The Role Of ELF3/MAPK14 Signaling Pathway In Autophagy-Mediated Podocyte Injury

Background and aims:Diabetic kidney disease(DKD)is one of the microvascular complications of diabetes and the great causes of end-stage renal disease.Although the validity of existing treatments,the residual risk of onset and progression of diabetic kidney disease remains significant.autophage is a process of clearing abnormal and dysfunctional molecules and organelles of cells,while podocytes are highly specialized epithelial cells located on the surface of glomerular capillaries that have high levels of basal autophagy.E74 Like ETS Transcription Factor 3(ELF3),a subgroup of epithelial-specific ETS transcription factors,is mainly expressed in organs containing secreted epithelial cells.It has been shown that the expression of ELF3 can be induced by direct stress of high glucose and/or AGEs in podocytes,leading to diabetic glomerulosclerosis.Mitogen activated protein kinase 14(MAPK14)is involved ina wide range of cellular processes,such as differentiation,proliferation,survival and apoptosis,transcriptional regulation and development,and can inhibit autophagy under basic conditions.Therefore,we speculate that the ELF3/MAPK14 signaling pathway can regulate podocyte autophagy in high-glucose induced DKD,leading to podocyte injury.In this study,a model of high sugar induced podocyte injury was constructed to observe whether high sugar could regulate the level of autophagy in podocyte,and further explore the role and mechanism of ELF3/MAPK14 signaling pathway in podocyte injury.Methods:To establish the model of podocyte damage because of high sugar.1)To observe the effectiveness of different concentrations of high glucose(5.5 mM/L、30 mM/L,40 mM/L,50 mM/L)on autophagy of podocytes(MPC5);2)mRNA expression profile of podiatocytes treated with high glucose was screened byRNA-seq technique,and the expression of ELF3 was significantly up-regulated;Through the intersection analysis of mRNA expression profiles of autophagy database,Cistrome database and high-glucose treated podocytes,it was found that MAPK14 expression was significantly up-regulated in high-glucose treated podocytes,and the transcription factor binding prediction of MAPK14 promoter region was further conducted with bioinformatics technology.We found the transcriptional binding site of ELF3 in the MAPK14 promoter region.The expression levels of ELF3 and MAPK14 in high-glucose induced podocytes and DKD kidney tissues were detected.The spatial localization of ELF3 and MAPK14 in cells was observed by immunofluorescence.3)To observe the role of ELF3/MAPK14 in inducing podocyte loss and autophagy:RNA interference knockout ELF3 and gene transfection knockout ELF3 were used to detect the expression of podocin、desmin and autophagy markers by co-transfection of overexpression ELF3 and knockout MAPK14.The expression level of ELF3 in DKD kidney tissue was detected by immunohistochemical method.The expressions of podocin 、 desmin and autophagy markers(LC3,Beclin1,P62)were detected by Western blot and RT-PCR.Results:1)In the cell model,it was found that high glucose inhibited autophagy and enhanced podocin injury.The expressions of LC3,Beclin1 and Podocin showed a gradient decrease in concentration,while the expressions of P62 and desmin showed a gradient increase in concentration.2)The expressions of ELF3 and MAPK14 were increased in podocytes under the stimulation induced by high glucose,and the ELF3 positive area in DKD kidney tissue was significantly increased;Both ELF3 and MAPK14 were expressed in the nucleus by immunofluorescence colocalization.3)After ELF3 gene silencing,LC3 and Beclin1 increased significantly,P62 expression decreased,autophagy level increased,podocin expression increased,Desmin expression decreased,podocin injury decreased,and MAPK14 expression decreased;After transfection with overexpression of ELF3 plasmid,LC3 and Beclin1 were significantly decreased,P62 expression was increased,autophagy level was decreased,podocin expression was decreased,desmin expression was enhanced,podocin injury was enhanced,and MAPK14 expression was enhanced.In the recovery experiment,overexpression of ELF3 reduced the level of autophagy and enhanced the injury of podocytes.After MAPK14 gene silencing,the damage of podocytes decreased and the of autophagy increased.Nevertheless,after their cotransfection,the extent of autophagy and podocytes damage tended to be normal.Conclusion:The results of this study indicate that high sugar can lead to podocyte injury by inhibiting podocyte autophagy,and the mechanism may be that ELF3/MAPK14 signaling pathway inhibits podocyte autophagy related expression,resulting in kidney injury,which provides a new idea for the research on the mechanism of podocyte autophagy in the progression of DKD.