results: 1 Compared with NG group, the activity of SOD markedly decreased in HG group (P<0.01). GSH markedly increased the activity of SOD in dose-dependent manner (P<0.05). Compared with NG group, the contents of MDA markedly increased in HG group(P<0.01).These over expressions were downregulated by GSH in dose-dependent manner(P<0.01).2 Compared with NG group, the expressions of NF-κBmRNA significantly increased in HG group(P<0.01).GSH markedly inhibited the expression of dose-dependent manner(P<0.05).3 Compared with NG group, the contents of OPN enhanced significantly in HG group. These over expressions were downregulated by GSH in dose-dependent manner (P<0.01).4 SOD was negative related with MDA, NF-κB and OPN. There was a positive correlation among MDA, NF-κB and OPN. 5 Furthermore, Our study demonstrated GSH can inhibit apoptosis of HK-2 cells induced by high glucose effectively.Conclusions: 1 High glouse induces the expression of ROS, NF-κB and OPN in HK-2 cells. 2 GSH can suppress the overexpression of ROS, NF-κB and OPN , which may prevent the progression of diabetic nephropathy. 3 GSH can effectively inhibit HK-2 cells apoptosis induced by high glucose. .