High Glucose Induces The Expression Of Osteopontin Through Activation Of The PI3K/AKT/mTORC1Pathway In Human Renal Tubular Epithelial Cells

Objective (1) To explore the expression of osteopontin (OPN) induced by high glucose in human renal tubular epithelial cells (HK-2), and to investigate whether high glucose by PI3K/AKT/mTOR pathway regulating OPN expression.(2) To investigate rapamycin which the specific inhibitors of mTOR on the expression of OPN in diabetic animal model.Methods After stimulation with high-glucose (25mmol.L-1) culture medium, HK-2cells are then treated with the specific inhibitors or siRNA to inhibit the activity of PI3K/AKT/mTOR pathway. Subsequently, Real-time PCR is used to investigate the mRNA level of OPN, Western blot is performed to detect the protein expression of OPN, p-AKT, p-S6, Raptor and Rictor. Using the immunohistochemical detection of type2diabetic animal model (db/db mice) the expression of OPN in kidney tissue.Results (1) The expression level of OPN is increased in a time-dependent manner in HK-2cells followed by high-glucose stimulation. The mRNA level of OPN begin to increase at12hours, and reach a peak at48hours is1.60±0.03times higher than in the control group (P<0.05). The mRNA expression levels of72hours is significantly lower than before. Western blot analysis also show the protein expression of OPN begin to increase from12hours and reach the highest level at72hours. Compared with48hours, but the difference has not statistically significant.(2) Meanwhile, inhibition of the PI3K/AKT/mTOR pathway by LY294002and/or rapamycin led to significant down-regulation of OPN (P<0.05), and the combination has more significant effect.(3) Moreover, the treatment with Raptor siRNA (inhibit mTORCl), but not Rictor siRNA (inhibit mTORCl) result in reduction of OPN expression.(4) In type2diabetic nephropathy in mice (db/db) found rapamycin inhibit the expression of OPN in renal tubular.Conclusions High glucose increases the expression of OPN through the activation of PI3K/AKT/mTORCl signaling pathway in HK-2cells. And rapamycin can inhibit the expression of OPN in kidney of type2diabetic model.