金属蛋白酶1组织抑制剂在慢性肾衰竭大鼠钙化动脉中的表达

Expression of tissue arterial calcification inhibitor of metalloproteinases 1 in chronic renal failure rats with Department of Nephrology China

目的探讨金属蛋白酶1组织抑制剂（TIMP-1）是否参与慢性肾衰竭大鼠动脉钙化形成。方法雄性Wistar大鼠按随机数字表法分组，其中健康对照组8只，实验组36只。用2％腺嘌呤250mg·kg^-1·d^-1灌胃制备动脉钙化模型。von Kossa染色观察主动脉、股动脉、肾动脉和冠状动脉钙化情况。RT—PCR和Western印迹检测主动脉TIMP-1表达。免疫组织化学方法检测TIMP-1、骨桥蛋白（OPN）、核心结合因子α1（Cbfα-1）在主动脉的表达。结果实验组大鼠给药2周后出现明显慢性肾衰竭表现，BUN、Scr、血磷、钙磷乘积和全段甲状旁腺素（iPTH）显著高于对照组（P〈0．01）；给药6周后主动脉、股动脉、肾动脉和冠状动脉中层出现不同程度的钙化。RT—PCR和Western印迹结果显示，实验组大鼠主动脉TIMP-1表达较对照组上调（P〈0．05），随时间延长呈上升趋势。免疫组化的结果显示，给药后第2、4、6、8周主动脉平滑肌细胞TIMP-1蛋白表达均显著高于对照组（P〈0．05）；钙化主动脉出现Cbfα-1的阳性表达；OPN表达显著增多（P〈0．01）。TIMP-1的表达和OPN及Cbfα-1表达均呈正相关（r=0．317，P=0．000；r=0．485，P=0．000）。结论大鼠腺嘌呤肾衰竭模型血管钙化的病理变化与人类慢性肾衰竭血管钙化相似，是研究慢性肾衰竭血管钙化周期较短、较好的动物模型。TIMP-1高表达和慢性肾衰竭动脉血管钙化相关。

Objective To examine whether tissue inhibitor of metalloproteinases-1 （TIMP-1） is involved in arterial calcification of chronic renal failure （CRF） rats. Methods CRF model was induced in male Wistar rats by garage daily with 2% adenine 250 mg/kg. The calcification of aorta, femoral artery, renal artery and coronary artery was evaluated histomorphometrically by von Kossa-stained sections at 2-, 4-, 6- and 8-week respectively. RT-PCR and Western blot were used to observe the expressive levels of TIMP-1 mRNA and protein. Expressions of TIMP-1, osteopontin （OPN） and core binding factor α1 （Cbfα-1） protein were analyzed by immunhistochemistry. Results Serum urea nitrogen, creatinine, inorganic phosphate, calcium-phosphorus product and intact parathyroid hormone （iPTH） increased significantly in the model animals compared with control group after 2 weeks （P〈0.01）. Medial calcification was found in above four arteries of model groups after 6 weeks. RT-PCR and Western blot showed that TIMP-1 expression of model group was significantly higher than that of control group （P〈 0.05）, and obviously elevated in a time-dependent manner. The expression of TIMP-1 and OPN in calcified aortic smooth muscle cells increased obviously （P〈0.05）, and positive immunostaining of Cbfc^-I was found. The expression of TIMP-1 was positively correlated with OPN and Cbfct-1 （r=0.317, P=0.000; r=0.485, P=0.000）. Conclusions The pathology of arterial calcification in CRF rats induced by adenine is similar to CRF patients, which may serve as a useful model of CRF with arterial calcification. The up-regulation of TIMP-1 seems to participate in the formation and development of vascular calcification in CRF.