| Background and Objective Congenital hydronephrosis(CHN)is one of the common congenital malformations in pediatric urology.It is mainly caused by pelvic-ureteral junction obstruction(PUJO),accounting for about 90%.The incidence of CHN is common,accounting for about 1% of newborns,boys more than girls,left more than right,bilateral 10%-40%.With the aggravation of hydronephrosis,dilatation of renal pelvis,calyces and tubules,thinning of renal parenchyma,and severe impairment of renal urine concentration and dilution ability in children,failure to remove obstruction in time can lead to urinary tract infection,renal parenchymal atrophy and even renal failure.CHN is an important cause of end-stage renal failure in infants and children.It has been found that the down-regulation of aquaporin-1(AQP-1)expression in kidney caused by ureteral obstruction is related to the decline of urine concentration function.AQP-1 is a channel protein that efficiently transports water.It is mainly distributed in the apical plasma membrane of epithelial cells of the proximal convoluted tubules and the descending branche of the medullary loop.It plays an important role in the Isoabsorptive process of the proximal convoluted tubule and urine concentration,especially in the descending branch of the medullary loop and the countercurrent doubling mechanism.It has been reported in the literature that the function of renal urine concentration has not been fully restored even after early pyeloureteroplasty,which is related to the level of AQP-1 expression in the kidney after relieving obstruction.In recent years,a large number of reports have reported the protective effect oferythropoietin(EPO)on kidney injury,which has become a hot issue in research.Stoyanoff et al found that EPO exerts anti-apoptotic effects and renal protection by up-regulating EPO-R expression and regulating mitochondrial apoptosis pathways..Sang et al.found that rh EPO could reduce serum levels of TNF-?,IL-6,iNOS and KIM-1 in sepsis rats and then regulate inflammatory response.It had a protective effect on sepsis-induced acute kidney injury.It was also found that rHuEPO had a dose-dependent protective effect on the kidney of sepsis rats.Wen et al.used psoas major muscle embedding method to establish a model of left partial ureteral obstruction in young rats.EPO intervention was given after operation.It was found that EPO could promote the expression of AQP-2 in the kidney of partial ureteral obstruction from the level of mRNA and protein.Wang et al.found that EPO can promote the expression of AQP-2 m RNA,protein and function in kidney of young rats with bilateral ureter obstruction-release(BUO-R).However,whether EPO can promote the recovery of AQP-1 expression in unilateral ureter obstruction-relief,and whether it can promote the recovery of AQP-1 expression and the recovery of renal function and water balance regulation in kidney of BUO-R young rats have not been reported in the literature.The purpose of this study was to establish UUO-R model and BUO-R model respectively,and to investigate the effects of EPO on the expression of AQP-1 in kidney of UUO-R young mice and the effects of EPO on the expression of AQP-1,renal function and water and water balance regulation in kidney of BUO-R young mice.Part One Effect of EPO on AQP-1 expression in kidney of UUO-R young ratsMethods 24 young male SD rats aged 6-7 weeks were randomly divided into unilateral ureteral obstruction(UUO)group,unilateral ureteral obstruction-relief(UUO-R)group,UUO-R+EPO group and sham group(Sham group),with n=6 in each group.The UUO model was built through bilateral ureteral ligation.UUO group was executed 24 hours after obstruction.UUO-R group and UUO-R+EPO group wererelieved after obstruction of 24 h.In the UUO-R+EPO group,EPO was intraperitoneally injected(500 IU/Kg)at 1h,1d,3d,and 5d after the obstruction was removed,and the UUO-R group was given the same dose of 9 g/L saline at the same time point..In Sham group,the ureter was separated without ligation.The rats were sacrificed one week after relieving the obstruction.The left kidney specimens were taken.The expression of AQP-1 protein in the kidney tissues of each group was detected by immunohistochemistry and Western blot.ResultsImmunohistochemistry results showed that the AQP-1 positive cells were brown-yellow,mainly concentrated in proximal convoluted tubules and located in the apical plasma membrane of epithelial cells.The staining intensity of Sham group was the strongest,followed by UUO-R + EPO group,UUO-R group again,UUO group was the weakest.Histologically,the wall of proximal convoluted tubules in UUO group,UUO-R group and UUUO-R+EPO group became thinner and the lumen enlarged,Western blot results shows that AQP-1 expression was highest in Sham group,next in UUO-R + EPO group,third in UUO-R group and lowest in UUO group(P<0.05).Part Two Effect of EPO on AQP-1 expression and renal function in kidney of BUO-R young ratsMethods48 young male SD rats aged 6-7 weekswere randomly divided into bilateral ureteral complete obstruction group(BUO,n=6),BUO-R group(n=12),BUO-R+EPO group(n=12)and Sham group(n=18).The experimental group established the bilateral ureteral obstruction model by ureteral ligation.BUO group was killed after 24 h,and BUO-R and BUO-R+EPO groups were relieved after obstruction of 24 h.EPO(500 U/ kg)was given to BUO-R+EPO rats at 1h after release of BUO,and then repeated 1d,3d and 5d thereafter and the same volume of 9 g/ L saline was simultaneously given to BUO-R rats.The Sham group was preparedin parallel by laparotomy and free dissection of bilateral ureters but not ligated,Both side kidneys and blood samples were harvested at 3d and 7d(24 h,3d,7d for Sham group)after release of BUO.The urine samples were collected by using metabolic cage before death.The plasma osmotic pressure,creatinine and urea nitrogen in the plasma of young rats were detected.The expression of AQP-1 protein in all groups of kidney tissues was detected by immunohistochemistry and Western blot.Results At day 3 after release of BUO,24 h water intake and urine volume of BUO-R+EPO group was higher than that of Sham group,but lower than that of BUO group(P < 0.05),Urine osmotic pressure was highest in Sham group,BUO-R+EPO group was the second,BUO-R group was the lowest(P<0.05),plasma osmotic pressure,Cr and BUN of BUO-R+EPO group was higher than that of Sham group,but lower than that of BUO group(P<0.05),but all of them lower than group BUO(P<0.05).At day 7 after release of BUO,no obvious change in Sham group,and the indexes of BUO-R group and BUO-R+EPO group gradually recovered,but they still did not reach the normal level(P<0.05).The difference between group BUO-R and BUO-R+EPO group was statistically significant(P < 0.05).The immunohistochemical results showed that the expression of AQP-1 in collecting duct in BUO group was significantly down-regulated compared with that in Sham group,whereas,it was slightly weaker in BUO-R group and BUO-R+EPO group than Sham group(P<0.05).Compared with 3 days after release of BUO,the staining intensity of BUO-R+EPO group and BUO-R group was enhanced,still lower than that of the Sham group.These results were further confirmed by adopting Western blot,BUO group was also the lowest of the four groups,BUO-R+EPO group was higher than that of BUO group,but lower than that of Sham group(P<0.05).Conclusions1.EPO could promote the recovery of AQP-1 protein expression in kidney of UUO-R young rats.2.EPO could promote the recovery of AQP-1 protein expression,renal functionand water balance regulation in the kidney of BUO-R young rats. |
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