Dysregulation Of LncRNA GM5524 And GM15645 Involves In High-glucose Induced Podocyte Apoptosis And Autophagy In Diabetic Nephropathy

Background:Diabetic nephropathy(DN)is a common chronic complication of diabetes,which is characterized by increased urinary albumin excretion(microalbuminuria)and is the leading cause of end-stage renal disease(ESRD)However,the mechanism of Diabetic nephropathy pathogenesis process remains unclear.Recently,long noncoding RNAs dysregulation are regarded to contribute to the occurrence and development of various human diseases,but the function of lncRNAs in human diabetic nephropathy are poorly understood.Our previous study using microarray analysis revealed that hundreds of lncRNAs are dysregulated in DN,but these lncRNAs function were not demonstrated.Among those dysregulated lncRNAs,Gm5524 is significantly up-regulated in response to diabetic nephropathy,while Gm15645 is significantly down-regulated in response to diabetic nephropathy. Aims:To explore the potential functions of Gm5524 and Gm15645 in DN.Methods:In the kidney tissues of diabetic nephropathy mice,Gm5524 was significantly up-regulated and Gm 15645 was significantly down-regulated by microchip screening.In this study we first validated these results in diabetic nephropathy mice kidney tissues and podocytes treated with high glucose through qRT-PCR.We then down-regulated or over-expressed Gm5524 and Gm15645 expression respectively in mouse podocyte to further observe the change of cell autophagy and apoptosis in normal conditions and high glucose conditions.Finally we used Western Blot method to detect the effects of Gm5524 and Gm15645 on the apoptosis and autophagy related factors to preliminarily explore possible mechanisms.Results:In this study,we validated the microarray result by qRT-PCR in DN and control tissues,and the results showed that Gm5524 is indeed up-regulated and Gm 15645 is down-regulated in DN tissues as well as the microarray data.We then down-regulated or over-expressed Gm5524 and Gm 15645 expression in mouse podocyte.Flow cytometry analyses of these cells showed that podocytes had higher apoptotic rate after knockdown of Gm5524 expression and up-regulation of Gm15645 expression under normal condition,while the apoptotic rate is more higher in high-glucose treated podocytes.Then the electron microscopy analysis showed that podocytes present higher number of electrodense inclusions and lipidic granules after over-expression of Gm5524 or knockdown of Gm15645,which is more obvious in high-glucose treated podocytes.Finally the western blot assays in podocytes showed that knockdown of Gm5524 and over-expression of Gm15645 increased cleaved Caspase3,Bax and LC3 I expression,while decreased LC3 ?,Atg5,Atg7,and Bcl2 expression.Conversely,Gm5524 over-expression and Gm15645 down-regulation decreased cleaved Caspase3,Bax and LC3 I expression,and increased LC3 ?,Atg5,Atg7,and Bcl2 protein levels.Conclusion:Our findings reveal the roles of lncRNA Gm5524 and Gm15645 in the high-glucose induced podocyte apoptosis and autophagy during diabetic nephropathy,which may further our understanding of the involvement of lncRNAs in diabetic nephropathy,and provide a potential novel therapeutic target for this disease.