Studies Of Function And Regulatory Mechanism Of ?-arrestin-1 In The Development Of Renal Fibrosis

Background:Renal tubulointerstitial fibrosis is the common pathway from chronic kidney disease(CKD)to end-stage renal disease(ESRD).Regardless of the underlying disease,once the chronic kidney disease progresses,the initiation of fibrosis results in massive accumulation of extracellular matrix and extensive scar formation in the kidney tissue,eventually leading to the destruction of renal parenchymal cells and renal failure.Therefor,it is significant to find a way to delay the progression of renal fibrosis.Interstitial fibrosis and glomerulosclerosis are the main pathological features of renal fibrosis.The pathological process is complicated,which involves a variety of signaling pathways and cytokines,eventually resulting in excessive extracellular matrix and impaired renal function.Despite substantial progress being made in understanding the mechanisms contributing to the pathogenesis of renal fibrosis,such as fibroblast accumulation,epithelial-to-mesenchymal transition(EMT),infiltration of inflammatory cells,there is only a few therapys available to treat or prevent renal fibrosis in clinical use today.The G protein-coupled receptor(GPCR)family is the largest membrane protein family,which participate in the pathogenesis of a variety of tissue fibrosis.More than 30 percent of drugs use GPCRs as drug targets.?-Arrestins,which are the negative regulator of GPCR,not only mediate the desensitization and endocytosis of G protein-coupled receptors,but also serve as scaffold proteins for the downstream pathways.Arrestins consists of four family members,arrestinl(visual arrestin)and arrestin4(cone arrestin)are distributed in retina,which responsible for regulating visible signal;arrestin2(?-arrestin-1)and arrestin3(?-arrestin-2)which share 78%of the amino acid sequence,widely distributed in various tissues and organs,play an important role in a variety of diseases.The combination of ?-arrestins and GPCR can inhibit the dissociation of a subunit from ?? subunit,thereby terminating the downstream signaling.Recent studies also found that ?-arrestins can act as scaffold proteins alone,recruiting intracellular protein molecules to regulate complex signaling pathways such as mitogen-activated protein kinase(MAPK),c-Jun N-terminal kinase(JNK),NF-?B and so on.Our previous studies demonstrated that?-arrestin-1 and ?-arrestin-2 were up-regulated in the diabetic nephropathy model of mice and in human tissue sections;?-arrestins negatively regulated autophagy of podocytes by inhibiting ATG12-ATG5 binding.However,the role of ?-arrestins in the development of renal fibrosis is still unclear.Therefore,this study devoted to clarify the role of ?-arrestins in the process of renal fibrosis.The up-regulation of ?-arrestin-1 in renal interstitial fibrosis was found for the first time.The EMT level and the degree of renal fibrosis in ?-arrestin-1 knockout mice were significantly reduced,and the specific molecular mechanism was explored by in vitro experiments.Objective:1.Determine the expression pattern of P-arrestins in renal interstitial fibrosis.2.Investigate the role of ?-arrestin-1 in renal interstitial fibrosis.3.Discuss the action of p-arrestin-1 on Wnt/?-catenin pathway in renal fibrosis.Methods:Part 1 ?-Arrestin-1 promotes the development of renal fibrosis1.Studies on the mice with UUO nephropathy:Male C57BL/6J mice were randomly divided into 4 groups.The mice model of UUO(unilateral ureteral obstruction)nephropathy was established by ligation of unilateral ureter and inducing urinary tract obstruction.We observed the expression pattern of ?-arrestin-1 and ?-arrestin-2 in the kidney from mice with UUO nephropathy by Real-time RT-PCR,Western-blot(WB)and Immuno-histochemistry(IHC).To define the tubular segment specificity of ?-arrestin-1 expression in the kidney,we used double immunostaining for ?-arrestin-1(green)and various tubular markers(red)in the kidney.2.Human renal biopsy samples:The human renal fibrosis section samples were supplied from the Department of Pathology,Shandong University School of Medicine,including normal control,diabetic nephropathy samples,polycystic kidney samples and uronephrosis samples.IHC staining was used to observe the expression pattern of ?-arrestin-1 in the samples from people with different renal disorders.3.Studies on ?-arrestin-1 deficiency(?-arrestin-1-/-)mice with UUO nephropathy:Male C57BL/6J mice and male ?-arrestin-1-/-mice,each divided into two groups,randomized choose one group for UUO modeling.The degree of renal interstitial fibrosis was evaluated by PAS(Periodic Acid-Schiff),MASSON and Sirius Red staining.Real-time RT-PCR,WB and IHC methods were used to detect the expression levels of fibrosis markers,including collagen I,?-SMA and fibronectin.Immunofluorescence method was used to observe the degree of inflammatory cells infiltration in renal cortex,and the expression levels of inflammatory factors such as TGF-?,IL-1? and IL-6 were detected by Real-time RT-PCR.We to examined the level of EMT in the kidney by double-labelling immunofluorescence and assessed the expression level of E-cadherin by WB.In addition,we explored the downstream signaling pathway of ?-arrestin-1.The expression levels of Wntl,Wnt2b,Wnt3 and Wnt4 were detected by Real-time RT-PCR,and the raltive protein level of active-?-catenin was detected by WB.Part 2 The role of ?-arrestin-1 in fibroblast cells and renal epithelial cells1.Regulation of fibrosis-related proteins expression by ?-arrestin-1:Normal rat kidney fibroblast(NRK-49F)cells were cultured in vitro and stimulated by transforming growth factor-?1(TGF-?1)with concentration gradient.WB was used to detect the expression level of ?-arrestin-1 in different cell groups.NRK-49F cells were transfected with small interfering RNA(siRNA)of?-arrestin-1,and the efficiency of gene silencing was detected by WB.The relative protein levels of collagen I,?-SMA and fibronectin were assessed by WB,in NRK-49F cells with ?-arrestin-1 deficiency under TGF-?1 stimulation.2.The regulating action of ?-arrestin-1 on Wnt/?-catenin Pathway:The expression of Wntl and active-?-catenin was examined by WB in NRK-49F cells with ?-arrestin-1 deficiency under TGF-?1 stimulation.Then,we added SKL2001,the agonist of Wnt/?-catenin to cultured NRK-49F cells,and detected the expression levels of active-?-catenin,a-SMA and fibronecitn.For further prove,we compared the expression of active-?-catenin,a-SMA and fibronectin,between gene silencing of Wntl with the addition of Wnt/?-catenin inhibitor Dkkl under the stimulation of TGF-?1.3.Impact of ?-arrestin-1 on renal tubular epithelial cells:Human renal proximal tubular cells(HK-2 cells)were cultured in vitro under TGF-?1 stimulation,and the expression pattern of ?-arrestin-1 was evidenced by WB.HK-2 cells were transfected with siRNA-?-arrestin-1,and the efficiency of gene silencing was detected by WB.Then,we detected the relative protein levels of EMT-related proteins,including E-cadherin,vimentin,slug and a-SMA,and Wnt pathway-related proteins,including Wntl and active-?-catenin.Results:Part 1 ?-Arrestin-1 promotes the development of renal fibrosis1.?-Arrestin-1 is induced in the kidney from mice with UUO nephropathy:Analysis by Real-time RT-PCR and WB showed that ?-arrestin-1 was induced more significantly than ?-arrestin-2 in the kidney from mice with UUO,which was also confirmed in paraffin-embedded sections of kidney tissue by IHC.We used double immunostained for ?-arrestin-1(green)and various tubular markers(red)in the kidney.The results showed that the upregulation of ?-arrestin-1 is mainly in proximal tubules.2.Upregulation of ?-arrestin-1 in human renal biopsy samples:IHC results showed that P-arrestin-1 was induced significantly in the kidney from patients with renal fibrosis,including diabetic nephropathy,polycystic kidney disease or urinary tract nephropathy.3.?-Arrestin-1 deficiency alleviated renal damage in mice with UUO nephropathy?-Arrestin-1 deficiency alleviated renal interstitial fibrosis:PAS,MASSON and Sirius red staining showed that ?-Arrestin-1 deficiency reduced the degree of interstitial fibrosis in the kidney from mice with UUO nephropathy.We aslo found that fibrosis-related proteins,including collagen I,?-SMA and fibronectin,were decreased in ?-arrestin-1-/-mice with UUO nephropathy by Real-time RT-PCR,WB and IHC.P-Arrestin-1 deficiency reduced inflammatory responses:Real-time RT-PCR showed that the expression of inflammatory cytokines,including TGF-1,IL-1 and IL-6 were decreased in ?-arrestin-1-/-mice with UUO nephropathy compared to WT mice.Immunofluorescence showed that macrophage infiltration was significantly reduced in ?-arrestin-1-/-mice with UUO nephropathy compared to WT mice,rather than neutrophil infiltration.?-Arrestin-1 deficiency inhibited EMT:Double immunofluorescence showed that the EMT was suppressed in?-arrestin-1-/-mice with UUO nephropathy compared to WT mice,which manifested as the upregulation of E-cadherin and downregulation of ?-SMA.WB results also showed the sham expression pattern of E-cadherin.?-Arrestin-1 deficiency negatively regulated Wnt/p-catenin pathway:Mechanically,we found that the expression of Wnt1 was decreased in?-arrestin-1-/-mice with UUO nephropathy compared to WT mice,rather than Wnt2b,Wnt3 and Wnt4.To examine the biologic consequence of Wnt1 induction,we next investigated the activation of ?-catenin.Interestingly,active-?-catenin expression was dramatically reduced in ?-arrestin-1-/-mice with UUO nephropathy compared to WT mice.Thereby,we speculated that ?-arrestin-1 promoted renal fibrosis by activating Wnt1/?-catenin pathway.Part 2 The role of ?-arrestin-1 in fibroblast cells and renal epithelial cells1.?-Arrestin-1 promots fibroblast activation:We observed the upregulation of ?-arrestin-1 in NRK-49F cells with TGF-?1 stimulation.WB results showed that gene silencing of ?-arrestin-1 significantly abolished NRK-49F cell activation under TGF-?1 stimulation,with the down-regulation of ?-SMA,collagen I,and fibronectin.2.?-Arrestin-1 activated Wnt/?-catenin pathway:To reveal the mechanism by which ?-arrestin-1 impacted the activation of fibroblast,we detected the expression of Wnt/?-catenin pathway in NRK-49F cells.Interestingly,TGF-?1 increased the expression of Wntl and the activation of ?-catenin in NRK-49F cells,which suppressed by gene silencing of ?-arrestin-1.After adding Wnt/(3-catenin agonist,the expression of active-p-catenin restored,and the expression of fibrin a-SMA and fibronectin increased.To further certify the mechanism,Wnt/?-catenin pathway inhibitor Dkkl was used to stimulate fibroblasts.The effect of Wntl gene silencing and Dkkl stimulation both down-regulated the expression of active-?-catenin,a-SMA and fibronectin.Therefor,we demonstrated that p-arrestin-1 facilitated renal fibroblast activation by accelerating Wntl/?-catenin signaling pathway.3.Gene silencing of ?-arrestin-1 reduced EMT in HK-2 cells:We found that the relative protein level of ?-arrestin-1 was dramatically increased in TGF-?1-stimulated HK-2 cells.Gene silencing of ?-arrestin-1 reduced the expression of vimentin,slug and ?-SMA,and recovered the expression of E-cadherin.Further study showed that ?-arrestin-1 deficiency suppressed the EMT by negatively regulating Wntl and active-p-catenin expression.Conclusion and innovation:1.Our study for the first time demonstrated that ?-arrestin-1 plays a significant role in the development of renal fibrosis.?-Arrestin-1 was up-regulated in the mice with UUO nephropathy,and the deficiency of ?-arrestin-1 ameliorated renal damage by inhibiting the activation of fibroblasts,reducing EMT and inflammatory responses.2.Mechanically,both in vitro and in vivo experiments revealed that ?-arrestin-1 accelerated Wnt/?-catenin pathway,by up-regulated the expression of Wntl and activated ?-catenin,thereby promoting fibroblast activation and epithelial cell mesenchymal transition.3.Interfering ?-arrestin-1 could significantly ameliorate renal fibrosis by inhibit Wnt/?-catenin pathway,which provides a new molecular biological proof for renal fibrosis treatment.