The Role And Mechanism Of Ubiquitin-conjugating Enzyme E2S (UBE2S) In Renal Tubular Epithelial To Mesenchymal Transition

Objective Aims: Renal interstitial fibrosis(RIF)is the necessary pathway for most chronic kidney disease(CKD)to progress to end-stage renal failure.A large number of clinical and experimental studies have shown that the degree of RIF damage is similar to that of CKD patients.The degree of renal damage is obviously related,as an important indicator to reflect the severity of renal function decline and judge the prognosis of patients.Therefore,identifying the key molecular mechanisms involved in the pathogenesis of renal interstitial fibrosis will provide new therapeutic strategies for the treatment of CKD patients.Previous studies have shown that epithelial mesenchymal transition(EMT)is one of the important factors leading to renal interstitial fibrosis.EMT may also be experienced in the epithelium of mature tissues,such as inflammation and injury,leading to fibrogenesis.EMT can be caused by various conditions,including in vitro stimulation of transforming growth factor-β1(Transforming growth factor-β1,TGF-β1),animal kidney fibrosis models,and human obstructive nephropathy.Ubiquitination is a way of post-translational modification of proteins.As one of the key roles in catalyzing this process,ubiquitin-conjugating enzyme(E2)is involved in cell proliferation,differentiation and migration.Ubiquitin-conjugating enzyme E2S(UBE2S,also known as E2-EPF)is one of the important members of the E2 protein family.It can regulate transcriptional silencing induced by DNA damage,and promote the EMT process of tumor cells by regulating cell cycle and differentiation,thereby participating in tumorigenesis.However,the role of UBE2 S in renal interstitial fibrosis and renal tubular EMT is still unknown.This topic first constructed a unilateral ureteral obstruction(UUO)model through in vivo experiments to explore the relationship between UBE2 S and EMT involved in renal interstitial fibrosis;And apply the anti-fibrosis drug Pirfenidone(PFD)treatment intervention to explore the role of UBE2 S in rat kidney fibrosis.Then in vitro experiments EMT was induced in human proximal renal tubules epithelial cells(HK-2 cells)by TGF-β1 to explore the relationship between UBE2 S and EMT.Finally,the possible molecular mechanism of UBE2 S affecting TGF-β1 induced EMT in renal tubules was studied in depth.Methods: In vivo experiment: There are 48 male SD rats(SPF grade)aged 8-10 weeks,weighing 280-320,randomly divided into 8 groups(n=6): sham operation group(SHAM),operation 3 days group(UUO3),operation 7 days group(UUO7),operation 14 days group(UUO14),treatment 3 days group(PFD3),treatment 7 days group(PFD7),placebo 3 days group(-PFD3),placebo 7 days group(-PFD7).The rats were sacrificed on the 3d,7d and 14 d by group,and the ligated kidneys were taken.The treatment group and placebo group were given PFD(500mg/kg/d)and normal saline(500mg/kg/d)respectively on the first day after the operation,until they were terminated after 3days or 7days.HE and Masson staining were used to observe the morphological changes in the kidney tissues of each group.Immunohistochemical methods were used to detect the expression of UBE2 S,α-SMA and vimentin in kidney tissue of rats in the groups.Western blot and Real-time PCR were used to detect the the expression of UBE2 S,HIF-1α(hypoxia-inducible factor1α),α-SMA and vimentin in kidney tissue of rats in the groups.In vitro experiment: HK-2 cells were stimulated with 5ng/ml TGF-β1,and the cell morphology change was studied.The expressions of UBE2 S,HIF-1α,α-SMA and vimentin were detected by Western blot;UBE2S shRNA lentiviral vector and UBE2 S overexpression lentiviral vector were constructed respectively to transfect HK-2 cells,then we cultured transfected cells with TGF-β1 to detect the expression of UBE2 S,HIF-1α,α-SMA,vimentin,and study the effect on cell transdifferentiation.At the same time,through overexpression and knockdown of UBE2 S,we explore the expression of HIF-1α in EMT stimulated by TGF-β1,and study the mechanism of UBE2 S affecting EMT induced by TGF-β1.Results: 1.Successfully established a UUO rat model: In HE staining,renal tubules in UUO group were significantly expanded,epithelial cells were necrotic and shedding,and renal tubule damage was obvious as the obstruction time prolonged;In Masson staining,UUO7 and UUO14 groups showed a large number of blue-stained fibrotic lesions;EMT marker proteins,α-SMA and vimentin,were significantly increased in UUO groups,and there was a tendency to increase with the obstruction time;UBE2S expression increased in the UUO group in western blot,and reached a peak in UUO3 and UUO 7 days;The expression of HIF-1α in UUO groups increased significantly.After PFD intervention,the expression levels of UBE2 S,HIF-1α and EMT marker proteins,α-SMA and vimentin in the treatment group were significantly lower than those in the placebo group;2.Lentiviral overexpression UBE2 S was transfected into HK-2 cells and stimulated with 5ng/ml TGF-β1 for 24 h,the expression of α-SMA and vimentin was significantly higher than that of the control group;and overexpression of UBE2 S increased cell migration ability stimulated by TGF-β1.Lentiviral sh UBE2 S was transfected into HK-2 cells and stimulated with 5ng/ml TGF-β1 for 24 hours,the expression of α-SMA and vimentin was significantly lower than that of the control group.3.Under the induction of TGF-β1,UBE2 S overexpression up-regulates HIF-1α;knocking down UBE2 S down-regulates HIF-1α,with a increasement expression of VHL.Conclusion: 1.UBE2 S participates in the renal fibrosis induced by the rat UUO model,and co-regulates EMT with HIF-1α;PFD anti-fibrosis drugs may alleviate renal fibrosis caused by EMT by inhibiting UBE2S;2.UBE2 S can promote EMT in HK-2 cells induced by TGF-β1;3.UBE2 S promotes TGF-β1 induced EMT in HK-2 cells by up-regulates HIF-1α.