慢性间歇性缺氧通过调控线粒体自噬介导的NLRP3炎症小体活化加重肾小管上皮细胞焦亡

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中文摘要

阻塞性睡眠呼吸暂停（OSA）所致的肾损伤由慢性间歇性缺氧（CIH）触发。我们此前的文章已揭示CIH通过激活NLRP3炎症小体触发肾小管上皮细胞焦亡，而CIH激活NLRP3炎症小体的机制仍不明确。将大鼠和HK-2细胞暴露于CIH以建立CIH模型。采用组织病理学染色、血尿素氮（BUN）和血清肌酐（Scr）水平检测评估肾损伤。采用Western blot和免疫荧光实验评估与NLRP3炎症小体、焦亡和线粒体自噬相关的蛋白。检测线粒体膜电位以及ROS和ATP的产生以评估线粒体功能。采用CCK-8、LDH释放和ELISA实验评估细胞损伤和炎症。应用线粒体分裂抑制剂1（Mdivi-1）进行挽救实验。CIH在体内诱导肾损伤并抑制线粒体自噬，表现为病理特征恶化、Scr和BUN水平升高、Tom20与LC3B共定位减少，以及LC3-II/I比值、Atg5、PINK1和Parkin表达下调。PINK1过表达通过增强线粒体与LC3B的共定位、上调LC3-II/I比值和Atg5恢复线粒体自噬，并通过增强线粒体膜电位、减少ROS产生和增加ATP含量恢复CIH暴露HK-2细胞的线粒体功能。PINK1过表达减轻了CIH暴露HK-2细胞的细胞活力下降、炎症和NLRP3炎症小体介导的焦亡，而Mdivi-1部分消除了这些作用。PINK1部分通过恢复线粒体自噬和线粒体功能减轻CIH暴露HK-2细胞中NLRP3炎症小体介导的焦亡，为OSA相关肾脏疾病提供了潜在的治疗策略。

英文摘要

Obstructive sleep apnea (OSA)-induced renal injury is triggered by chronic intermittent hypoxia (CIH). Our previous article has revealed that CIH triggers the pyroptosis of renal tubular epithelial cells through the activation of NLRP3 inflammasome, whereas the mechanism by which CIH activates NLRP3 inflammasome remains elusive. Rats and HK-2 cells were exposed to CIH to establish CIH models. Histopathological staining, blood urea nitrogen (BUN) and serum creatinine (Scr) levels were examined to assess renal injury. Western blot and immunofluorescence assay were used to assess proteins related to NLRP3 inflammasome, pyroptosis and mitophagy. Mitochondrial membrane potential and production of ROS and ATP were examined to evaluate mitochondrial function. CCK-8, LDH release and ELISA assays were performed to assess cell injury and inflammation. Mitochondrial division inhibitor 1 (Mdivi-1) was applied for a rescue assay. CIH induced renal injury and suppressed mitophagy in vivo, evidenced by worsening pathological features, elevated Scr and BUN levels, reduced colocalization of Tom20 with LC3B, and downregulated expression of LC3-II/I ratio, Atg5, PINK1 and Parkin. PINK1 overexpression restored mitophagy by enhancing colocalization of mitochondria with LC3B, upregulating LC3-II/I ratio and Atg5, and restored mitochondrial function of CIH-exposed HK-2 cells through enhancing mitochondrial membrane potential, reducing ROS production and increasing ATP content. PINK1 overexpression alleviated cell viability, inflammation and NLRP3 inflammasome-mediated pyroptosis in CIH-exposed HK-2 cells, which were partly abolished by Mdivi-1. PINK1 attenuated NLRP3 inflammasome-mediated pyroptosis in CIH-exposed HK-2 cells partially through restoring mitophagy and mitochondrial function, providing potential therapeutic strategies for OSA-related kidney diseases.