Effects Of CUEDC2 In The Pathogenesis Of Renal EMT In Diabetic Nepropathy And Its Mechanism

Diabetic nephropathy(DN)is common and refractory microvascular complication of diabetic.Glomerulosclerosis,deposition of extracellular matrix(ECM)and fibrosis in tubular interstitial are the main features of DN,and tubulo-interstitial fibrosis(TIF)is reported to be pivotally involved in the onset and progression of various kidney diseases to End Stage Renal Disease,including DN.Compelling evidence demonstrated the crucial important role of tubular epithelial to mesenchymal transdifferentiation(EMT)in renal fibrogenesis.While the role of EMT in renal fibrosis has been recognized,their precise molecular mechanism of regulation in process of DN needs to be further clarified.Transforming growth factor-?1(TGF-?1)has been recognized as a key mediator in DN,and directly induces the tubular EMT.TGF-?1 may up-regulate the expression of ECM proteins,simultaneously,enhances the expression of inhibitors of ECM-degrading enzymes,such as plasminogen activator inhibitor type 1(PAI-1).More importantly,TGF-?1/smad signaling is reported as a major and canonica pathway leading to renal fibrosis.CUE domain-containing protein 2(CUEDC2)is a novel multi-functional protein,which is over expressed in a variety of cancers and plays critical roles in many biological processes,such as cellcycle,inflammation and tumor genesis.The CUE domain,known as a small ubiquitin-binding motif of approximately 40 amino acids,is able to recognize both mono-and poly-ubiquitin and facilitate intermolecular mono-ubiquitination.CUEDC2 was initially proved to interact with progesterone receptor(PR)and promote progesterone-induced ubiquitination and degradation of receptors.Also,increasing evidence suggested that CUEDC2 played an important role in tumorigenesis.CUUEDC2 was found to inhibit the phosphorylation of IkB kinase a(IKKa)and IKK? by recruiting the protein phosphatase 1(PP1)to the IKK/CUEDC2 complex and allowing PP1 to dephosphorylate IKKa and IKK?.However,the impact of CUEDC2 expression in the development of DN has not been elucidated yet.To identify the effect and mechanism of CUEDC2 in the Pathogenesis of Renal EMT in DN,we designed the project as follows:Party I Effects of CUEDC2 in renal function and tubulo-interstitial fibrosis of diabetic nephropathy.To investigate the effects of CUEDC2 on renal interstitial fibrosis and renal biochemical function in diabetic nephropathy,we first detected the expression and localization of CUEDC2 in the kidney of both clinical tissues and db/db mice.Then,we examined whether overexpress of CUEDC2,by the ultrasound-microbubble gene transfer technique,improved renal function and interstitial fibrosis in db/db mice.We obtained the following results:1.CUEDC2 was mainly located in the medulla tubules epithelial cells of Clinical tissues and db/db mice.The expression of CUEDC2 was down-regulated in DN renal tissue and db/db mice.2.Up-regulation of CUEDC2 in vivo significantly reduced the bodyweight,blood glucose,cholesterol and urinary albumin excretion in db/dbmice.CUEDC2 could down-regulate the expression of Fibronectin and collagen I in vivo,inhibit the renal fibrosis,and have the therapeutic effect.Party II Effects of CUEDC2 in the Pathogenesis of Renal EMT in DN and its mechanismIn order to explore the role and possible mechanism of CUEDC2 in renal tubular epithelial cell EMT in DN,we cultured human renal tubular epithelial cells(HK2)in vitro and investigated the effect and possible mechanism of CUEDC2 protein in the Pathogenesis of Renal EMT.We obtained the following results:1.Overexpression of CUEDC2 could down-regulate the expression of p-Smad2 and p-Smad3,and down-regulate the expression of mesenchymal markers:Fibronectin and vimentin,while up-regulation of E-cadherin expression.On the contrary,knockdown CUEDC2 expression,presented the the opposite results,which showed that overexpression/interference CUEDC2 could inhibit/promote TGF-?/Smads signaling pathway and TGF-?1-induced HK2 EMT.2.CUEDC2 and Smad7 have endogenous and exogenous interactions,TGF-?1stimulation can promote this interaction,indicating that CUEDC2 can mediate Smad7 TGF-P type I receptor(T?RI)dephosphorylation inactivation,Which negatively regulates TGF-?/Smads signaling pathway,and inhibits the role of EMT in diabetic nephropathy.