Atg5介导肾小管上皮细胞自噬功能低下调控肾脏炎症反应的作用和机制

G2/M-arrested tubular epithelial cells (TECs) after injury showed increased release of inflammation and contributed to renal fibrosis. Our previous study demonstrated that Atg5 deficient proximal tubular epithelial cells enhanced cell cycle arrest at G2/M, and this Atg5-mediated effect was autophagy-dependent. Moreover, kidney tissue of Atg5-/- mice showed a significant increase in protein levels of inflammatory cytokines as compared with those in wild-type mice, suggesting autophagy deficiency aggravate renal inflammation. However, the mechanism(s) were unknown. In this proposal, to further study the relationship among TECs autophagy, inflammation and renal fibrosis, mice models of chronic kidney nephropathy was induced in littermate Atg5+/+ and Atg5-/- mice with or without rapamycin treatment. Primary TECs from Atg5+/+ and Atg5-/- mice, upregulating/downregulating/mutation Atg5 in HK-2 cells, and regulating cell cycle in HK-2 cells were used to investigate the effects of Atg5-mediated TECs autophagy in the release of inflammation, activation of NF-κB/NLRP3 signal pathways induced by Ang II, and explore whether autophagy play a role in degradation key molecules of NF-κB/NLRP3 signal pathways. Finally, we will prospectively investigate correlation between autophagy and pathology and clinical outcome in chronic kidney disease. Our study will provide a scientific basis and new target site for prevention and treatment of renal inflammation and fibrosis.

肾小管上皮细胞（TECs）受损后可停滞于G2/M期，释放炎症因子，促进肾脏纤维化。我们前期研究表明特异性近端TECs的Atg5基因失活可增加G2/M期停滞，且依赖于Atg5的自噬功能。此外，Atg5基因失活小鼠的肾脏炎症因子水平明显高于野生型小鼠，提示TECs自噬缺陷可加重炎症反应。但其机制尚不清楚。本项目拟利用上述小鼠及雷帕霉素增强自噬的慢性肾病模型，进一步证实TECs自噬、肾脏炎症反应与纤维化的关系；分别应用上述小鼠原代TECs，上调/沉默/变异HK-2细胞Atg5和药物改变细胞周期等方法，探讨Ang II刺激下炎症因子合成和NF-κB/NLRP3信号通路活化及协同效应，研究Atg5介导的TECs自噬靶向降解和抑制炎症信号通路的关键分子；前瞻性观察自噬与慢性肾脏病临床及病理的联系。本研究有助于揭示TECs自噬调控肾脏炎症反应，减缓纤维化的作用和机制，为临床防治肾脏纤维化提供科学依据。

肾脏的炎症反应不仅涉及免疫细胞的浸润活化，肾小管上皮细胞（TECs）也参与炎症反应的进展。我们既往的研究表明，Atg5介导的TECs自噬缺失，通过自噬依赖作用促进肾小管皮细胞G2/M期阻滞，加重小鼠UUO所致肾间质纤维化。但是，自噬是否通过调控肾脏炎症反应而发挥保护作用，尚不清楚。因此，本项目研究的主要内容包括：①利用特异性敲除TECs自噬相关基因Atg5小鼠，建立UUO模型，探讨Atg5介导的TECs自噬在调控肾脏纤维化过程中炎症反应的作用；②通过高表达、沉默和基因突变的技术，调节HK-2细胞株Atg5的表达水平，并提取小鼠原代TECs，应用Ang II刺激，探讨自噬通过调控NF-κB通路，抑制炎症反应的机制。. 我们的研究结果发现：①与Atg5+/+小鼠相比，Atg5-/-小鼠的肾间质损伤更为严重，炎症细胞（F4/80+巨噬细胞及CD3+T细胞）浸润增多，炎症因子（IL-1β、IL-6及TNF-α）mRNA及蛋白表达水平增加。此外，NF-κB信号通路活化，p-IκBα表达水平增多，伴随IκBα的降解，p-p65表达上调；敲除Atg5基因，促进NF-κB信号通路进一步活化。②在Ang II刺激下，HK-2细胞IL-1β蛋白表达水平呈时间依赖性逐渐增多；肾小管上皮细胞NF-κB通路活化，p-p65水平呈时间依赖性改变。抑制自噬上调Ang II介导的炎症因子（IL-1β、IL-6及TNF-α）mRNA水平以及IL-1β蛋白表达水平。高表达Atg5，可抑制Ang II诱导的上述炎症因子的水平；转染Atg5-K130R质粒，不能改变细胞的自噬水平。与空载体组相比，转染Atg5-K130R质粒细胞的上述炎症因子水平无明显改变。敲除 Atg5 基因下调自噬，p-IκBα蛋白表达水平增多，IκBα的降解，p-p65表达上调；Ang II促进p65蛋白向细胞核内转移，抑制自噬增加其核易位；应用JSH-23抑制NF-κB通路活化，LC3-II表达减少及p62的积聚。 . 本研究阐明了TECs自噬可通过抑制NF-κB信号通路活化，减轻肾脏炎症反应和炎症因子水平，从而抑制肾脏纤维化。