Effect Of SIS3 On Renal Interstitial Fibrosis Induced By Unilateral Ureteral Obstruction In Mice

Objective: Renal interstitial fibrosis is the main cause of end-stage renal failure(ESRD).There is no effective drug for renal interstitial fibrosis for the prevention and treatment of CKD yet now.TGF-?/Smads signaling is the master pathway regulating renal interstitial fibrosis pathogenesis in which Smad3 directly regulates the expression of various fibrosis genes and acts as integrator of various pro-fibrosis signals.These indicate that TGF-?/Smads signaling is an important therapeutic target in renal interstitial treatment.In this study,based on the establishment of UUO mouse model,we investigated the role of SIS3,a specific inhibitor of Smad3,in renal interstitial fibrosis by investigating its possible mechanism of action,and provided clinical anti-renal interstitial fibrosis treatment theoretical and experimental basis.Methods: Forty BALB/c male mice(6 weeks old,20 ± 2 g)randomly divided into 4 groups(n=10 for each group)including Sham,UUO,SIS3 low dose(0.2 mg/kg)and SIS3 high-dose group(2 mg/kg).The UUO mouse model was established by dissociating and ligating the left ureter,and the sham group only dissociating left ureter without ligating.One day after UUO surgery,mice of each group were intraperitoneally injected with different dosage of SIS3(in 5 % dimethyl sulfoxide)for 1 week.Sham and UUO group received vehicle injection(5 % dimethyl sulfoxide)instead.On the 8th post-operation day,blood samples were harvested for the detection of IL-1? byenzyme-linked immunosorbent assay(ELISA);UUO or sham kidney tissues were stained by hematoxylin-eosin staining(HE staining),Masson's trichrome staining(MASSON staining)and periodic acid-Schiff staining(PAS staining)to observe the renal gross structure injury and fibrosis;Immunohistochemistry(IHC)was used to detect the expression of fibronectin(FN)and ?-smooth muscle actin(?-SMA)in kidney tissues;Western-blot was used to detect the expression of TGF-?1,?-SMA,Vimentin,T-Smad2,T-Smad3,p-Smad2,p-Smad3,cleaved-caspase 3,tumor necrosis factor-?(TNF-?)and cyclooxygenase-2(COX-2)in kidney tissues;Real-time quantitative PCR(RT-PCR)was used to detect the expression of ?-SMA,collagen I and collagen III in kidney tissues;and apoptotic cells were counted by TUNEL staining.Results: 1.Kidney pathology results: Compared with the sham group,the UUO group had tubular dilatation,glomerular atrophy and epithelial cell shedding(P<0.05);Compared with the UUO group,SIS3 treatment(low dose and high dose group)in dosage-dependently relieved the renal gross structure injury and tubular necrosis in UUO kidney(P<0.05);2.Results for Masson staining,IHC and RT-PCR showed: Compared with the UUO group,SIS3 treatment significantly decreased ECM deposition,fibronectin staining intensity and the m RNA level of Collagen I and Collagen III in UUO kidney(P<0.05);SIS3treatment also remarkably decreased number of ?-SMA positive cells and decreased expression level of ?-SMA and Vimentin in UUO kidney(P<0.05);3.The TGF-?/Smads signaling activity analysis showed: Compared with the UUOgroup,SIS3 treatment inhibited the p-Smad3(P<0.05)but not p-Smad2(P>0.05)and decreased the protein level of TGF-?1 in a dose-dependent manner(P<0.05);4.Inflammatory factor analysis results showed: Compared with the UUO group,SIS3 treatment also ameliorated the increased pro-inflammatory TNF-? and COX2 in UUO kidney(P<0.05)and circulated IL-1?(P<0.001)in UUO mouse;5.The apoptotic index showed that the number of TUNEL positive cells in SIS3-treated group was significantly decreased(P<0.05)and the expression of cleaved-caspase 3 was significantly decreased compared with the UUO group(P<0.05).Conclusion: 1.SIS3 treatment suppressed the activation of myofibroblast and thus reduce renal interstitial fibrosis.2.SIS3 can effectively improve renal inflammation and tubular epithelial cell apoptosis caused by UUO by inhibiting the activity of TGF-? / Smad3 signal pathway.