西罗莫司对双侧输尿管梗阻大鼠水电解质代谢紊乱的保护作用

目的 探讨西罗莫司对双侧输尿管梗阻(BUO)大鼠水电解质代谢紊乱及肾脏上皮钠通路γ(γ-ENaC)、Na+-K+-ATP酶及水通道蛋白2(AQP2)蛋白的影响.方法 Wistar大鼠48只,随机分为假手术组、BUO组和西罗莫司组,每组16只.BUO组和西罗莫司组大鼠结扎双侧输尿管制作BUO模型,假手术组仅游离双侧输尿管后缝合.BUO组及西罗莫司组双侧输尿管梗阻24 h后解除梗阻.西罗莫司组每天西罗莫司口服液(2.5 ml/次)灌胃,假手术组及UUO组用同体积生理盐水灌胃.于术后4、7d测量3组大鼠尿量并留取尿液进行生化分析;术后4、7d抽取动脉血化验,同时取出双侧肾脏,采用免疫组化及蛋白质印迹法检测肾脏γ-ENaC、Na+-K+-ATP酶及AQP2蛋白的表达.结果 BUO组大鼠输尿管梗阻解除后4、7d尿量分别为(85.31±13.15)、( 66.39±10.56)ml,显著多于假手术组( 35.36±7.74)、(33.90±8.03)ml及西罗莫司组(69.81±10.70)、(48.57±9.01)ml;尿钠浓度(42.17±7.35)、(43.63±18.39) mmol/L,显著低于假手术组(170.56±18.39)、(172.52±7.35) mmol/L及西罗莫司组(76.18±13.20)、(134.28±13.20) mmol/L,3组间比较差异均有统计学意义(P＜0.05).西罗莫司组4、7d肾脏γ-ENaC表达量分别为2.09±0.32、2.27±0.35,Na+ -K+ -ATP酶分别为2.41±0.48、2.67±0.43,AQP2分别为2.17±0.45、2.63±0.28,显著高于同时间点的BUO组(1.28±0.21、1.45±0.17,1.99±0.28、2.18±0.24,0.93±0.22、1.31±0.16),低于同时间点假手术组(2.58±0.51、2.60±0.56,2.89±0.53、2.97±0.66,3.05±0.63、3.10±0.67),3组间比较差异均有统计学意义(P＜0.05).结论 γ-ENaC、Na+-K+-ATP酶及AQP2蛋白水平降低可能是泌尿系梗阻后肾小管性钠回吸收障碍、低渗性尿量增多的主要原因之一.西罗莫司可以通过抑制肾小管γ-ENaC、Na+-K+-ATP酶及AQP2蛋白丢失,减轻术后肾脏钠回吸收障碍及低渗性尿等水电解质代谢紊乱,保护肾脏功能.

Protective effect of sirolimus on water-electrolyte imbalance following bilateral ureteral obstruction in rats

Objective To explore the molecular mechanism of renal function defects after urinary obstruction and investigate the effect of sirolimus on the expression of γ-ENaC,Na + K + ATPase and AQP2,and its mechanism of renal Water-Electrolyte imbalance following bilateral ureteral obstruction (BUO) in rat kidneys.Methods Forty-eight rats were randomly divided into a sham operation group ( sham group),a BUO group,and a sirolimus treatment after BUO group.Bilateral ureters were exposed and occluded with ligature in the BUO and sirolimus treatment groups.Twenty-four hours later,the obstructed ureters were decompressed by removal of the ligature.The sham animal group underwent identical surgical procedures,but the ureter was simply dissected without removal of the ligature.The sirolimus treatment groups was given sirolimus intragastricly 0.4 ml per day (2 mg/kg · d) from the day before surgery until the rats were scari fled.The sham and BUO groups were given the same volume of intragastric saline.The urine and blood were collected at 4 d,7 d after surgery,and the functional data were observed.The expression of γ-ENaC,Na+K + ATPase and AQP2 were examined by immnohistochemistry and immunoblotting.Results On day four and seven post ureteral obstruction release,urine volume in the BUO group were (85.31 ± 13.15,66.39 ±10.56 ml),significantly higher than that of sham operation (35.36 ± 7.74,33.90 ± 8.03 ml) and sirolimus treatment groups (69.81 ± 10.70 ml,48.57 ± 9.01 ml) (P ＜ 0.05 ).Urine sodium concentrations in the BUO group were (42.17 ± 7.35 mmol/L,43.63 ± 18.39 mmoL/L),significantly lower than that of sham operation ( 170.56 ± 18.39 mmoL/L,172.52 ± 7.35 mmol/L) and sirolimus treatment groups (76.18 ± 13.20 mmol/L,134.28 ± 13.20 mmol/L),P ＜ 0.05.Immunoblotting assay showed that,on day four and seven post rats ureteral obstructions were released,integral optical density of γ-ENaC (2.09 ±0.32,2.27 ±0.35),Na+ K+ATP enzyme (2.41 ±0.48,2.67 ±0.43) and AQP2 (2.17 ±0.45,2.63 ±0.28) in the sirolimus treatment group were significantly higher than those of BUO group ( 1.28 ± 0.21,1.45 ±0.17) (1.99 ±0.28,2.18±0.24) (0.93 ±0.22,1.31 ±0.16),but still lower than the sham group (2.58±0.51,2.60±0.56) (2.89±0.53,2.97 ±0.66) (3.05 ±0.63,3.10±0.67).There were significant differences among all the three groups ( P ＜ 0.05 ).Conclusions The downregulation of γ-ENaC,Na + K + ATPase and AQP2 expression after BUO may contribute to the impaired renal tubular sodium reabsorption,decreased urinary concentration,and postobstructive polyuria.Sirolimus treatment significantly prevents impairment in renal function and also prevents downregulation of y-ENaC,Na + K+ ATPase and AQP2during BUO,demonstrating a marked renoprotective effect of sirolimus treatment in conditions with urinary tract obstruction.