Study On The Relationship Between Treg And Obstructive Nephropathy By A Modified RUUO Model

Objective:To,develop a improved mouse model of relief of unilateral ureteral obstruction(RUUO);To explore the effect of losartan on fibrosis of the kidney after release of obstruction and the changes of regulatory T cells in this process.Methods:C57BL/6 mice were randomly divided into RUUO + losartan group(n=6),RUUO group(n=6)and sham group(n=6).7 days after left ureteral obstruction(UUO),the obstruction was released by Replantation + Catheter way in RUUO+Losartan group and RUUO group.Losartan was given to the mice in RULUO+Losartan group after replantation.5 days post-UUO,4 days and 7 days post-RUUO,the percentage of regulatory T cells(Treg)in peripheral blood was detected by flow cytometry;7 days post-RUUO,kidneys were harvested for HE and Masson staining,as well as immunohistochemistry of a-SMA expression,expression of Foxp3 protein in kidney was detected by Western blot.Results:There was no hydronephrosis in kidneys of the RUUO +Losartan group and RUUO group.These kidneys with pale color were smaller than those of the contralateral side,and there was no obvious hydronephrosis in the renal pelvis.HE showed renal tubular disorder,brush border loss,lumen expansion,tube formation in RUUO group;sham group showed clear renal tubular structure and intact epithelium;compared with RUUO group,pathology changes in RUUO + losartan group were mild.Tubular damage score in RUUO group(3.9±0.74)was significantly higher than that in RUUO + losartan group(2.4±0.48)(p<0.05),and the score of renal tubular injury in sham group(0.5±0.45)was lower than the other two group(p<0.05).As for Masson,there was no obvious blue staining areas in sham group while RUUO group showed obvious blue areas,mainly distributed in renal interstitium,and RUUO +losartan group had reduced blue areas compared with RUUO group.The proportion of collagen area in RUUO + losartan group,RUUO group and sham group were 5.33±0.64%、9.60±1.49%and 1.17±0.41%,respectively.There was statistical difference between every two groups(p<0.05).Immunohistochemical staining of renal a-SMA showed that the expression of a-SMA in sham group was low and mainly expressed in the small vessel wall as well as in the periphery of glomerulus and renal tubule.The expression of a-SMA was significantly increased in RUUO group,mainly in the renal tubular interstitium.The expression of RUUO + losartan group was significantly lower than that of the RUUO group.The average optical density of RUUO + losartan group for a-SMA immunohistochemistry staining was 0.0138±0.0011,lower than operation group(0.0203±0.0012)(P<0.05).Average optical density of a-SMA in sham group was 0.0038±0.0003 and was significant lower than the other two groups(P<0.05).At 5 days post-UUO,4 days and 7 days post-RUUO,the percentage of Foxp3+Treg/CD4+T cell in peripheral blood showed no significant difference between RUUO + losartan group,RUUO group and sham group.Western blot showed the expression of Foxp3 in kidney in RUUO group was significantly reduced.The integral optical density of Foxp3 to GAPDH ratio in RUUO group is 0.0664±0.0080,lower than RUUO + losartan group as 0.5987±0.2543(P<0.05)and sham group as 0.9910±0.1484(P<0.05),respectively;expression of Foxp3 protein in RUUO + losartan group was lower than that in sham group(P<0.05).Conclusion:Using the method of Replantation + catheter,a reliable mice model for relief of unilateral ureteral obstruction can be got;regulatory T cell marker,Foxp3,was reduced in the kidney of this mice model;losartan can improve renal fibrosis after relief of unilateral ureteral obstruction and improve the expression of Foxp3 protein in kidney.