螺内酯对ROCK1在单肾切除糖尿病大鼠肾组织中表达及活性的影响

目的 观察小G蛋白Rho相关蛋白激酶1(ROCK1)在单肾切除糖尿病大鼠肾组织中的表达及活性改变,探讨螺内酯对糖尿病肾病(DN)的保护机制.方法 制作单肾切除加链脲菌素( 45 mg/kg)诱导的大鼠糖尿病模型,随机分为假手术组(N)组、单肾切除对照?组、单肾切除糖尿病模型(D)组、单肾切除糖尿病螺内酯干预(S)组.分别于给药4周和8周后观察各组大鼠各项生化指标、肾组织病理形态学改变；免疫组织化学和实时PCR法检测肾皮质ROCK1、结缔组织生长因子(CTGF)水平；Western印迹法检测肾皮质肌球蛋白磷酸酶目标亚单位1(p-MYPT1)表达情况.结果 与N组、C组比较,实验第4周和第8周D组血糖、肾质量指数(肾质量/体质量,KWI)、24 h尿蛋白量(Upro)、收缩压(SBP)均明显增高(均P＜0.01)；肾小球细胞外基质(ECM)沉积增多；肾小管肿胀变性；8周时血清白蛋白(Alb)明显下降、Scr明显上升(均P＜0.05).随时间延长,D组肾皮质中ROCKI、CTGF表达逐渐增强,p-MYPTI蛋白表达逐渐增加.ROCK1表达和活性和CTGF表达呈正相关(r=0.874,P=0.000；r=0.858,P=0.000).与同期D组比较,S组24 hUpro、KWI、肾组织病理改变、肾皮质ROCK1、CTGF和p-MYPT1表达等均得到明显改善,而两组血糖、Scr、Alb、SBP间差异无统计学意义.结论 ROCK1表达和活性在单肾切除糖尿病大鼠肾脏中均上调,并和CTGF表达呈正相关.螺内酯可能通过下调ROCK1表达及活性,降低CTGF表达,从而发挥保护早期糖尿病大鼠肾脏的作用.

Impact of spironolactone on the expression and activity of ROCK1 in renal tissues of uninephrectomized diabetic rats

Objective To investigate the expression and activity of Rho-associated kinase 1 (ROCK1) in renal tissues of uninephrectomized diabetic rats, and to explore the possible mechanism of renal protection of spironolactone. Methods The model rats were established by a single intraperitoneal injection of streptozotocin (STZ) after uninephrectomy and randomly divided into sham operation group (N), uninephrectomized control group (C), uninephrectomized diabetic group (D), and spironolactone treated group (S). Four and 8 weeks later, biochemical indexes and renal morphology were detected. Expressions of ROCK1 and connect tissue growth factor (CTGF) were examined by immunohistochemistry and real-time PCR. Protein expression of p-MYPT1 was examined by Western blotting. Results After 4 and 8 weeks, compared with group N and C, blood glucose, systolic blood pressure, 24-h urinary protein, kidney weight/body weight (KWI) were significantly increased(P<0.01), and extracellular matrix proliferation and basement membrane thickening were found in group D. After 8 weeks in group D, Alb significantly decreased and Scr significantly increased(P<0.05). In group D, protein and mRNA expression of ROCK1 and CTGF increased significantly and protein expression of p-MYPT1 increased significantly as well with time. Treatment with spironolactone could partially reverse those changes. Conclusions Expression and activity of ROCK1 increase in renal tissues of uninephrectomized diabetic rats and are positively correlated with the expression of CTGF. Spironolactone can protect the kidney of diabetic rats in early stage probably through decreasing the expression of ROCK1, CTGF and inhibiting the activation of ROCK1.