Effects Of CUEDC2 In Renal Interstitial Fibrosis And Its Mechanism

Renal Interstitial Fibrosis(RIF) is the commom pathological basis and final outcome of almost all chronic kidney diseases which leads to end stage renal failure,and leads to poor prognosis and renal dysfunction.The mechanism of Renal Interstitial Fibrosis has not been elucidated completely.Inflammatory response, epithelial mesenchymal transition and excessive deposition of extracellular matrix proteins may induce the develpoment of RIF.Inflammatory reaction is an initial part of the mechanisms in response to renal injury,is a major initial force in the develpoment of RIF.Inflammatory cell infiltration especially the macrophages can directly promote renal fibrosis by Macrophage myofibroblast transition(MMT) process, and induce the expression of pro-inflammatory factors,future aggravated infiltration of inflammatory cell and increased extracellular matrix protein.Inflammatory factors also plays a importment role in Renal Interstitial Fibrosis.Inflammatory factors which can facilitate the infiltration of monocytes/macrophages and lymphocytes,the differentiation and proliferation of renal interstitial cells,and can stimulate the expression of pro-inflammatory and pro-fibrotic cell factors,acclerate the synthesis and accumulation of extracellular matrix,result in severe inflammatory responseand lead to Renal Interstitial Fibrosis.Epithelial mesenchymal transition(EMT) is a process that involves the loss of inherent epithelial adhesion molecules and increased expression of mesenchymal protein markers,in addition to present a fibroblastic appearance and acquire invision and migratory activities. Furthermore, secretion of many cytokines contribute to the progression of inflammation response and the develpoment of renal intersisitial fibrosis. Transforming growth factor-β1(TGF-β1) has a pivotal role in EMT process, and is thought to be a key regulatory factor. Most of effects of TGF-p are mediated through the Smad signaling pathway. TGF-β is a powerful multifunctional cytokine involved in a variety of signaling pathway and many biological processes.TGF-β1 induces EMT via Smad-dependent and Smad-independent signal transduction signaling pathways in tubular cells.TGF-β is a growth regulator factor, which can inhibit most cell types growing, including tubular epithelial cells but stimulate mesenchymal cells growing to promote extracellular matrix deposition.Smads are cytoplasmic signal transducer proteins superfamily members, including receptor-activated Smad proteins (Smad2 and Smad3), inhibitory Smad protein (Smad7),and common mediator (Smad4). TGF combine with TβRI receptor kinase to phosphorylate Smad2 and Smad3, then combine with Smad4, and the compound translocate to the nucleus to redulate translation.Therefore, epithelial mesenchymal transition and renal interstitial fibrosis can be prevented by blocking the TGF-β/Smad signaling pathway.There are a number of protein can regulate epithelial mesenchymal transition process involved in negative regulation response. Smad7 protein is a inhibitin regulature protein in TGF-β/Smad signaling pathway, plays an important role in tubulointerstitial fibrosis by enhancing ubiquitin-dependent degradation. Smad7 modulated the ubiquitin-proteasome degradation pathway are involved in E3 ubiquitin ligases.Smad7 modulates the TβRI for dephosphorylation by combining with the PP1 holoenzyme.Some researches showthat the level of HGF can upregulate the expression of smad2/3 transcriptional co-repressor by restraining the TGF-β1-initiated EMT.Moreover,BMP7 protein alsoplays a important role of negative regulatory in EMT.The anti fibrotic role of BMP7 is almostly mediated throuhg balancing the profibrotic effect of TGF-β.BMP-7 can inhibit inactivating matrix-producing cells and facilitating mesenchymal-to-epithelialtransition to reduce extracellular matrix protein formation.BMP-7 also can promote the extracellular matrix protein degradation.And others researches show that HSP72 protein inhibites EMT via reducing the phosphorylation of Smad3 and p-Smad3 and nuclear translocation and confirmes that HSP72 can interacte with both Smad3 and p-Smad3. Therefore, HSP72 also can inhibit the expression of mesenchyme protein,such as a-SMA and Fibronectin by attenuating the phosphorylation and nuclear translocation of STAT3 protein, which is induced by TGF-β,decreasing the expression of its downstream protein.CUE domain-containing 2 (CUEDC2) is a multi-functional protein, which regulates cell cycle, growth factor signaling and inflammation and tumorigenesis. CUEDC2 acts as a novel regulator of PR and promotes PR degradation through the ubiquitin-proteasome pathway.CUEDC2 impairs the reactivity of breast cancer with endocrine therapies by reducing the expression of estrogen receptor-a. CUEDC2 can express in many tumor,such as breast cancer,kidney cancer(high expression) or lung adenocarcinoma(low expression). CUEDC2 has an inhibitory role in NF-κB signaling pathway, which plays a key role in inflammatory responses and CUEDC2 plays a crucial role in modulating macrophage function.The activion of NF-κB is related with the mechanism of UUO.So if the signaling pathway was blocked,the inflammatory response was inhibited.But the mechanism of renal interstitial fibrosis is not cleared,the aim of our experiment is to build the renal interstitial fibrosis model to discuss the effects and mechanisms of CUEDC2 protein.1.Effects of CUEDC2 on Inflammation in rats with Unilateral Ureteral ObstructionObjective:To reserch the effects of CUEDC2 on inflammation response in rats with unilateral ureteral obstruction.Methods:To set up the model of unilateral ureteral obstruction:30 balb/c rats were purchased from the Laboratory Animal Center of Sun Yat-Sen University and were randomly distributed into sham operation group(sham-vector),uuo operation group(uuo-vector) and cuedc2 treatment group after uuo(uuo-cuedc2).Each group has 10rats.UUO operation group and treatment group with CUEDC2 plasmid transfection were performed the operation of unilateral ureteral obstruction.To dissociate the right ureter,then to ligation and dividing.While the sham operation group only free right ureter, don’t ligation and dividing.Using ultrasound-microbubble gene transfer technique to transfect the plasmid, respectively in the 1st day before the uuo operation, and the 1st day and the 7th day after the uuo operation. Vector plasmid was transfeced into sham operation group and uuo operation group,while the CUEDC2 plasmid was transfeced into cuedc2 treatment group.All plasmids were injected via tail vein of rat. At 7th day and 14th day after uuo operation,5 rats of each group were respectively executed.Taken the tissue samples, and collected blood of each group to centrifuge and to keep the supernatant,saved at-80 ℃ for reserve. After perfusion and swill of kidney, when kidney bleach,we carefully peeling the right renal and removing the capsule. Kidney tissues can be divided into different small pieces to stain with immunohistochemical or used to extract protein.Inflammation factors including intercellular cell adhesion molecule-1,monocyte chemotactic protein-1,IL-1 and IL-8 were quantified by ELISA.Immunohistochemistry was performed to measure the expression of CUEDC2 and the surface antigen of monocyte/macrophage.Results:Immunohistochemical result of CUEDC2 shows that the expression of CUEDC2 protein was decreased at postoperation, after plasmid transfection, the expression increased(P< 0.05).At 7 and 14d after operation,the expression of ICAM1,MCP1,IL1,IL8 and CD68 increased, with the extend of obstruction time,the trend is more obvious(p< 0.05).But the group was treated by CUEDC2, expression of ICAM1,MCP1,IL1,IL8 and CD68 showed a marked decrease when compared to uuo-vector(p< 0.05).Conclusions:CUEDC2 protein can inhibit the monocyte/macrophage infiltration, reduce the expression of inflammation factors in the renal fibrosis tissue, relieve the inflammation response of renal and inhibit the occurrence of renal interstitial fibrosis.2.The role of CUEDC2 in renal interstitial fibrosis and its possible mechanism(1)study in virtoObjective:to study role of CUEDC2 in EMT of HK2 cell and its possible mechanismMethods:5 ng/ml TGF-β1 stimulates HK2 cell to induce the formation of EMT model,a total of six points. The Kit was used to extract the vector and cuedc2 plasmid.Preparation of slow virus to build a stable expression of plasmid HK2 line with calcium phosphate transfection method.TGF-β1 was also used to stimulate cells, four time points. Western blot was used to detected the expression of CUEDC2, Fibronectin, E cadherin, Vimentin, p-smad2, p-smad3 and smad2 and smad3.Results:the level of E-cadherin protein decreased,the expression of Fibronectin rised after TGF-β1 stimulated HK2 cells, along with the prolonged stimulation time, the trend was more obvious. EMT model has been successfully established by stimulate with TGF-β.At this point, the expression of CUEDC2 protein gradually decreased with the long playing obstruction.TGF-β1 stimulate vector-hk2 and cuedc2-hk2, respectively, the expression of cuedc2 protein was significantly increased in cuedc2-hk2 group, this outcome shows that stable expression cell line has been builded successfully.In cuedc2-hk2 group,at each time,E-cadherin protein expression increased, Fibronectin, Vimentin protein and p-smad2 and p-smad3 protein expression decreased.Conclusions:CUEDC2 protein expression was decreased in TGF-β1 induced EMT process.when, overexpression of CUEDC2, mesenchymal phenotype protein expression can be reduced, epithelial phenotype protein expression was increased, smad2 and smad3 protein phosphorylation was inhibited, inhibition the EMT induced by TGF-β.Therefore,CUEDC2 may participate in the negative regulation of EMT process.(2)study in vivoObjects:To investigate the effects of CUEDC2 protein in renal interstitial fibrosis in obstructive nephropathy and its possible mechanism.Methods:UUO model establishment and tissue collection were same as above method.Hematoxylin-eosin and Masson staining were used to measure renal pathology.Western blot was used to detected the expression of CUEDC2, Fibronectin, E cadherin, Vimentin, p-smad2, p-smad3 and smad2 and smad3.Results:CUEDC2 plasmid transfection treatment group,7 d and 14 d after UUO, renal tubule degeneration,necrosis, interstitial inflammatory cells infiltration and fibrosis significantly reduced compared with uuo group.and the area of renal interstitial collange deposition is significantly reduced in comparison with UUO group.In UU0-CUEDC2 group, CUEDC2 protein expression, E-cadherin protein expression significantly increased, while the expression of Fibronectin, Collagen I, p-smad2, p-smad3 decreased.Conclusions:In uuo model,CUEDC2 protein can relieve the damage of renal tubular, reduce the interstitial collagen deposition, inhibition the synthesis of extracellular matrix, inhibition expression of p-smad2 and p-smad3 protein and slow down the progress of renal interstitial fibrosis.Summary:The level of CUEDC2 was decreased in EMT of HK2 cell and uuo model of rat,so we can conclution that CUEDC2 protein involves in counteregulatory responses. And CUEDC2 can inhibit the renal interstitial fibrosis via inhibiting the inflammatory cell infiltration, reducing the expression of inflammatory cytokines, to weaken the inflammatory reaction, and reducing the amount of interstitial collagen deposition, inhibiting smad2 and smad3 protein phosphorylation, increasing the smad7 protein expression to Inhibit the activation of TGF/smad signaling pathways, inhibit the EMT process.In conclusion,CUEDC2 reduce the level of renal fibrosis,and CUEDC2 has a certain function of kidney protection.