广谱基质金属蛋白酶抑制剂对高血压肾纤维化的影响

Effects of Broad-Spectrum Matrix Metalloproteinases Inhibitor on Hypertensive Renal Fibrosis in Stroke-prone Spontaneously Hypertensive Rats

目的研究多西环素(DOX)对卒中易感型自发性高血压大鼠(SHR-SP)肾脏基质金属蛋白酶(MMPs)及金属蛋白酶组织型抑制剂(TIMPs)活性的影响,探讨DOX对高血压肾纤维化病理转归的作用机制。方法选用7周龄雄性SHR-SP随机分为药物干预组和对照组,定期观察大鼠体重、测量无创鼠尾压、尿量、尿肌酐、尿蛋白变化。观察终止时进行血流动力学分析;测定血清肌酐、尿素氮浓度;采用组织病理、明胶酶谱和逆明胶酶谱等方法观察各组大鼠肾脏组织学改变和MMP-2、MMP-9、TIMP-1、TIMP-2等活性变化。结果收缩压、脉压、血清肌酐浓度在DOX组与对照组之间无统计学差异,但DOX组有升高趋势;从19周龄起,DOX组大鼠尿蛋白浓度与尿肌酐浓度比值即高于对照组[(8·3±6·4vs3·3±2·0)mg/μmol,P<0·05];病理染色显示肾小管损伤平均指数和胶原容积分数均高于对照组(损伤平均指数:3·14±0·47vs2·69±0·38,P<0·05;胶原容积分数:10·5%±2·7%vs6·6%±1·9%,P<0·01);与血压正常大鼠相比,高血压大鼠肾脏MMP-2、MMP-9、TIMP-1和TIMP-2的活性升高;药物干预后MMP-2、MMP-9活性降低(MMP-2:2·62±0·75vs3·39±0·92;MMP-9:2·89±1·13vs4·01±0·91,P<0·05),而TIMP-1、TIMP-2活性则显著升高(TIMP-1:2·89±1·76vs1·54±0·86,P<0·05;TIMP-2:1·69±0·47vs1·06±0·47,P<0·01)。结论广谱基质金属蛋白酶抑制剂DOX可以抑制SHR-SP肾脏中MMP-2、MMP-9活性,加重高血压造成的肾脏损害。在肾脏损害发生后,抑制MMPs活性可能会加重高血压肾脏纤维化的进展。

Objective To investigate the effect of doxycycline（DOX） on the activity of renal matrix metalloproteinases and tissue inhibitors of metalloproteinases in stroke-prone spontaneously hypertensive rats （ SHR-SP）, and the mechanism by which effects on the pathological evolution of hypertensive renal fibrosis in rats. Methods Male SHR-SPs of 7 weeks old were randomly divided into vehicle-treated group and doxycycline-treated（30 mg/kg · d, po, n=20） group. Body weight, SBP, urine volume, urinary protein, urinary creatinine were continuously measured. After the study the haemodynamics analysis was performed. Plasma creatinine and urea nitrogen were measured, kidneys section were embedded for pathological analysis. The activity of MMP-2 and MMP-9 was determined by gelatin zymography and the activity of TIMP-1 and TIMP-2 by reverse gelatin zymography. Results Compared with control group, doxycycline caused an elevated tendency in SBP, PP and creatinine. Obvious difference of protain/creatinine between DOX group and control group from the age of 19 weeks （8. 3 ±6.4 vs control：3.34-2. 0 mg/μmol, P〈0. 05） was found. Both the kidney injury score and the collagen volume fraction of tubule were higher in DOX group than control group （Kidney injury score：DOX：3. 14±0. 47 vs control：2.69± 0.38, P〈0.05 ~;Collagen volume fraction：DOX ： 10.5 ± 2.7 % vs control： 6.6 ± 1.9 %, P〈0.01 ）. The activity of MMP-2 and MMP-9 was lower in the SHR-SPs received doxycycline（MMP-2：DOX：2. 62±0. 75 vs control：3.39 ± 0.92; MMP-9 ：DOX：2.89 ±1.13 vs control：4.01±0.91, P〈0.05）, while the activity of TIMP-1 and TIMP-2 in doxycycline-treated group was higher than those of vehicle-treated group （TIMP-1 ： DOX.- 2.89± 1.76 vs control：1. 54±0.86, P〈0.05;TIMP-2：DOX：1. 69±0. 47 vs control：1. 06±0.47, P〈0. 01）. Conclusion The present study showed that doxycycline inhibit MMP-2 and MMP-9 activities in the kidney of SHR-SPs, and accentuated the damage of renal structure and function suggesting inhibition of MMPs may lead to acceleration of interstitial fibrosis and ongoing renal function impairment.