促红细胞生成素对双侧输尿管梗阻解除后幼鼠肾脏水通道蛋白-1表达及功能的影响

Effect of erythropoietin on the expression and function of renal aquaporin-1 after release of bilateral ureter obstruction in young rats

目的探讨促红细胞生成素(EPO)对双侧输尿管梗阻-梗阻解除(BUO-R)后幼鼠肾脏水通道蛋白-1(AQP-1)表达及功能的影响。方法幼年雄性SD大鼠48只,随机号码表法分为双侧输尿管完全梗阻(BUO)组(n=6)、双侧输尿管梗阻-梗阻解除(BUO-R)组(n=12)、BUO-R+EPO组(n=12)及假手术组(Sham组)(n=18)。实验组采用输尿管结扎法建立双侧输尿管完全梗阻模型,BUO组梗阻24 h后处死;BUO-R组和BUO-R+EPO组梗阻24 h后解除梗阻,BUO-R+EPO组于解除梗阻后1 h、1 d、3 d、5 d给予EPO腹腔注射(500 IU/kg),BUO-R组相同时间点给予同等剂量的9 g/L盐水,分别在解除梗阻后3 d、7 d处死幼鼠;Sham组仅分离输尿管,不予结扎,分别于梗阻术后24 h、解除梗阻后3 d、7 d处死幼鼠,各组幼鼠处死前取双肾标本并采集血液标本。解除梗阻后将幼鼠放置于代谢笼中,记录24 h饮水量和尿液量,并收集尿液用于检测尿液渗透压的变化。检测各组幼鼠血浆渗透压、血肌酐(Cr)、尿素氮(BUN)水平,采用免疫组织化学、Western blot检测各组肾脏组织中AQP-1蛋白表达水平。结果BUO-R后3 d,24 h饮水量和尿液量BUO-R组最多,BUO-R+EPO组次之,Sham组最少(P<0.05);尿液渗透压Sham组最高,BUO-R+EPO组次之,BUO-R组最低(P<0.05);血浆渗透压、Cr、BUN BUO-R组最高,BUO-R+EPO组次之,Sham组最低,但均低于BUO组(P<0.05)。梗阻解除后7 d,Sham组无明显变化,BUO-R组和BUO-R+EPO组以上各项指标逐渐恢复,但仍未达到正常水平(P<0.05),BUO-R组与BUO-R+EPO组比较差异有统计学意义(P<0.05)。免疫组织化学结果显示Sham组染色强度最强,BUO-R+EPO组次之,BUO-R组再次,BUO组最弱;解除梗阻后7 d与解除梗阻后3 d相比,BUO-R+EPO组和BUO-R组染色强度均增强,仍低于Sham组。经Western blot进一步验证,各组幼鼠肾脏AQP-1表达量Sham组最高,BUO-R+EPO组次之,BUO-R组再次,BUO组最低(P<0.05);解除梗阻后7 d与解除梗阻后3 d相比,BUO-R+EPO组、BUO-R组表达量均增高,但仍低于Sham组(P<0.05)。结论EPO能够促进BUO-R幼鼠肾脏AQP-1蛋白表达及功能恢复。

Objective To investigate the effect of erythropoietin(EPO)on the expression and function of aquaporin-1(AQP-1)in the kidney of young male SD rats after release of bilateral ureter obstruction(BUO-R). Methods Forty-eight young SD rats were randomly divided into bilateral ureteral complete obstruction(BUO) group (n=6), BUO-R group (n=12), BUO-R+ EPO group (n=12) and Sham group (n=18). The BUO model was built through bilateral ureteral ligation.BUO group were killed after 24 h, and BUO-R group and BUO-R+ EPO group were relieved after obstruction of 24 h. EPO(500 U/kg)was given to BUO-R+ EPO rats at 1 h after release of BUO, and then repeated 1 d, 3 d and 5 d thereafter and the same volume of 9 g/L saline was simultaneously given to BUO-R rats.The Sham group was prepared in parallel by laparotomy and free dissection of bilateral ureters but not ligated, both side kidneys and blood samples were collected on 3 d and 7 d(24 h, 3 d, 7 d for Sham group) after release of BUO.The urine samples were collected by using metabolic cage before death.The plasma osmotic pressure, creatinine(Cr) and urea nitrogen(BUN) in the plasma of young rats were detected.The expression of AQP-1 protein in all groups of kidney tissues was detected by adopting immunohistochemistry and Western blot. Results On day 3 after release of BUO, 24 h water intake and urine volume of BUO-R+ EPO group were higher than those of Sham group, but lower than those of BUO group(P<0.05), the urine osmotic pressure of BUO-R+ EPO group was higher than that of BUO group, but lower than that of Sham group (P<0.05), while plasma osmotic pressure, Cr and BUN of BUO-R+ EPO group were higher than those of Sham group, but lower than those of BUO group(P<0.05), and they were all of lower than BUO group (P<0.05). On day 7 after release of BUO, there was 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 BUO-R group 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 that of 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, but still lower than that of the Sham group.These results were further confirmed by adopting Western blot, and BUO group was also the lowest of the four groups, and BUO-R+ EPO group was higher than that of BUO group, but lower than that of Sham group (P<0.05). Conclusion EPO can promote not only the recovery of AQP-1 protein expression but also the recovery of renal function in young BUO-R rats.