The Protect And Perhaps Mechanism Of Nestin In Injury Of LN Podocyte Skeletal Protein

Objectives: As the most common complication of systemic lupus erythematosus,lupus nephritis has high prevalence and low cure rate.Proteinuria is the typical clinical manifestations,and most proteinuria patients were discovered accompanying with podocytes injury.Nestin is a type of VI intermediate filament,and the knockdown of nestin had been proved to result in podocyte injury and proteinuria.However,the mechanism remains poorly understood.So in this study,we discussed the relationship of nestin,nephrin and mitochondrial autophagy to confirm the protect role of nesitn in LN podocyte injury,which would provide the theoretical basis for the prevention and treatment of LN.Methods: Mouse podocytes were cultured at 33°C in humidified atmosphere of 5% CO2 in DMEM-F12 media containing 10% fetal bovine serum(FBS),penicillin/streptomycin(100 U/ml and 100 ?g/ml,respectively)and 2-3 drops Mouse Interferon ?(10u/ml)to promote propagation.After being passaged,cells had been transfered to 37°C,DMEM-F12 media containing 10% FBS and penicillin/streptomycin(without Interferon ?)to promote differentiation.The cells were divided into control group,LN group,LN+shnestin and LN+3MA group.Plasma from patients with LN,shnestin and 3-MA,a specific inhibitor of autophagy,were used to infer the role of nestin in podocyte of LN.The expression of nephrin,a specific slit diaphragm protein of podocyte,was detected by western blot to assess the integrity of podocyte skeleton;the mitochondrial autophagy level were measured with the expression of PINK1,LC3II/I,P62 by western blot and immunofluorescence;the injury of mitochondrion were detected by JC-1.Results:1.The expression of nestin in podocyte was upregulated by LN plasmaWestern blot showed that the expression of nestin in podocyte was upregulated transiently by LN plasma.2.The expression of nephrin in podocyte was upregulated by LN plasmaWestern blot showed that the expression of nephrin in podocyte was upregulated transiently by LN plasma in 24 h and decreased in 48 h.3.Knockdown of nestin blocked the transient upregulation of nephrin induced by LN plasmaWestern blot showed that the expression of nephrin in LN+shnestin group was decreased compared with LN plasma group.4.A transient mitochondrion injury and mitochondrial autophagy activation was induced by LN plasmaThe result of JC-1 showed that the podocyte mitochondria membrane potential showed a transient revert in LN plasma group.Western blot showed that the expressions of PINK1 and LC3II/I in podocyte were upregulated transientlly by LN plasma.5.Knockdown of nestin blocked the transient upregulation of mitochondrial autophagy activation induced by LN plasmaThe results of western blot and immunofluorescence showed that the expression of PINK1 in LN+shnestin group was decreased compared with LN plasma group.6.Inhibition of autophagy blocked the transient upregulation of nephrin induced by LN plasmaWestern blot showed that the expression of nephrin in LN+3-MA group was decreased compared with LN plasma group.Conclusion:1.The expression of nephrin was upregualted transiently in podocyte by nestin in LN plasma group.2.Nestin upregulated the expression of nephrin in podocyte partly by activating mitochondrial autophagy in LN.