血管内皮生长因子及其受体Flk-1在糖尿病大鼠肾脏的表达及厄贝沙坦的干预(英文)

Interventional effect of irbesartan on vascular endothelial growth factor and its Flk-1 receptor expressions in the kidney of diabetic rats

背景：已有实验提示，血管内皮生长因子及其受体系统可能参与糖尿病肾病的发生发展。目的：实验进一步验证血管内皮生长因子及其受体Flk-1在糖尿病鼠肾脏中的表达变化及厄贝沙坦对其影响和可能的机制。设计：随机对照动物实验。单位：四川大学华西医院。材料：雄性封闭群SD大鼠18只，体质量150-200g，标准化饲养。方法：实验于2006-08／2007-04在四川大学华西医院实验室完成。建立糖尿病肾病大鼠模型，分为糖尿病肾病组、厄贝沙坦干预组和正常对照组，每组6只大鼠。采用10g／L链脲霉素按55mg／kg体质量一次性腹腔内注射诱导造模，对照组给予相同剂量的枸橼酸缓冲液，干预组在成模后按3mg／（kg·d）给予厄贝沙坦。采用反转录聚合酶链式反应技术以及免疫组织化学技术检测血管内皮生长因子和Flk-1的表达，检测尿蛋白、肾小球面积和体积，并分析数据相关性。主要观察指标：①肾脏病理以及尿蛋白、肾小球面积和体积。②肾脏血管内皮生长因子与Flk-1 mRNA表达。③肾脏免疫组织化学检查。④相关分析。结果：①肾脏苏木精-伊红染色病理检查显示糖尿病肾病组出现明显肾小球增大，系膜基质增多，系膜细胞增多，小管扩张，干预组病变轻于糖尿病肾病组。糖尿病肾病组尿蛋白显著高于对照组（P〈0．01）；糖尿病肾病组肾重，体质量、肾小球面积和体积明显高于对照组（P〈0．01），干预组蛋白尿、肾重，体质量、肾小球面积和体积均低于糖尿病肾病组（P〈0．01）。②糖尿病肾病组16周时肾脏血管内皮生长因子与Flk-1 mRNA表达均明显上调，显著高于对照组（P〈0．05）；干预组16周时肾脏血管内皮生长因子与Flk-1 mRNA表达也有上调，高于对照组（P〈0．05），但低于糖尿病肾病组（P〈0．05）。③糖尿病肾病组血管内皮生长因子着染明显强于对照组（P〈0．01），干预组着染也强于对照组（P〈0．01），但与糖尿病肾病相比较，干预组着染明显减少（P〈0．01）。Flk-1着染也有类似变化。④血管内皮生长因子、Flk-1与尿蛋白、肾小球面积和体积呈正相关（P〈0．05）。结论：血管内皮生长因子及其受体Flk-1在糖尿病肾病发病机制中起重要作用，过度表达导致肾脏损伤，血管紧张素Ⅱ受体拮抗剂厄贝沙坦具有通过抑制血管内皮生长因子和Flk-1异常表达这一非血流动力学的肾脏保护作用。

BACKGROUND： Some studies have presented that vascular endothelial growth factor and its receptor system may take part in onset and development of diabetic nephropathy （DN）. OBJECTIVE： To further verify the interventional effects of irbesartan on vascular endothelial growth factor （VEGF） and its Flk-1 receptor expressions in kidney of diabetes mellitus （DM） rat, and the possible mechanism of irbesartan. DESIGN： Randomized control animal study. SETTING： West China Hospital of Sichuan University. MATERIALS： Eighteen male closed colony SD rats weighing 150-200 g were cared in standardization. METHODS： This study was performed at Laboratory of West China Hospital of Sichuan University from August 2006 to April 2007. All rats were randomly divided into a DN group, an irbesartan group and a normal control group, with 6 rats in each group, 10 g/L streptozotocin （55 mg/kg） was intraperitoneally injected to establish models; rats in the control group were administrated with the same dosage of citric acid buffer solution; rats in the irbesartan group were administrated with 3 mg/（kg·d） irbesartan after model establishment. VEGF and Flk-1 expressions were detected using reverse transcription polymerase chain reaction （RT-PCR） and immunohistochemistry technique; while, urine protein level, area and volume of renal glomerulus were detected, and correlations of data were analyzed. MAIN OUTCOME MEASURES： （1） Renal pathological indicators, urine protein level, area and volume of renal glomerulus; （2） VEGF and Flk-1 expressions; （3） renal immunohistochemical examination; （4） correlation analysis. RESULTS： （1） Renal pathological examination by HE staining indicated that renal glomerulus was remarkably enlarged in the DN group; mesangial matrix was increased; mesangial cells were also increased; renal tubule was expanded. The lesions in the irbesartan group were milder compared to the DN group. Urine protein level in the DN group was significantly higher compared to the control group （P 〈 0.01）; renal weight/body mass, area and volume of renal glomerulus were significantly higher compared to the control group （P 〈 0.01）; urine protein level, renal weight/body mass, area and volume of renal glomerulus in the irbesartan group were significantly lower compared to the DN group （P 〈 0.01）. （2） By the 16^th week, VEGF and Flk-1 expressions in the DN group were significantly up-regulated compared to control group （P 〈 0.05）; while, VEGF and Flk-1 expressions in the irbesartan group were also up-regulated compared to the control group by the 16^th week （P 〈 0.05） but down-regulated compared to the DN group （P 〈 0.05）. （3） VEGF staining in the DN group was darker compared to control group （P 〈 0.01）, while the staining in the irbesartan group was also darker compared to the control group （P 〈 0.01）, but the staining in the irbesartan group was lighter compared to the DN group （P 〈 0.01）. Flk-1 staining was similar to the VEGF. （4） VEGF and Flk- 1 were positively correlated with urine protein level, area and volume of renal glomerulus （P 〈 0.05）. CONCLUSION： VEGF and its Flk-1 receptor play important roles in DN pathogenesis. Over expressions may cause renal injury, but irbesartan （angiotensin Ⅱ receptor antagonist） has the protective effects on kidney through inhibiting abnormal expression of VEGF and Flk-1.