Mechanism Of Uhrf2 And Ube2v1 In High Glucose-induced Epithelial-mesenchymal Transition Of Renal Tubular Epithelial Cells

As one of the major complications of diabetes,diabetic nephropathy(DN)has become a serious disease endangering human life and health,and it is the leading cause of end-stage renal disease.Diabetic nephropathy can cause kidney fibrosis,edema,high blood pressure,kidney failure,and severe cases can lead to death.Renal tubular epithelial cells damage and fibrosis will occur in the course of diabetic nephropathy,which is a serious pathogenesis of diabetic nephrotic fibrosis.Epithelial-mesenchymal Transition(EMT)causes epithelial cells to lose polarity,lose connection with the basement membrane,and gain higher migration ability,which is a hallmark process of the renal tubule interstitial fibrosis.Ubiquitination is an important pathway for intracellular signal transmission,E1 activation enzyme,E2 binding enzyme and E3 ligase enzyme are the main members.K63 ubiquitination mainly participate in cell signaling,DNA repair and protein activity regulation.It was reported that UBE2V1,as the only E2 enzyme to take part in the K63 ubiquitination,it was involved in diabetic nephropathy,and UHRF2 is a E3 ubiquitin ligase,which participates in the ubiquitination of K63.But the specific mechanism of ube2vl involved in diabetic nephropathy remains unclear.Currently,uhrf2 research mainly focus on the fields of DNA methylation and tumor,and the relationship between uhrf2 and diabetic nephropathy has not been reported.Therefore,in this study,human embryonic kidney epithelial cells(HKC)are used to research the mechanism of uhrf2 and ube2vl in EMT,and preliminary inquire the relationship between the two genes.The aim is to reveal the pathogenesis of diabetic nephropathy fibrosis and provide new ideas and targets for the treatment of diabetic nephropathy.The main findings are as follows:1.High glucose promotes the expression of uhrf2 in HKC cells and lead to its EMT transformation.Uhrf2 mRNA transcription level was significantly upregulated in HKC cells,post 1h high glucose culture(D-glucose 30mmol/L).Immunofluorescence staining showed that UHRF2 was mainly located in the cytoplasm of HKC cells,and no significant changes were observed after treated with high glucose.At the same time,EMT process was simulated and the migration rate of HKC cells was detected by scratch experiment.The results showed that high glucose culture or uhrf2 overexpression could accelerate cell migration,and uhrf2 knock down decrease cell motility.Immunofluorescence and western blot experiments showed that with high-glucose culture,?-SMA,ZEB1 significantly up-regulated at protein level,and E-cadherin was down-regulated,but uhrf2 knock down could inhibit the up-regulation of ?-SMA.ZEB1 and weaken the down-regulation of E-cadherin.The results showed that high-glucose cultured HKC cells could induce the expression of uhrf2,and uhrfl up-regulation could further promote the EMT process and accelerate the process of diabetic nephropathy fibrosis.2.High glucose promotes the expression of ube2vl in HKC cells and lead to its EMT transformation.When HKC cells were treated with high glucose(D-glucose 30mmol/L),ube2vl mRNA transcription level was significantly upregulated at 1h.At the same time,EMT process was simulated and the migration rate of HKC cells was detected by scratch experiment.The results showed that high glucose treatment or ube2v1 overexpression could accelerate cell migration,and ube2v1 knock down decrease cell motility.In addition,immunofluorescence and western blot experiments showed that HKC cells under high glucose could significantly up-regulate the protein levels of?-SMA,ZEB1 and significantly down-regulate the protein level of E-cadherin,but knockdown of ube2v1 could inhibit the up-regulation of ?-SMA and ZEB1,and weaken the down-regulation of E-cadherin.The results showed that high-glucose cultured HKC cells could induce the expression of ube2v1,and ube2v1 up-regulation could further promote the EMT process and accelerate the process of diabetic nephropathy fibrosis.3.Uhrf2 and ube2v1 may participate the EMT transformation process of HKC cells by synergistic actions.Real-time fluorescence quantitative PCR showed that the expression of uhrf2 significantly decreased when ube2vl was knocked down,while the expression of ube2v1 did not change significantly when uhrf2 was knocked down.Immuno-fluorescence assay showed that UHRF2 remain localized in the cytoplasm when ube2v1 was knocked down.Besides,immunoprecipitation experiments showed that high glucose would increase the ubiquitination level of K63 in HKC cells,and UHRF2 would occur K63 ubiquitination under high-glucose.The results showed that ube2v1 mediates uhrf2 transcription up-regulation in HKC cells under high glucose,and may promote the K63 ubiquitination of UHRF2 to participate the EMT transformation process of HKC cells,thus accelerating the fibrosis process of diabetic nephropathy,but the specific mechanism needs to be further studied.In conclusion,the experimental results showed that both uhrf2 and ube2vl were involved in the EMT of HKC cells under high glucose,and they may participate in this process through synergistic effects,thus promoting the occurrence of diabetic nephropathy fibrosis and providing new ideas and targets for the treatment of diabetic nephropathy.