G蛋白耦联受体激酶4对肾脏胃泌素受体的调节及其在高血压发生中的作用

Role of G protein-coupled receptor kinase 4 in regulation of renal gastrin receptor in hypertension

目的研究G蛋白耦联受体激酶4(GRK4)对肾脏胃泌素受体(CCKBR)的调节作用及对高血压发病的影响。方法以WKY(Wistar-Kyoto)大鼠及自发性高血压大鼠(SHR)为研究对象,采用大鼠单肾胃泌素灌注模型,分析胃泌素诱导的尿钠排泄作用在二者的差异。分别利用免疫印迹法和免疫沉淀法检测不同表型大鼠肾脏CCKBR的蛋白表达和磷酸化的差异。比较用GRK4siRNA敲降WKY大鼠及SHR肾脏近曲小管(RPT)上皮细胞的GRK4后CCKBR磷酸化和CCKBR介导的Na^+-K^+-ATP酶活性抑制作用的变化;通过免疫共沉淀的方法分析GRK4和CCKBR结合在WKY大鼠和SHR RPT细胞上的差异。结果胃泌素呈剂量依赖性地促进WKY大鼠肾脏的尿钠排泄,这一作用在SHR丧失。与WKY大鼠相比,SHR肾脏CCKBR磷酸化水平升高(WKY大鼠组0.85±0.14比SHR组1.11±0.16,P<0.05)。胃泌素(10^(-9)mol/L,15 min)激活WKY大鼠RPT细胞CCKBR后,Na^+-K^+-ATP酶活性显著下降,而这一作用在SHR RPT细胞上丧失;利用siRNA降低SHR RPT细胞上GRK4表达后,胃泌素可以明显抑制Na^+-K^+-ATP酶活性。同时,GRK4siRNA降低了SHR RPT细胞CCKBR磷酸化水平(对照组1.19±0.14比干扰组0.85±0.13,P<0.01)。免疫共沉淀发现GRK4和CCKBR的结合在SHR RPT细胞上增加(WKY大鼠RPT细胞0.35±0.12比SHR RPT细胞0.57±0.15,P<0.05)。结论 GRK4对CCKBR磷酸化和功能具有调节作用,此作用影响了水钠排泄。

Objective To identify the regulation of G protein coupled receptor kinase 4 （GRK4） to gastrin receptor （cholecystokinin receptor type B, CCKBR） in kidney and its role in the pathogenesis process of hypertension. Methods Wistar-Kyoto （WKY） rats and spontaneously hypertensive rat （SHR） were used in this study. The gastrin induced natriuresis and diuresis in different strains of rats was measured by gastrin infusion test. And immunoblot and immunoprecipitation were used to detect the WKY and SHR kidney CCKBR protein expression and phosphorylation level. The effect of GRK4 siRNA on CCKBR phosphorylation and CCKBR-associated Na^＋-K^＋- ATPase activity in WKY and SHR renal proximal tubule （RPT） cells were checked. The difference of interaction between GRK4 and CCKBR in WKY and SHR RPT cells was studied via co-immunoprecipitation （co-IP）. Results Gastrin infusion induced the natriuresis and diuresis in WKY rats in a dose-dependent manner, and the effect of gas trin was abrogated in SHR. As compared with WKY, the phosphorylation level of CCKBR in SHR kidney was increased （WKY 0.85±0.14 vs SHR 1.11±0.16, P〈0.05 ）. Na^＋-K^＋-ATPase activity was significantly decreased in kidney from WKY rats with gastrin （10^-9 mol/L, 15 rain） treatment, whereas gastrin did not decrease the Na^＋-K^＋ ATPase activity in SHR RPT cells. After reducing GRK4 expression by siRNA, gastrin suppressed the Na^＋- K^＋-ATpase activity via activating CCKBR. And GRK4 siRNA also decreased the level of CCKBR phosphorylation （control 1.19±0. 14 vs siRNA group 0.85±0.13, P〈0.01）. In addition to immunoprecipitation, the interaction of GRK4 and CCKBR was increased in the RPT cells of SHR （WKY 0.35±0.12 vs SHR 0.57±0.15, P〈0.05）. Conclusion GRK4 can regulate phosphorylation and function of CCKBR, which might involve in the regulation of diuresis and natriuresis.