TIMP-2通过与MT1-MMP相互作用激活ERK1/2-NF-κB信号通路参与慢性移植肾失功发病机制的研究

In our previous studies, it has been proved that ERK1/2-NF-κB signaling pathway is involved in the pathogenesis of inflammatory infiltration in the early stage of chronic renal allograft dysfunction (CRAD)，but what remained unclear is the up-stream signals to activate above signaling pathway, especially the extracellular signal molecules. In above studies, we also observed the up-regulated expression of tissue inhibitor of metalloproteinase-2 (TIMP-2) in the renal tissue of CRAD rat model and human transplant recipients. It is suggested by the results of our initial studies that TIMP-2 might activate ERK1/2-NF-κB signaling pathway via its interaction with membrane-type 1 matrix metalloproteinase (MT1-MMP) and play key roles in CRAD. This project is designed to prove that up-regulated TIMP-2 expression in CRAD kidney tissue is tightly correlated with the development of CRAD and confirm that TIMP-2 is a key extracellular molecule in activating ERK1/2-NF-κB signaling pathway by means of molecular cloning，RNAi and Co-IP techniques，and therefore to elucidate the mechanism for TIMP-2 to interact with MT1-MMP and activate ERK1/2-NF-κB signal pathway, providing new idea for the prevention and treatment in the early stage of CRAD.

我们前期证实ERK1/2-NF-κB信号通路参与慢性移植肾失功（CRAD）早期炎症细胞浸润发生机制，但对CRAD过程中ERK1/2-NF-κB信号通路激活的上游信号事件特别是胞外信号分子尚不清楚。我们发现临床CRAD患者和大鼠移植肾组织基质金属蛋白酶抑制剂-2（TIMP-2）表达上调，结合我们的预实验结果推测TIMP-2可能是通过与膜型基质金属蛋白酶（MT1-MMP）相互作用激活ERK1/2-NF-κB信号通路并进而在CRAD中发挥了关键作用。本项目拟利用分子克隆、RNAi和Co-IP等分子生物学技术在HK-2细胞和CRAD大鼠模型进一步证实CARD肾组织TIMP-2表达上调与CARD发生发展密切相关；证明TIMP-2是激活ERK1/2-NF-κB信号通路的关键胞外分子；探索TIMP-2与MT1-MMP相互作用并进而激活ERK1/2-NF-κB信号通路的机制，为CRAD早期防治提供新思路。

项目采用CRAD大鼠模型，应用TIMP2-shRNA慢病毒作为干预手段,在国内外率先在CRAD体内实验证实TIMP2表达上调激活ERK1/2-NF-κB信号通路，促进移植肾早期炎症细胞浸润、VSMCs移行，促进慢性移植肾IF/TA。在本项目的资助的研究中我们发现，CRAD肾组织中存在着TIMP2表达上调，干预实验证实其上调可以激活ERK1/2-NF-κB信号通路，进而介导移植肾早期炎症因子表达，进而促进炎症细胞浸润和VSMCs移行；而采用TIMP2-慢病毒干预后，ERK1/2-NF-κB信号通路活化得到抑制，结果移植肾炎症浸润得到改善，CRAD的进展得到一定程度控制；MT1-MMP转染HK2细胞实验显示，在转染了MT1-MMP的质粒后，蛋白印迹初步结果显示TIMP-2的表达上升、p-ERK1/2、pp-65、ICAM1的蛋白表达均上调，初步说明MT1-MMP的过表达可以上调ERK信号通路，继而激活NF-κB信号通路，导致CRAD早期炎症细胞浸润。本研究在国际上首次探讨了TIMP2参与慢性移植肾失功发病机制的具体机制及其信号通路，为未来临床应用TIMP2阻断剂治疗慢性移植肾失功提供了实验基础，有助于提高移植肾长期存活率。在执行本研究计划中我们共发表标注本基金的SCI论文4篇。