整合素连接激酶在移植肾间质纤维化TGF-β1/Wnt途径交互作用的机制研究

Renal interstitial fibrosis and tubular atrophy are common in transplant kidneys, which severely affect long-term renal function and induce medical resource consumption. Wnt/β-catenin and TGF-β1 signaling pathway are involved in multiple EMT associated processes such as embryogenesis, fibrosis and tumor progression, where Integrin Linked Kinase (ILK) plays a vital role in the extensive cross-talk. ILK mediating the cross-talk between two pathways in the field of renal transplantation has not been reported before without clear results regarding the mechanisms. In this proposed research project we aim to clarify the key role of ILK in the cross- talk between the two pathways in three aspects: (1) Utilizing Chromatin Immunoprecipitation-The Next Generation Sequencing and Duolink Proximity Ligation Assay (PLA) to explore the interaction between ILK and key transcription factors. (2) Evaluating Living-donor (LD) and Donation after Cardiac Death (DCD) allografts in mice renal transplantation models. (3) Applying Lentivirus infection to interfere gene expression so as to evaluate the potential treatment target of ILK in allograft fibrosis.

间质纤维化病变及小管萎缩于移植肾中常见，并严重影响长期肾功能的维持，造成了医疗资源的严重消耗。整合素连接激酶(Integrin Linked Kinase, ILK)作为重要的交互位点，介导TGF-β1/Smad和Wnt/β-catenin途径协同作用于胚胎发育、纤维化和肿瘤进展等领域，但ILK促纤维化进程在移植肾领域尚未报道。本项目力求阐明ILK在慢性移植肾纤维化发生和进展中的关键作用。具体研究内容包括三个方面：(1)利用染色质免疫共沉淀-下一代测序联用和邻位连接等技术，明确ILK在慢性移植肾纤维化的作用机制以及与TGF-β1和Wnt/β-catenin途径转录因子的相互作用；(2)以活体或心脏死亡的ILK条件性敲除小鼠作为供肾来源，探讨不同类型供肾纤维化的异同；(3)利用慢病毒在体转染技术，介导野生型小鼠肾脏ILK表达沉默，评估ILK在移植肾进程性纤维化防治中的潜在应用价值。

整合素连接激酶（ILK）在移植肾中表达与移植时间和纤维化发展程度呈现正相关趋势。对ILK进行下调可TGF-β1/Smad和Wnt/β-catenin途径激活水平下降；使利用免疫共沉淀-质谱联用技术对移植肾进行我们发现Rel同源结构域（RHD）转录因子在TGF-β1/Smad和Wnt/β-catenin途径中均有参与，而ILK家族中较多成员受RHD调控，因此推测ILK活性和TGF-β1和Wnt途径激活存在着相互正反馈效应；对ILK进行抑制可使移植肾纤维化进程减慢，说明ILK可作为移植肾纤维化治疗潜在靶点进行进一步研究探讨。