NEK7在慢性肾脏病小管间质纤维化中的作用及机制研究

Renal fibrosis is the common pathological features of various chronic renal diseases, which contributes to the progression of CKD to end-stage renal disease. NEK7, a member of NIMA-related kinase (Nek) family, plays an important role in the process of cell proliferation. As a recently identified possible component of NLRP3 inflammasome, NEK7 is involved in the activation of NLRP3 inflammasome. However, the role of NEK7 in renal disease is unclear. Analysis from an online biological database revealed that NEK7 was higher in the kidneys of CKD patients. Our preliminary study also showed that the expression of NEK7 and NLRP3 were all upregulated in UUO mouse model and TGF-β1-treated renal tubular epithelial cells and renal interstitial fibroblasts. Moreover, the expression of NLRP3 and the extracellular matrix level were decreased when NEK7 was inhibited by a siRNA approach, indicating a potential role of NEK7 in activating the NLRP3 inflammasome and promoting the fibrotic response in kidney. Bioinformatic analysis indicated that there were nine binding sites of the transcription factor ASH1 on the promoter region of NEK7. Furthermore, ASH1 was induced by TGF-β1 in RTECs and silencing ASH1 reduced NEK7 expression. Therefore, we hypothesized that ASH1-regulated NEK7 could contribute to the pathogenesis of renal tubulointerstitial fibrosis through activating NLRP3 inflammasome and/or modulating cell cycle progression and cell proliferation. In the current proposal, we will employ CKD patients’ samples and models of animals, cells, and molecules to define the role of NEK7 in modulating renal tubulointerstitial fibrosis, as well as the underlying mechanisms, which could bring the new insights into the understanding and intervention of CKD.

肾脏纤维化是多种慢性肾脏疾病（CKD）进展到终末期肾病的共同病理基础。NEK7属于NEK蛋白激酶家族，在细胞增殖过程中起重要作用。近期研究又显示NEK7可能是NLRP3炎症小体的新组分，参与炎症小体活化，但NEK7在肾脏疾病中的作用尚无研究。在线生物数据库分析发现NEK7在CKD患者肾脏高表达。预实验也显示在UUO模型及TGF-β1刺激的肾小管上皮细胞及肾间质成纤维细胞，NEK7与NLRP3同时上调。干扰NEK7可下调NLRP3并减少细胞外基质。生物信息学分析显示转录因子ASH1与NEK7启动子区有9个结合位点，且TGF-β1可上调ASH1，而敲低ASH1可下调NEK7。由此推测：转录因子ASH1启动NEK7表达，进而通过调控NLRP3炎症小体和/或细胞增殖介导肾小管间质纤维化。本课题将在整体、细胞和分子层面探讨NEK7调控肾小管间质纤维化的作用及机制，为防治CKD提供新思路。

慢性肾脏病（CKD）是指各种原因引起的持续至少3个月的肾脏损害或肾功能下降，严重危害人类健康。缺血再灌注损伤引起的急性肾损伤（AKI）是导致CKD的重要原因之一。肾小管间质纤维化是许多CKD进展为终末期肾病的共同途径。肾小管上皮损伤及其诱发的一系列生化反应是启动和推进肾小管间质纤维化的主要因素。而肾小管上皮细胞不仅是损伤刺激的靶细胞，也是肾脏炎性反应的参与者。因此，阻断炎症介质的产生是减轻肾小管上皮细胞损伤、抑制肾脏成纤维细胞活化的重要环节。NEK7属于NIMA相关激酶家族，主富集于细胞中心体区域，调控纺锤体的正确组装及有丝分裂进程，在细胞增殖过程中起到重要作用。近年研究报道显示，NEK7也是NLRP3炎症小体活化必不可少的组成成分，但目前尚无NEK7在肾脏中的相关报道。.本研究应用肾小管上皮细胞条件性NEK7敲除鼠、尾静脉高压注射过表达NEK7，及在肾小管上皮细胞中干预NEK7探讨其在缺血再灌注（IR）及缺氧/复氧和TGF-β1诱导的CKD模型中的作用及机制。结果显示，NEK7在CKD患儿、IR诱导的小鼠肾组织及TGF-β1诱导的肾小管上皮细胞中表达上调。敲除NEK7可改善IR诱导的肾功能下降，肾脏炎症及纤维化反应。反之，体内过表达NEK7，可加重这一反应。体外结果显示，肾小管上皮细胞干预NEK7可获得与体内一致的结论。此外，单侧缺血再灌注损伤后，肾组织中NLRP3炎症小体激活。而敲除或干涉NEK7可显著抑制NLRP3炎症小体的活化，反之亦然。进一步利用NEK7 cKO小鼠构建IR模型，同时给予NLRP3抑制剂MCC950处理，发现抑制NLRP3并没有进一步减轻炎症和纤维化反应。而过表达NEK7后构建IR模型再给予MCC950处理后，则发现NEK7可显著加重肾脏纤维化，而抑制NLRP3则可减轻上述反应。免疫共沉淀结果也显示HK2细胞中NEK7-NLRP3存在相互作用。由此可知，NEK7可通过激活NLRP3炎症小体加重IR诱导的肾间质纤维化。