Rap1b Inhibit High Glucose-induced Human Renal Tubuler Epithelial Cells Apoptosis Through Glycogen Synthase Kinase3β-β-Catenin Signaling

Objective To detect the expression of Raplb-GTP in human renal proximal epithelial cell lines (HK-2) and investigate the role of Rap1b in the development of high glucose induced apoptosis in HK-2.Methods The human renal tubular epithelial cells (HK-2) line was cultured according to routine protocol. Stimulate HK-2cells with different concentration of D-glucose (5mM、10mM、20mM、30mM)and30mM D-glucose at different time points. The expression of Raplb-GTP protein was examined by Western Blot. Transfect pcDNA3.1/Rap1b or the empty pcDNA3.1vector into HK-2cells and evaluate the expression of phosphorylation level of GSK-3beta protein, nuclear beta-catenin protein and apoptosis protein caspase-3by Western Blot. The expression of suvivin and Bcl-2were evaluated by Real Time PCR method. Furthermore, treated HK-2cells with LiCl or wortmannin to inhibit or activate GSK-3β And the expression level of the nuclear beta-catenin protein and apoptosis protein caspase-3under high glucose condition were evaluated by Western Blot, and the expression of suvivin and Bcl-2were evaluated by Real Time PCRo Results (1) The expression of Raplb-GTP in HK-2cells was significantly decreased under high-glucose2hours treatment group.(2) The over-expression of Rap1b under high glucose ambience elevating the phosphorylation level of GSK-3beta protein and promoting beta-catenin nuclear transcription, increasing anti-apoptotic gene survivin and Bcl-2expression, inhibiting of apoptosis protein activation.(3) GSK-beta inhibitor LiCl promoting beta-catenin nuclear transcription increasing the expression of anti-apoptotic genes survivin and Bcl2, inhibiting of apoptosis protein activation. However, GSK-3beta indirect inhibitor wortmannin inhibiting the transcription of beta-catenin nuclear, reducing expression of anti-apoptotic genes survivin and Bcl-2, promoting the activation of apoptosis proteins.Conclusion (1) High glucose inhibits HK-2cells Rap1b-GTP activity, and this is associated with HK-2cells apoptosis.(2) Over-expression of Rap1b can reverse the activation of apoptosis proteins induced by high-glucose in HK-2cells.(3) Rap1b modulate the downstream gene expression through promoting the phosphorylation of GSK-3βand the nuclear transcription of β-catenin.