Department of Nephrology, the First Affiliated Hospital, Sun Yat-Sen University, Guangzhou, China.
NHC Key Laboratory of Nephrology, Guangzhou, China.
Cell Death Dis. 2019 Mar 15;10(4):253. doi: 10.1038/s41419-019-1483-7.
G2/M-arrested proximal tubular epithelial cells (TECs) after renal injury are linked to increased cytokines production. ATG5-mediated autophagy in proximal TECs has recently been shown to protect against G2/M cell cycle arrest and renal fibrosis. However, the impacts of autophagy in regulating inflammatorily response mounted by injured TECs remains largely unknown. In the present study, we investigated whether ATG5 acts as an innate immune suppressor in proximal TECs during kidney injury. Using the unilateral ureteric obstruction model in proximal tubule-specific autophagy-deficient mice, we demonstrated that ablation of epithelial ATG5 genes markedly impaired autophagy, resulting in enhanced nuclear factor κB (NF-κB) activation, macrophage and lymphocyte infiltration, and proinflammatory cytokines production in obstructed kidneys, as compared with wild-type mice. Following stimulation with angiotensin II (Ang II), siRNA silencing of ATG5 in cultured HK-2 cells or ATG5-deficient primary proximal TECs produced more cytokines, including IL-1β, IL-6, and TNF-α than did their control cells. Overexpressed ATG5, but not the autophagy-incompetent ATG5 mutant K130R in HK-2 cells, rendered resistant to Ang II-induced inflammatory response. Immunofluorescence assay indicated that ATG5 and p65 colocalized in the nucleus and cytoplasm, and their interaction was verified in immunoprecipitation assay from HEK-293T cell extracts. Genetic downregulation of endogenous ATG5 increased Ang II-induced phosphorylation and nuclear translocation of p65 and transcriptional activity of NF-κB, whereas the overexpressed ATG5, rather than ATG5 mutant K130R, hampered activation of NF-κB signaling, suggest an autophagy-dependent anti-inflammatory effect of ATG5. Further, pharmacological manipulation of autophagy yielded similar results both in vivo and in vitro. Additionally, JSH-23, a specific inhibitor of NF-κB nuclear translocation, rescued Ang II-driven IL-1β production in ATG5 siRNA-treated cells and decreased the proportion of cells in G2/M phase. In conclusion, ATG5-mediated autophagy in tubules targets NF-κB signaling to protect against renal inflammation.
肾损伤后 G2/M 期阻滞的近端肾小管上皮细胞（TECs）与细胞因子产生增加有关。最近研究表明，近端 TECs 中的 ATG5 介导的自噬可防止 G2/M 细胞周期阻滞和肾纤维化。然而，自噬在调节受损 TECs 引发的炎症反应中的作用在很大程度上仍然未知。在本研究中，我们研究了 ATG5 在肾损伤期间是否作为近端 TECs 中的先天免疫抑制剂发挥作用。使用近端小管特异性自噬缺陷小鼠的单侧输尿管梗阻模型，我们证明上皮 ATG5 基因的缺失明显削弱了自噬，导致 NF-κB（NF-κB）激活、巨噬细胞和淋巴细胞浸润以及促炎细胞因子产生增强在梗阻肾脏中，与野生型小鼠相比。用血管紧张素 II（Ang II）刺激后，HK-2 细胞或 ATG5 缺陷型原代近端 TECs 的 siRNA 沉默 ATG5 比其对照细胞产生更多的细胞因子，包括 IL-1β、IL-6 和 TNF-α。过表达 ATG5，但不是自噬无能的 ATG5 K130R 突变体，使 HK-2 细胞抵抗 Ang II 诱导的炎症反应。免疫荧光分析表明，ATG5 和 p65 在核和细胞质中共定位，并在从 HEK-293T 细胞提取物进行的免疫沉淀测定中验证了它们的相互作用。内源性 ATG5 的遗传下调增加了 Ang II 诱导的 p65 磷酸化和核易位以及 NF-κB 的转录活性，而过表达的 ATG5，而不是 ATG5 K130R 突变体，阻碍了 NF-κB 信号通路的激活，表明 ATG5 具有依赖自噬的抗炎作用。此外，体内和体外的自噬药理学操作均产生了类似的结果。此外，NF-κB 核易位的特异性抑制剂 JSH-23 挽救了 ATG5 siRNA 处理细胞中 Ang II 驱动的 IL-1β 产生，并减少了处于 G2/M 期的细胞比例。总之，管腔中的 ATG5 介导的自噬靶向 NF-κB 信号通路以防止肾脏炎症。
