Cdc42-mediated Podocyte Apoptosis And Impaired β-cell Insulin Secretion:A Preliminary Study On The Mechanism Of Renon-pancreatic Communication

Objectives:Progressive impairment of beta cell function may result in the prolongation and progression of Type 2 diabetes mellitus(T2DM).From the holistic theory of TCM and the discovery of renal injury secondary to pancreatitis,there is a potential communication between the kidney and pancreas.This study sought to clarify whether Cdc42 affects podocyte apoptosis and further impinges on islet cell function.This study firstly verified that high glucose could induce the up-regulation of Cdc42 expression and the apoptosis of MPC5 in podocyte mouse podocytes,and the regulation of Cdc42 could affect the apoptosis of podocytes.After that,the in vitro co-culture model of MPC5 and β-TC6 cells and the in vitro co-culture model of MPC5 and primary mouse islet cells were preliminarily established.After CDC42 was regulated in MPC5S,insulin secretion and content of β-TC6 and mouse islet primary cells were detected.Methods and materials:1.Verification of mouse podocyte apoptosis Firstly,TUNEL assay,Western blot and PCR were used to verify that the high-glucose environment(25mmol/L)could induce the apoptosis of podocytes and the up-regulation of Cdc42 and Bax expression.The role of Cdc42 in MPC5 apoptosis was verified.2.Verification of insulin secretion of β-TC6 cells and primary islet cells in mice The co-culture model of MPC5 cells with β-TC6 cells and primary islet cells in mice was established.To verify the regulation of Cdc42 expression in MPC5 on insulin secretion of β-TC6 cells and primary islet cells in mice.Result:1.High glucose induced apoptosis of and up-regulated expression of Cdc42 and Bax of MPC5 cells:TUNEL assay showed that high glucose(25mmol/1)increased apoptosis of MPC5 cells(Figure 3)(P<0.05).Western blot and PCR results showed that the expression of Cdc42 and Bax in MPC5 cells was up-regulated in high glucose environment(25mmol/1)(Figure 1-2)(P<0.05)2.Modulate the expression of Cdc42 in MPC5 cellsWestern blot and PCR were used to verify the transfection effect.The expression of Cdc42 and Bax were up-regulated in the MPC5 cells transfected with Cdc42 plasmid(Figure 4)(P<0.05),The expression of Cdc42 and Bax were down-regulated in the MPC5 cells transfected with siCdc42(Figure 4)(P<0.05).3.Modulating the expression of CDC42 in MPC5 cells affects the apoptosis of MPC5 cellTUNEL results showed that apoptosis increased in MPC5 cells transfected with Cdc42 plasmid compared control(Figure 5)(P<0.05),apoptosis of MPC5 cells transfected with siCdc42 was reduced.4.The expression of CDC42 in MPC5 cells affected insulin secretion and insulin content in β-TC6 cellsInsulin secretion and insulin content of β-TC6 cells were detected by insulin secretion test(GSIS)and immunofluorescence.Compared with control,insulin secretion and insulin content of β-TC6 cells decreased after co-culture with MPC5 cells transfected with Cdc42 plasmid(Figure6-7)(P<0.05),insulin secretion and insulin content ofβ-TC6 cells were increased after co-culture with MPC5 cells transfected with siCdc42(Figure6-7)(<0.05).5.Effects of Cdc42 expression in MPC5 cells on insulin content in primary islet cells of miceThe secretion of insulin in primary islet cells of mice was detected by immunofluorescence.Compared with control,after co-culture with MPC5 cells transfected with Cdc42 plasmid,there was no significant change in insulin content of primary islet cells,the insulin content of primary islet cells increased after co-culture with MPC5 cells transfected with siCdc42(Figure 9)(P<0.05).Conclusion:1.High glucose(25mmol/L)can induce the expression of Cdc42 and Bax in MPC5 cells,and induce the apoptosis of MPC5 cells2.The expression of Cdc42 in MPC5 cells was positively correlated with the expression of Bax in MPC5 cells and the apoptosis of MPC5 cells3.The co-culture system proved that the expression of Cdc42 in MPC5 cells could affect the secretion and content of insulin in β-TC6 cells and primary mouse islet cells,and the relationship was negative.