The Efficacy And Mechanism Of Fluorofenidone On Renal Interstitial Fibrosis In Rats With Unilateral Ureteral Obstruction

Objectives:The present study aims to investigate the antifibrotic effects of different doses of Fluorofenidone (AKF-PD) on tubulointerstitial fibrosis in rats with obstructive nephropathy caused by unilateral ureteral obstruction (UUO) and to explore the working mechanisms of AKF-PD on tubulointerstitial fibrosis. The antifibrotic effects of AKF-PD were compared with that of Pirfenidone (PFD) and enalapril in this rat UUO model.Methods:180 male Sprague-Dawley(SD) rats were randomly subdivided into UUO model group by ligation the left ureter (UUO group, n=140),Sham-operated rats (n=20) without ureteral ligation and normal rats receiving AKF-PD (500 mg/kg,n=20). After 3 day following the operation, the UUO rats were randomly divided into six groups, as follows:UUO with drug vehicle treatment, UUO treated with AKF-PD (62.5 mg/kg,125 mg/kg,500 mg/kg,1000 mg/kg), UUO treated with PFD (500 mg/kg), and UUO treated with enalapril (10 mg/kg).The number of rats were 20 in each group. All drugs and vehicle treatment groups were administered by oral gavage once daily after 3 day of the operation. All rats were sacrificed on day 14 after surgery and their obstructed kidneys were collected for HE and Masson staining to observe the pathologic change and collagen deposition in renal tissue; immunohistochemically staining to detect the protein expression of typeⅠcollagen (collagenⅠ), typeⅢcollagen (collagenⅢ), connective tissue growth factor (CTGF), a-smooth muscle actin (a-SMA); Real-time PCR to detect the mRNA expression of collagenⅠ, collagenⅢ, transforming growth factor-β1 (TGF-β1), CTGF, platelet-derived growth factor (PDGF)-B, a-SMA, matrix metallopeptidase-9 (MMP-9), tissue inhibitor of metalloproteinase-1 (TIMP-1); Western blot to detect the protein expression of PDGF-BB, MMP-9 and TIMP-1.Results:The tubulointerstitial injury scores, the interstitial collagen deposition score, the protein and mRNA expression of collagen I and collagenⅢin the obstructed kidneys of UUO group significantly increased compared with sham-operated group (p<0.05). There was not any significant between sham-operated rats and normal rats receiving AKF-PD (p>0.05); The tubulointerstitial injury scores exhibited that AKF-PD(125mg/kg>500mg/kg>1000mg/kg), enalapril and PFD treatment significantly attenuated the progression of tubulointerstitial injury compared with UUO group (p<0.05), the tubulointerstitial injury scores in the AKF-PD (500 mg/kg) group was significantly lower than that in PFD(500 mg/kg) group (p<0.05); The interstitial collagen deposition score exhibited that AKF-PD(62.5mg/kg、125mg/kg、500mg/kg>1000mg/kg), enalapril and PFD treatment significantly reduced the collagen deposition compared with UUO group (p<0.05); Immunohistochemical staining for collagenⅠand collagenⅢshowed that AKF-PD(62.5mg/kg、125mg/kg、500mg/kg、1000mg/kg), enalapril and PFD treatment resulted in a significant reduction of interstitial collagenⅠand collagenⅢexpression compared with UUO group (p<0.05), AKF-PD(500 mg/kg) exerted a stronger inhibition than PFD and enalapril on interstitial collagenⅠand collagenⅢprotein expression (p<0.05); collagenⅠandⅢmRNA expression were detected by Real-time PCR showed that AKF-PD(62.5mg/kg、125mg/kg、500mg/kg、1000mg/kg), enalapril and PFD treatment had significant inhibition effects on collagenⅠandⅢmRNA expression in the renal tissue of UUO rats (p<0.05), AKF-PD(500 mg/kg) exerted a stronger inhibition than PFD and enalapril on mRNA expression of collagenⅢ(p<0.05).The mRNA and protein expression of CTGF, PDGF, a-SMA, TIMP-1 and the mRNA expression of TGF-β1 in UUO rats were prominently higher than in sham-operated rats (p<0.05).The mRNA and protein expression of MMP-9 were significantly lower in the UUO group than in sham-operated rats (p<0.05); AKF-PD (62.5mg/kg、125mg/kg、500mg/kg>1000mg/kg), enalapril and PFD treatment resulted in a significant reduction of TGF-β1 mRNA expression compared with UUO group (p<0.05), TGF-β1 mRNA levels in the AKF-PD (500mg/kg、1000mg/kg) groups were significantly lower than that in the enalapril group (p<0.05); AKF-PD(62.5mg/kg、125mg/kg、500mg/kg、1000mg/kg), enalapril and PFD treatment markedly inhibited CTGF mRNA levels compared with UUO group (p<0.05); Immunohistochemical staining revealed that CTGF protein expressed in UUO kidney were significantly reduced by AKF-PD (125mg/kg、500mg/kg>1000mg/kg), enalapril and PFD treatment (p<0.05); AKF-PD(125mg/kg、500mg/kg、1000mg/kg), PFD, and enalapril all significantly attenuated both PDGF-B mRNA and PDGF-BB protein e xpression levels otherwise increased in UUO kidney (p<0.05), and all treatment groups had no significant difference (p>0.05); Compared with the UUO group, the AKF-PD(62.5mg/kg、125mg/kg、500mg/kg、1000mg/kg), PFD, and enalapril groups exerted a significant reduction of a-SMA mRNA and protein levels in renal tissue(p<0.05); None of AKF-PD(62.5mg、125mg/kg>500mg/kg、1000mg/kg),PFD, or enalapril exerted significant regulation effects on MMP-9 mRNA and protein expression as compared to that in UUO kidneys (p>0.05),and the AKF-PD, PFD, and enalapril treatments resulted in a depressed TIMP-1 mRNA and protein expression(p<0.05).Conclusions:AKF-PD attenuates renal interstitial fibrosis in the rat model of obstructive nephropathy and the effective dose range from 62.5mg/kg to1000mg/kg. AKF-PD also has the dose-dependent effect on the dose of 62.5mg/kg、125mg/kg、500mg/kg. There has no significant difference between the group of AKF-PD 500mg/kg and AKF-PD 1000mg/kg; The mechanism of antifibrotic effects of AKF-PD may due to it’s ability of reducing the overexpression of TGF-β1, CTGF and PDGF-B, inhibiting fibroblast dividing or tubular cell transdifferentiation, and preventing TIMP-1 production.