硝苯地平对糖尿病肾病大鼠肾脏保护作用机制的探讨

Study on renoprotection mechanism of nifedipine in diabetic nephropathy rats

目的探讨血管内皮生长因子（VEGF）、基质金属蛋白酶9（MMP-9）及金属蛋白酶1组织抑制剂（TIMP-1）在糖尿病肾病（DN）发病中的作用以及钙离子拮抗剂硝苯地平对肾脏保护作用的机制。方法大鼠随机分为3组：健康对照（N）组、DN组和硝苯地平治疗（T）组，每组10只。采用链脲菌素诱发DN大鼠模型，治疗组以硝苯地平控释片15mg·kg^-1·d^-1干预治疗，治疗第2周和4周，检测大鼠平均动脉压（MAP）、尿蛋白量（24h）、Scr水平；用免疫组化和Western印迹方法检测大鼠肾脏VEGF、MMP-9、TIMP-1的蛋白表达水平；RT-PCR法检测VEGF、MMP-9、TIMP-1mRNA的表达。结果与N组比较，DN组大鼠尿蛋白量、Scr水平和MAP均显著上升[分别为（78．87±17．62）比（6．30±1．91）mg／24h、（203．88±18．08）比（84．98±10．09）μmol／L、（141．1±8．7）比（99．8±3．8）mmHg，P均〈0．05]；肾组织VEGF、MMP-9、TIMP-1 mRNA和蛋白表达增强（P〈0．05），且MMP-9／TIMP-1比值明显下降（P〈0．05）。与DN组比较，T组尿蛋白量（24h）[（58．35±10．48）mg]、Scr水平[（165．81±15．86）μmol／L]和MAP[（102．6±4．4）mmHg]均显著下降（P均〈0．05）；肾组织VEGF表达下调（P〈0．05），MMP-9、TIMP-1mRNA和其蛋白表达水平显著增高（分别P〈0．05和P〈0．01），MMP-9／TIMP-1比值显著升高（P〈0．05）。DN大鼠肾组织VEGF mRNA水平与尿蛋白量（24h）呈正相关（r=0．748，P〈0．05），与MMP-9／TIMP-1的比值呈负相关（r=-0．711，P〈0．05）。结论VEGF、MMP-9和TIMP-1可能参与了DN的发病过程，硝苯地平可能通过影响VEGF、MMP-9及TIMP-1的表达而实现对肾脏的保护作用。

Objective To investigate the role of vascular endothelial growth factor （VEGF）, matrix metalloproteinase-9（MMP-9） and tissue inhibitor of metalloproteinase-1 （TIMP-1） in the progress of diabetic nephropathy（DN）, and to assess the effect of nifedipine （an calcium-channel blocker） on the expression of VEGF, MMP-9 and TIMP-1, thus to elucidate the renal protection mechanism of nifedipine against DN. Methods Thirty male Wistar rats were randomly divided into three groups： normal control（ N group ）, diabetic nephropathy group（ DN group ） and treatment group with nifedipine（T group, 15 mg·kg^-1·d^-1）. At day 14 and day 28 with nifedipine, five rats of each group were sacrificed to test glomerular hypertrophy, renal function, mean artery pressure （MAP） and 24 h urinary protein excretion. The expression levels of VEGF, MMP-9 and TIMP-1 were examined by immunohistochemical technique, RT-PCR and Western blot. Results Compared with N group, 24 h urinary protein excretion, Scr and MAP increased significantly in DN group[（78.87±17.62） vs （6.30±1.91） mg/24 h,（203.88±18.08） vs （84.98±10.09） μmol/L, （141.1±8.7） vs （99.8±3.8） mm Hg, respectively, all P〈0.05], meanwhile, mRNA and protein expression levels of VEGF, MMP-9 and TIMP-1 increased significantly in DN group （P〈0.05）, but the ratio of MMP-9/TIMP-1 decreased （P〈0.05 or P〈0.01）. Compared with DN group, after 4 weeks with nifedipine, 24 h urinary protein excretion[（ 58.35± 10.48 ） mg/24 h], Scr[ （ 165.81 ± 15.86 ） μmol/L] and MAP [（102.6 ±4.4）mm Hg] decreased significantly, VEGF expression down-regulated, the protein expression of MMP-9 and TIMP-1 up-regulated and MMP-9/TIMP-1 ratio increased （P〈 0.05 or P〈0.01） in T group. Conclusions VEGF, MMP-9 and TIMP-1 may play an important role in the progression of diabetic nephropathy. Nifedipine can suppress the expression of VEGF and increase the ratio of MMP-9/TIMP-1, thus preventing the progression of diabetic nephropathy.