血管紧张素转换酶抑制剂对糖尿病大鼠肾皮质细胞膜胰岛素受体的调节.doc

血管紧张素转换酶抑制剂对糖尿病大鼠肾皮质细胞膜胰岛素受体的调节血管紧张素转换酶抑制剂对糖尿病大鼠肾皮质细胞膜胰岛素受体的调节2010-09-06 吴永贵林善锬周江华 【摘要】目的研究血管紧张素转换酶抑制剂(ACEI)对糖尿病大鼠肾皮质细胞膜胰岛素受体表达的调节。方法实验大鼠随机分三组：单侧肾切除组(C组，6例)；糖尿病组(D组，7例)及糖尿病苯那普利治疗组(DB组，7例)。观察四周后各组血糖、血胰岛素及血肌酐水平和体重、肾重及肾重/体重之比的变化，测定血浆、肾皮质及髓质血管紧张素转换酶(ACE)活性，采用Western杂交分析肾皮质细胞膜胰岛素受体蛋白表达。结果苯那普利治疗四周后能够改善糖尿病大鼠血糖、血胰岛素及血肌酐水平的异常，对糖尿病肾脏肥大有显著抑制作用，对血浆、肾皮质及髓质ACE活性抑制分别达92.00%，88.77%与73.40%。肾皮质细胞膜胰岛素受体蛋白表达比对照组增加约2.10倍，苯那普利治疗四周后对此有明显的下调作用。结论苯那普利可下调糖尿病状态下肾皮质细胞膜胰岛素受体蛋白高表达，这可能是其对糖尿病肾脏保护作用的重要机制之一。 【关键词】血管紧张素转换酶抑制剂糖尿病肾皮质胰岛素受体大鼠 Regulation of renal cortex cell membranes insulin receptor in diabetic rats by ACEI WU Yonggui,LIN Shanyan,ZHOU Jianghua.Division of Nephrology,Hua Shan Hospital,Shanghai Medical Univsersity,Shanghai,200040 【Abstract】ObjectiveTo investigate into the regulation of renal cortex cell membranes insulin receptor in diabetic rats by angiotensin converting enzyme inhibitor(ACEI). MethodsThe rats were randomly divided into following groups: uninephrectomized rats (group C, n=6), streptozotocin-induced diabetic rats (group D, n=7) and diabetic rats treated with benazepril (an ACEI), (group DB, n=7). Blood glucose, serum insulin, serum creatinine level as well as body weight, kidney weight and kidney weight/body weight were observed after 4 weeks of study. ACE activities in plasma, renal cortex and medulla were measured by fluorimetric assay. The expression of renal cortex cell membranes insulin receptor were determined by Western blot analysis. ResultsAfter 4 weeks of treatment, benazepril could ameliorate hyperglycemia, decrease serum insulin and increase serum creatinine level. It could also significantly supress kidney hypertrophy in diabetic state. ACE activities in plasma, renal cortex and medulla were reduced by approximately 92.00%, 88.77% and 73.40%, respectively. Western blot analysis showed that the expression of renal cortex cell membranes insulin receptor was increased by about 2.10 folds in diabetic rats, however, benazepril might significantly down-regulate its expression. ConclusionBenazepril may down-regulate the increased expression of renal cortex cell membranes insulin receptor protein in diabetic rats, which may be one of the important mechanism of its nephroprotection. 【Key words】AngiotensinconvertingenzymeinhibitorsDiabetes mellitusRenal cortexInsulin receptorRat (Chin J Endocrinol Metab, 1999,15：86-89) 糖尿病肾病(DN)是糖尿病特别是1型糖尿病病程中常伴有的严重并发症之一，其发生机制均直接或间接与高血糖有关。研究证实糖尿病状态下肝脏糖代谢严重障碍，肾组织糖代谢却异常活跃1。血管紧张素转换酶抑制剂(ACEI)对DN的保护作用已为大量的临床和实验所证实，然而，有关它对DN的保护作用是否涉及到对肾组织糖代谢的影响尚未见文献报道。胰岛素的生物效应由其靶细胞膜上胰岛素受体介导，已有的研究表明糖尿病状态下肾组织细胞膜胰岛素受体的数目和亲和力均存在明显异常。为此，本研究初步观察了一种ACEI制剂苯那普利(Benazepril)对糖尿病大鼠肾皮质细胞膜胰岛素受体蛋白表达的调节作用，旨在进一步探讨ACEI对糖尿病肾组织糖代谢影响的机制。 材料和方法 一、糖尿病模型建立 纯种雄性成年Wistar大鼠20只(购自上海计划生育研究所)，体重180200g，所有大鼠均接受右侧肾切除术，2周后腹腔注射单剂量链脲佐菌素(STZ，Sigma公司)60mg/kg(溶解在10mmol/L枸椽酸缓冲液中，pH 5.5)，对照组仅注射等量的枸椽酸缓冲液，4872小时后尾静脉采血用血糖仪测定血糖，当血糖16.7mmol/L，同时尿糖在以上确定为糖尿病模型。 二、动物分组及给药 随机分：(1)对照组(C组，6例)；(2)糖尿病组(D组，7例)；(3)糖尿病苯那普利治疗组(DB组，7例)。苯那普利粉剂(北京诺华制药有限公司提供)10mg.kg-1.d-1灌服，C与D组仅给予等量自来水灌服。4周末大鼠在空腹下尾静脉采血待测血糖及血胰岛素水平。次日在3%水合氯醛麻醉下行右侧颈动脉插管收集血标本，保存于-70冰箱中待测血肌酐及ACE活性；肾脏在腹主动脉插管下注入4预冷的生理盐水反复灌洗后，称重、分离皮、髓质，保存于-70冰箱中待测肾组织血管紧张素转换酶(ACE)活性及肾皮质细胞膜总蛋白提取。血糖、血肌酐水平由日立7150型全自动生化分析仪测得；血胰岛素水平采用放射免疫法测得。 三、血浆及肾皮、髓质ACE活性测定 参考Unger等2方法略加改进，由日立F-4000荧光分光光度计测得(EX360nm、EW486nm)，所需底物马尿酰-组氨酰-亮氨酸(Hip-His-Leu)、荧光激发物邻苯二醛及标准品组氨酰-亮氨酸(His-Leu,HL)均购自Sigma公司，酶活性单位表示为mmol HL.min-1.ml-1(血浆)或nmol HL.min-1.mg-1(组织)。 四、肾皮质细胞膜制备 肾皮质加裂解液1% SDS，100mmol/L Tris(pH 7.4)，100mmol/L sodium pyrophosphate, 10