基质金属蛋白酶/组织金属蛋白酶抑制物表达失衡在衰老大鼠肾小管间质损害中的意义

Significance of imbalance between matrix metalloproteinases and tissue type inhibitor of metalloproteinases in renal tubulointerstitial lesions of aging rats

目的 研究基质金属蛋白酶 (MMP)及组织金属蛋白酶抑制物 (TIMP)在不同鼠龄的单侧输尿管梗阻 (UUO)大鼠肾小管间质中的表达变化 ,探讨其可能的作用。方法 选用 3月、2 6月龄大鼠制备UUO模型 ,采用组织病理及逆转录 聚合酶链反应 (RT PCR)、Western印迹等方法观察UUO术后3、7、14d不同鼠龄大鼠的肾脏组织学改变和MMP 2、MMP 9、TIMP 1、TIMP 2等在梗阻肾小管间质中的表达情况 ;用明胶酶谱法检测UUO术后不同时间点MMP 2、MMP 9的蛋白水解活性。结果 在对照组 ,TIMP 1、TIMP 2仅微弱表达于 3月龄肾脏中 ,而在 2 6月龄大鼠肾脏中其表达显著增加 (P <0 0 1) ;老年鼠肾组织中MMP 2、MMP 9的蛋白水解活性显著低于 3月龄大鼠 (P <0 0 1)。与对照组相比 ,3月龄、2 6月龄大鼠梗阻侧肾脏的肾小管间质纤维化面积随UUO术后时间的延长而增加 ,TIMP 1、TIMP 2及MMP 2、MMP 9的mRNA及蛋白质的表达在术后各时间点均显著增高 (P <0 0 1) ,MMP 2和MMP 9的蛋白水解活性随UUO术后时间的延长而逐渐下降。其活性的下降与TIMP 2及TIMP 1蛋白质表达的增加呈明显的负相关。与 3月龄鼠比较 ,2 6月龄鼠肾小管间质纤维化面积在UUO术后各时间点也明显增加 (P <0 0 1) ,TIMP 1、TIMP 2在肾组织中各时间点的mRNA及蛋白质表达均显?

Objective To explore the imbalance between the expression of metalloproteinases (MMPs) and that of tissue type inhibitors of metalloproteinase (TIMPs) in the renal tubulointerstitial lesions of aging rats and the potential role of this imbalance. Methods Forty eight male 26 month old Wistar rats were randomly divided into 2 groups of 24 rats: unilateral ureteral obstruction (UUO) group with the left ureter ligated and excised and false operation group used as control group. Forty eight male 3 month old Wistar rats were randomly divided into 2 groups of 24 rats: UUO group and false operation group just as in the 26 month old rats. Every group was randomly divided into 3 subgroups of 8 rats: 3 day group, 7 day group, and 14 day group to be killed 2, 7, and 14 days after the operation respectively. The 2 kidneys of each rat were taken out. Routine pathological examination and immunofluorescence (IF) examination were made to calculate the area of renal tubulointerstitial fibrosis and the expression of Ⅳ type collagen. RT PCR and Western blotting were used to detect the expressions of mRNA and protein of TIMP 1, TIMP 2, MMP 2, and MMP 9 in the kidney at different time points. Gelatin zymography was used to detect the proteolytic activity of MMP 2 and MMP 9. Results The renal interstitial lesion was more significantly in the 26 month old UUO rats than in the 3 month old UUO rats since the 3rd day after operation. Both the expression of MMP 2 mRNA and the expression of MMP 9 mRNA were not different between the 2 control groups. In the control groups, TIMP 1 and TIMP 2 Both MMP 2 mRNA and MMP 9 mRNA were expressed in both control groups, without significant differences between these 2 control groups. TIMP 1 mRNA and TIMP 2 mRNA were only weakly expressed in the renal tissues of the 3 omth old control group, however, the expressions of both TIMP 1 and TIMP 2 were significantly stronger in the 16 mionth old control group than in the 3 month old control group. The expressions of TIMP 1 mRNA and TIMP 2 mRNA at different time points were significantly stronger in the obstructed side than in the healthy side in the UUO groups, and significantly stronger in the 26 monh old rats than in the 3 month old rats. MMP 2 protein and MMP 9 protein were expressed in the 3 month old and 26 month old controls without difference between them. The expressions of MMP 2 protein and MMP 9 protein at different time points were significantly stronger in the obstructed side than in the healthy side in the UUO groups, without significant difference between the 26 month old UUO rats and the 3 month old UUO rats. The expressions of TIMP 1 protein and TIMP 2 protein were very weak at different time points in the renal tissues of the 3 month old controls and were significantly stronger in the 26 month old control rats. In the UUO rats the expressions of TIMP 1 protein and TIMP 2 protein in the renal tissues of the obstructed side at different time points were all significantly stronger for both age groups in comparison with the control groups of the same age and were significantly stronger in the 26 month old UUO rats than in the 3 month old UUO rats without significant difference between the 26 month old UUO rats and the 3 month old UUO rats. The MMP 2 activity and MMP 9 activity of the 26 month old rats were both significantly lower than those of the 3 month old control rats. The MMP 2 activity and MMP 9 activity at different time points of the 2 UUO groups were all significantly lower than those of the control groups of the same age, and those of the 26 month old were significantly lower than those of the 3 month old rats The MMP 2 activity and MMP 9 activity were negatively correlated with the expressions of TIMP 2 and TIMP 1. Conclusion The abnormal expression of MMPs/TIMPs including higher expression of TIMPs and decreased proteolytic activity of MMPs induced by aging may be one of the factors aggravating the renal tubulointerstitial lesions of aging rats.