Dickkopf相关蛋白1对自发性高血压大鼠尿钠排泄的作用及机制

Effect and mechanism of Dickkopf-related protein 1 on urinary sodium excretion in spontaneously hypertensive rats

目的探讨Dickkopf相关蛋白1(Dickkopf-related protein 1,DKK1)在高血压中的作用及对尿钠排泄功能的影响。方法 8~10周龄雄性正常血压大鼠(Wistar-Kyoto rats,WKYs;190~210 g)、雄性原发性高血压大鼠(spontaneously hypertensive rats,SHRs;190~210g)各12只,采用Western blot检测WKY和SHR大鼠肾脏DKK1和β-catenin蛋白表达。采用完全随机的方法,将12只WKY大鼠分成WKY+DKK1组和WKY对照组(n=6)、12只SHR大鼠分成SHR+DKK1组和SHR对照组(n=6);WKY+DKK1组和SHR+DKK1组微泵泵注重组蛋白DKK1[6 ng/(g·d)]。使用无创鼠尾测压仪测血压;代谢笼收集24 h尿量;计算24 h尿钠排泄情况,并用体质量校正。采用Western blot检测肾近端小管上皮(renal proximal tubule epithelial,RPT)细胞(WKY-RPT和SHR-RPT细胞)内DKK1和β-catenin蛋白表达;重组蛋白DKK1、DKK1 siRNA(siDKK1)和β-catenin信号抑制剂(inhibitor ofβ-catenin-responsive transcription,ICRT 14)、GSK3β抑制剂(6-bromoindirubin-3′-oxime,BIO,)干预细胞后,检测DKK1、β-catenin表达或钠-钾-ATP酶活性变化。结果 SHR大鼠肾脏DKK1表达水平较WKY大鼠低,而β-catenin升高(即Wnt/β-catenin激活);SHR+DKK1组与SHR组相比,在埋泵术后第2周大鼠收缩压显著下降[(145.21±2.83)vs(156.07±3.08) mmHg,P<0.05],24 h尿量增加[(37.40±3.76)vs(27.24±2.30)mL/kg,P<0.05],24 h尿钠排泄增多[(0.79±0.09)vs(0.51±0.09)mmol/kg,P<0.05]。WKY+DKK1组与WKY组间差异均无统计学意义。SHR-RPT细胞DKK1表达低于WKY-RPT细胞,而β-catenin升高;改变DKK1含量引起β-catenin改变(即Wnt/β-catenin通路活性改变)从而影响钠-钾-ATP酶活性,且钠-钾-ATP酶活性与DKK1呈负相关。结论 DKK1至少部分通过抑制Wnt/β-catenin通路降低钠-钾-ATP酶活性,促进尿钠排泄降低血压。

Objective To explore the role of Dickkopf-related protein 1(DKK1) in hypertension and its effect on urinary sodium excretion. Methods Twelve rats with normal blood pressure(Wister Kyoto rats, WKY), and 12 primary hypertension rats(spontaneously hypertensive rats, SHRs), all male, at an age of 8~10 weeks, weighing 190~210 g, were employed in this study. The expression of DKK1 and β-catenin in the kidney of WKY and SHR rats was detected by Western blotting. Then 12 WKY rats were randomly divided into WKY+DKK1 group and WKY control group(n=6), and so were the 12 SHR rats. Recombinant protein DKK 16 ng/(g·d) was pumped into the rats from the DKK1 groups via micro-syringe. The blood pressure of all the rats was measured by non-invasive tail-cuff method. And the urine was collected by metabolic cages for 24-hour urinary volume and urinary sodium, which were adjusted with their weight. The expression of DKK1 and β-catenin protein in renal proximal tubule epithelial(RPT) cells(WKY-RPT and SHR-RPT) was detected by Western blotting. After intervention with recombinant protein DKK1, DKK1 siRNA(siDKK1), and β-catenin nuclear signal inhibitor(inhibitor of β-catenin-responsive transcription, ICRT 14), inhibitor of GSK3β(6-bromoindirubin-3′-oxime, BIO), the expression of DKK1 and β-catenin and the activity of Na^+-K^+-ATPase were measured. Results The expression level of DKK1 was lower, while that of β-catenin(indicating Wnt/β-catenin was activated) was higher in the renal tissue of SHR rats than of WKY rats. When the SHR+DKK1 group was compared with the SHR group, the systolic blood pressure was significantly decreased at 2 weeks after implantation(145.21±2.83 vs 156.07±3.08 mmHg, P<0.05), and both urine volume(37.40±3.76 vs 27.24±2.30 mL/kg, P<0.05) and urinary sodium excretion(0.79±0.09 vs 0.51±0.09 mmol/kg, P<0.05) within 24 h were increased. There were no significant differences in above indexes between WKY+DKK1 group and WKY group. The expression of DKK1 was lower in the SHR-RPT cells than the WKY-RPT cells, and that of β-catenin was opposite. Intervention of DKK1 expression would cause the change of β-catenin(indicating the alteration of Wnt/β-catenin pathway), and thus affect the activity of Na^+-K^+-ATPase, and the activity of Na^+-K^+-ATPase was negatively correlated with DKK1. Conclusion DKK1 can reduce Na^+-K^+-ATPase activity at least partially by inhibiting the Wnt/β-catenin pathway, promote urinary sodium excretion and lower blood pressure.