The Functional Role And Mechanism Of WISP-1 In Renal Fibrosis

objective:Renal fibrosis is a kind of renal pathological damage.characterized by excessive accumulation and deposition of extracellular matrix(ECM).It is the common pathway of various chronic kidney diseases(CKDs)to end-stage kidney disease(ESKD).The pathogenesis of renal fibrosis is complex and there are no effective prevention measures.Our previous study found that the expression level of WNT1-inducible signaling pathway protein 1(WISP-1)was elevated in renal tissue and serum of CKD patients with renal fibrosis and in the in vivo and in vitro renal fibrosis models.Moreover,the expression of serum WISP-1 increased with the degree of renal fibrosis in renal tissue of CKD patients,suggesting that WISP-1 may be a pro-fibrotic factor and participate in the development of renal fibrosis.This study aimed to clarify the functional role of WISP-1 in renal fibrosis both in vitro and in vivo,and further explore its possible molecular mechanisms to provide new ideas for the treatment of renal fibrosis.Methods:1.The expression of WISP-1 in rat renal tubular epithelial cells(NRK52E)was up-regulated or interfered with over-expression WISP-1 plasmid and WISP-1 siRNA.Quantitative real-time polymerase chain reaction(qRT-PCR)and Western blot analysis were used to detect the expression of WISP-1 and renal fibrosis markers include collagen I(Col I)and fibronectin(FN)after 24 hours of stimulation with transforming growth factor-?1(TGF-?1).2.An in vivo model of renal fibrosis was constructed using a unilateral ureteral obstruction(UUO)mouse model,and monoclonal and or polyclonal antibodies against WISP-1 was intraperitoneally injected into the animal to inhibit the expression of WISP-1 in mouse kidney.The groups was divided ioto normal group,model group,UUO+IgG group and UUO+WISP-1 antibody with differently dosage groups(UUO+Ab1,UUO+Ab2,UUO+Ab3).The renal pathological and renal fibrosis changes were observed by routine pathological staining(HE,PAS,Masson trichrome staining).Immunohistochemistry(IHC)staining,qRT-PCR and Western blot were used to detect the WISP-1 and fibrosis indicators Col I,FN and ?-smooth muscle actin(?-SMA)expression.3.GFP-LC3 plasmid was transfected into NRK52E cells using lipofectamine 3000.The expression of GFP-LC3 spots representing autophagosome formation in cells were observed by laser confocal fluorescence microscopy.The changes of autophagy-related markers including anti-microtubule-associated protein 1 light chain 3(LC3),Beclin 1 and SQSTM1/p62 in renal tissues of each group were detected by Western blot.Results:1.The expression of WISP-1 was significantly higher in NRK52E transfected with WISP-1 plasmid than that in empty plasmid group(P=7.00×10-3),while WISP-1 of NRK52E transiently transfected with WISP-1 siRNA was less than that in normal group(P=7.25 × 10-4).2.After TGF-?1 tretment for 24 hours,Col I,FN and TGF-?1 in NRK52E were significantly increased.Compared with empty plasmid group,overexpression of WISP-1 further increased TGF-?1-induced expression of Col I and FN in NRK52E(P=3.26×10-4(Col I),9.50×10-5(FN)).Conversely,interference with WISP-1 expression in NRK52E cells further reduced TGF-?1-induced fibrosis markers Col I and FN production(P=2.23×10-4(Col I),1.70×10-5(FN)).3.After 7 days of intraperitoneal injection of anti-WISP-1 antibody in UUO mice,the mRNA(P=1.20×10-2(UUO+Ab1 group),3.00×10-5(UUO+Ab2 group),9.30×10-4(UUO+Ab3 group))and pretien levels(P=2.42×10-3(UUO+Abl group),3.46×10-2(UUO+Ab2 group),1.50×10-2(UUO+Ab3 group))of WISP-1 of UUO+WISP-1 antibody groups were significantly lower than UUO group protein;4.From the pathological manifestations,compared with UUO group and UUO+IgG group,renal interstitial fibrosis and ECM deposition in UUO+WISP-1 antibody groups were significantly reduced.5.The mRNA and protein levels of Col I,FN and ?-SMA in the kidney tissue of UUO+WISP-1 antibody groups were significantly lower than those of the UUO group(mRNA:UUO+Ab1 group:P=3.78×104(Col I),1.35×10-2(FN),1.98×10-2(?-SMA);UUO+Ab2 group:P=1.20×10-5(Col I),1.26×10-4(FN),1.06×10-3(?-SMA);UUO+Ab3 group:P=2.00×10-6(Col I),1.31×10-3(FN),4.89×10-3(?-SMA);protein:UUO+Ab1 group:P=9.15×10-4(Col I),1.67×10-2(FN),1.17×10-2(?-SMA);UUO+Ab2 group:P=2.23×10-2(Col I),5.80×10-3(FN),1.34×10-3(?-SMA);UUO+Ab3 group:P=2.76×10-2(Col I),1.55×10-1(FN),2.46×10-2(?-SMA)).6.The GFP-LC3 spots in the WISP-1 overexpression group were more than those in the TGF-?1 stimulation group in NRK52E cells.Conversely,interference with WISP-1 siRNA reduced the number of GFP-LC3 spots in TGF-?1-treated NRK52E cells.7.The expression of autophagy-related positive markers LC3 and Beclin 1 in UUO+WISP-1 antibody groups were lower than that in UUO group(UUO+Ab1 group:P=3.00×10-5(LC3),5.22×10-3(Beclin 1);UUO+Ab2 group:P=6.30×10-5(LC3),1.02×10-3(Beclin 1);UUO+Ab3 group:P=4.55×10-4(LC3),2.83×10-3(Beclin 1)),while the expression of negative marker SQSTM1/p62 were increased(UUO+Ab1 group:P=7.05×10-2,UUO+Ab2 group:P=1.25×10-2,UUO+Ab3 group:P=4.81×10-2).Conclusion:1.Overexpression of WISP-1 significantly aggravates renal fibrosis in rat renal tubular cells induced by TGF-?1,while reducing WISP-1 levels attenuates TGF-?1-induced renal fibrosis in rat renal tubular cells.2.Anti-WISP-1 treatment significantly reduces renal pathological damage and renal fibrosis in UUO mice.3.WISP-1 may mediate renal fibrosis by inducing autophagy in TGF-?1-treated rat renal tubular epithelial cells and UUO mouse models.4.WISP-1 as a pro-fibrotic factor may be a potential target for combating of renal fibrosis.