| Background:With the increasing incidence of chronic kidney disease,it has been a common concern how to reduce the morbidity and even to delay the progression of chronic kidney disease.As a common pathogenesis of end stage renal disease,renal interstitial fibrosis(RIF)is currently considered to be an effective entry point for solving this problem.Unilateral ureteral obstruction(UUO)model is one of the most commonly used models to study RIF.It was reported that the relief of unilateral ureteral obstruction(R-UUO)model is an ideal model for observing kidney injury and repairment after injury,and studying the therapeutic effect of medicine.But the experimental animals used in these studies are mainly rodents.The physiological structures are greatly different between rodents and mammals.Therefore,our team has proposed and successfully constructed a mammalian R-UUO model and found that UUO-induced renal damage,including pathological and molecular signal changes,can be restored after UUO for 5 days or 7 days.Nevertheless,the pathological variation between these two groups(UUO for 5 days and UUO for 7 days)presented no significant differences.In addition,it was difficult to recover renal function when recanalization was performed after 10 days of obstruction.These results indicated that UUO-induced renal injury could be self-repaired in a porcine model.However,the accurate procedure remains uncertain.Some important issues determined from the previous study were noted and improved.First,because the renal pathological changes were not obviously presented significant difference between the UUO 5-day group and the UUO 7-day group,we decided to analyze the animals at 3 days,7 days,and 10 days after UUO to replace 5 days,7 days,and 10 days.Second,we used a renal biopsy needle to instead of the traditional surgical resection methods,which presented severe renal injury or aggravated the distribution of RIF to isolate and analyze the kidney tissue.In this study,we observed the natural recovery process of the kidneys after obstruction for different days to eliminate the time node of reversible repairment of RIF caused by obstruction.In addition,we studied the anti-fibrosis effect of the compound F-86 with the R-UUO model and discussed the possible mechanism of the anti-fibrosis effect of F-86.Objectives:This study,on the one hand,aims to explicit the time point of reversible repairment of RIF caused by UUO in miniature pig,and to provide a theoretical basis for clinical prognosis of UUO-induced RIF;on the other hand,it is to investigate the anti-fibrosis effect and possible mechanism of compound F-86,which may provide a new therapeutic strategy for chronic kidney disease and anti-fibrosis treatment.Methods:This study was divided into two parts.In the first part,we established the UUO model to observe the spontaneous recovery of renal function and RIF before and after recanalization.Miniature pigs weighing 8-10 kg were randomly divided into the following three groups: recanalization after 3 days' obstruction group,recanalization after 7 days' obstruction group,recanalization after 10 days' obstruction group(5 miniature pigs per group).After 1 week's adaptive feeding,the miniature pig underwent UUO and recanalization at different time point.Contralateral vein,ipsilateral vein,and precaval vein blood samples were collected and the obstructed renal tissue samples were obtained by biopsy under direct vision on the 3 day,7 day,14 day and 28 day after recanalization.In the second part,the appropriate R-UUO time point is determined according to the experimental results of the first part.Then 25 male miniature pigs weighing about 8-10 kg were randomly divided into the following 5 groups: sham operation with recanalization after 7 days' obstruction group,sham operation with recanalization after 7 days' obstruction +F-86 group,recanalization after 7 days' obstruction group,recanalization after 7 days' obstruction + pirfenidone group,recanalization after 7 days' obstruction +F-86 group(5 in each group).We established the UUO 7 days recanalization model,and gave drugs by gavage treatment on the first day after recanalization.The F-86 treatment groups were given F-86 150mg/kg by gavage once a day.The pirfenidone treatment groups were administrated pirfenidone 500mg/kg once a day.Other groups received equivalent dose of solvent orally once a day.All the treatments last for 14 days.The miniature pigs were killed on the 28 th day.Contralateral vein,ipsilateral vein,and precaval vein blood samples were collected and the obstructed renal tissue samples were obtained by biopsy under direct vision on the 0 day of obstruction,0 day of recanalization,and 3rd day?7thday?14th day and 28 th day after recanalization.Blood samples were collected to measure the level of serum creatinine and blood urea nitrogen using the ELISA method in accordance with the respective test kit to evaluate renal function.Periodic acid–Schiff(PAS)and Masson staining were used to detect the histological pathology of tissues.Immunohistochemistry and Western blot methods were used to detect the expression of molecular biological indicators,such as E-cadherin,ZO-1,?-SMA,vimentin,collagen-?,BMP-7,Smad7 and TGF-?1 / Smads signaling pathway.Results:(1)The serum creatinine(Scr)and blood urea nitrogen(BUN)levels in each UUO group during R-UUO showed no significant differences between differenet sample sites including(precaval vein,ipsilateral vein,and contralateral vein;P > 0.05).Howereve,renal injury was presented different changes after R-UUO.Most RIF,as determined by pathological detection,dynamic epithelial-mesenchymal transition(EMT),interstitial collagen deposition,and transforming growth factor beta 1(TGF-?1)associated signals,was restored in the UUO 3-day group,partially moderated in the UUO 7-day group and not alleviated at all in the UUO 10-day group.(2)Compared with sham operation group,serum creatinine and blood urea nitrogen levels in sham operation +F-86 group showed no obvious change;compared with the obstruction recanalization group,PAS staining and Masson staining in the PFD and F-86 treatment group showed that PFD and F-86 can promote the repair of damaged renal tubules and significantly reduce collagen deposition,and renal tubular regeneration was more obvious in F-86 treatment group.(3)Immunohistochemistry showed that: compared with sham operation group,E-cadherin,ZO-1,?-SMA,vimentin,Collagen-?,TGF-?1 and BMP-7 appeared different rhythm and different change after UUO;And compared with obstruction recanalization group,with prolonged reperfusion time after relief of obstruction,F-86 and PFD treated group can promote the recovery of this process.(4)Western blot showed that up to 28 days after recanalization,the expression of E-cadherin,ZO-1,?-SMA,vimentin,Collagen-?,TGF-?1,BMP-7,Smad7 and down stream proteins of TGF-?/ Smads in renal tissues of each group show no statistical differences.Conclusions:(1)The reversible renal injury time node of miniature pig R-UUO model was between 7 and 10 days of obstruction.(2)F-86 reduced the EMT and extracellular matrix deposition caused by UUO,promoted the recovery of RIF in miniature pig R-UUOmodel.The anti-fibrosis effect of F-86 might be achieved by promoting the expression of BMP-7 and Smad7,and inhibiting the TGF-?1/Smads signal pathways. |
PDF Full Text Request