The Role Of Gsk-3β Inhibitor LiCl In The Injury Of Podocytes Caused By High Glucose

Background and AimsUnder the condition of high glucose, podocytes suffer from a series of injuries, including epithelial-mesenchymal transition (EMT),decreased cell vitality, and apoptosis. Glycogen synthase kinase-3β (GSK-3beta) of the podocytes, has a rich content and various functions, including cell adhesion, transcription, apoptosis, migration and so on,and therefore is involved in many kinds of pathological physiology process. Studies have found that GSK-3(3inhibitors induce epithelial cells in skins to obtain a mesenchymal phenotype,known as EMT. A research has shown that GSK-3β inhibitors ameliorate mesangial cell apoptosis induced by high glucose.Now the role of GSK-3β inhibitors is still controversial.Could GSK-3β inhibitors protect podocytes from injury caused by high glucose?The implications of the alteration of GSK-3β activity under high glucose are not clear. The experiment aims to investigate the role of GSK-3β inhibitor on podocytes EMT and apoptosis under high glucose condition by western blot, RT-PCR, MTT and flow cytometry.MethodsUsing mice glomerular podocyte cell line as an in vitro system.After podocytes were cultured in permissive condition at33℃,they were shifted to non-permissive condition at37℃and cultured for14days.We use western blot to detect Synaptopodin to ensure its full differentiation,then they were divided into4groups,(1) a normal glucose concentration group, podocyte culture medium contains5.6mmol/L glucose,(2) high glucose group, podocyte culture medium contains30mmol/L glucose,(3) high glucose+LiC1group, podocyte culture medium contains30mmol/L glucose and10mmol/L LiCl,(4) LiC1group, podocyte culture medium contains10mmol/L LiC1,48hours after treatment.We use the MTT method for the detection of podocyte survival rate,RT-PCR for the detection of Bcl-2, Bax’s mRNA level, flow cytometry for the detection of apoptosis, western blot for the detection of nephrin, α-SMA protein expression in different groups.Results1. MPC5have a fusiform,polygonal shape when they are in proliferation condition.And the cells have shorter,thicker and less foot processes. They have longer,thinner and much more foot processes when they are fully differentiated2. After podocytes were treated for48hours, MTT method were used to detect podocyte survival rate.The survival rate of high glucose group, high glucose+LiC1group dropped significantly compared with that of normal group (P＜0.05), LiC1group dropped slightly, high glucose+LiC1group had a lower survival rate compared with high glucose group (P＜0.05)3. RT-PCR detection:Bcl-2mRNA level of high glucose group was lower than that of the normal group (P<0.05), podocytes in high glucose+LiC1group did not express Bcl-2mRNA; Bax mRNA expression was increased in high glucose group, high glucose+LiC1group compared with the normal group (P＜0.05),and Bax mRNA expression was increased in high glucose+LiC1group compared with high glucose group (P<0.05)4. Flow cytometry detection:the apoptotic rate of podocytes was increased after exposure to high glucose for48h (P<0.05), LiC1increased the apoptotic rate of podocytes exposure to high glucose (P<0.05)5. Western blot detection:Nephrin expression in high glucose group, high glucose +LiC1group were significantly reduced compared with normal group (P<0.05),there was a lower expression in high glucose+LiCl group than in high glucose group (P<0.05); α-SMA expression was significantly increased in high glucose group, high glucose+LiC1group,and LiCl group compared with normal group (P<0.05), α-SMA expression in high glucose+LiCl group was higher than in high glucose group (P<0.05)Conclusion1. GSK-3β inhibitor LiC1do not increase podocyte survival rate under high glucose condition.2. High glucose condition can induce podocyte apoptosis, while LiC1can increase the opoptotic rate of podocytes in high glucose condition.3. GSK-3p inhibitor LiCl can lead to epithelial-mesenchymal transition in podocytes,and more prominent transformation of podocyte phenotype under high glucose induction.