阿米洛利抑制尿激酶受体表达及减少蛋白尿的机制

Amiloride reduces proteinuria and inhibits podocyte uPAR in the 5/6 nephrectomy rats

目的观察阿米洛利在经典的5/6肾切除大鼠模型中对蛋白尿的影响,探讨其影响蛋白尿的机制。方法构建经典的5/6肾切除大鼠模型,实验分为3组:假手术对照组(Sham)、5/6肾切除组(NTX)、5/6肾切除+阿米洛利干预组(NTX+阿米洛利)。采用考马斯亮蓝法检测各组24 h尿蛋白,利用免疫荧光共聚焦、实时荧光定量PCR等技术,检测足细胞尿激酶受体(uPAR)蛋白和uPAR mRNA的表达情况。结果第2周时,与假手术对照组(Sham)相比较,5/6肾切除组(NTX)24 h尿蛋白明显升高(47.50±28.05 mg vs 14.28±3.8 mg,P=0.023),有统计学意义;与NTX组相比,NTX+阿米洛利24 h尿蛋白无统计学意义(51.56±21.03 mg vs 47.50±28.05 mg,P=0.748)。第12周时,与NTX组(188.31±29.82)mg相比,NTX+阿米洛利组(121.37±31.14)mg、Sham组(21.32±8.59)mg 24 h尿蛋白增加量均进一步减少,具有统计学意义[P值分别为0.001,0.000]。激光共聚焦显微镜观察,与Sham组相比较,NTX组PLAUR mRNA表达明显增高(9.74±1.44 vs 1.01±0.13,P<0.01),有统计学意义;与NTX组相比较,NTX+阿米洛利mRNA表达明显降低(9.74±1.44 vs 5.01±1.36,P<0.01),有统计学意义。结论阿米洛利可能通过抑制受损足细胞uPAR的表达,从而起到降蛋白尿、延缓肾小球硬化的作用。

Objective To observe the effect of amiloride on the proteinuria of the 5/6 nephrectomy rats. Methods To establish the 5/6 nephrectomy rats model and divide the experiment into 3 groups, sham operated group（Sham）, 5/6 nephrectomy model group（NTX） and 5/6 nephrectomy with amiloride-treated group （NTX＋amiloride, n=15）. The concentration of protein and mRNA of uPAR and the change of podocytes motility were detected by coomassiebluestaining, immunofluorence method and real-time PCR. Results At second week, compared with Control group, the 24 h urine protein of NTX group was significantly increased （47.50±28.05 mg vs 14.28±3.8 mg, P=0.023）. There was no statistical significance in 24-hour urine protein between NTX＋amiloride group and NTX group （51.56±21.03 mg vs 47.50±28.05 mg, P=0.748）. The same situation was also observed at the time point of 12 week, comparing with NTX group, 24-hour urine protein decreased in Sham group （188.31±29.82 mg vs 21.32±8.59 mg, P=0.000） and NTX＋amiloride group （188.31±29.82 mg vs 121.37±31.14 mg, P=0.000）, with statistical significance when comparing with Sham group, the expression of uPAR mRNA in NTX group was significantly increased （9.74±1.44 vs 1.01±0.13, P=0.000）. In contrast, the expression of uPAR mRNA in NTX rats treated with amiloride was significantly lower than in NTX group （9.74±1.44 vs 5.01±1.36, P=0.000）. Conclusion Amiloride can reduce the proteinuria of the 5/6 nephrectomy rats model of transient proteinuria by inhibiting the induction of uPAR expression.