NLRP3炎症体与线粒体自噬cross-talk在对比剂所致急性肾损伤中的作用

Following the wide application of iodinated contrast media in disease diagnosis and treatment, the incidence of contrast-induced acute kidney injury (CI-AKI) markedly increases in both ambulatory and hospitalized patients. However, the underlying mechanism of CI-AKI is not fully understood. Our previous study implicated the NLRP3/caspase-1/IL-1β axis contributed to the pathogenesis of CI-AKI. Recent studies demonstrated that mitophagy, the autophagy process of damaged mitochondria, was a key mechanism in regulating the activation of NLRP3 inflammsome. Meanwhile, NLRP3 also blocked PINK1-mediated mitophagy. Besides, several studies have identified the protective role of mitophagy in kidney diseases, including ischemia-reperfusion induced acute kidney injury, diabetic kidney disease, and focal segment glomerular sclerosis, ect. NLRP3 inflammasome and mitophagy cross-talk in the pathogenesis of CI-AKI is remained to explore. .Our current study aims to investigate NLRP3 inflammasome and mitophagy cross-talk in the pathogenesis of CI-AKI in vitro and in vivo model of CI-AKI. The first part of the study was to suppress NLRP3 inflammasome by slicencing NLRP3 or Casp1, to identify the blocking effect of NLRP3 inflammasome on mitophagy in CI-AKI. The next step was to investigate whether altering the extent of mitophagy affect the activation of NLRP3 inflammasome and kidney injury in the process of CI-AKI by slicencing PINK1, BNIP3, NIX or by 3-MA, the autophagy inhibitor, or by enhancing mitophagy with rapamycin, the autophagy agonist. The third part was to evaluate the co-effect of supressing NLRP3 inflammasome and upregulating mitophagy in the pathogenesis of CI-AKI by overexpressing PINK1, BNIP3, or NIX in NLRP3-/- mouse..The evidence of the study will give us a deeper understanding of the role of NLRP3 inflammasome and mitophagy cross-talk in the pathogenesis of CI-AKI, which will facilitate us in identifying novel therapeutic strategies to preserve the structural integrity and function of the kidney after the administration of iodinated contrast media.

碘对比剂所致急性肾损伤（CI-AKI）是住院患者新发急性肾损伤的第三大原因。申请人前期研究发现，NLRP3/caspase-1/IL-1β炎症体通路激活参与CI-AKI发病机制。近期研究发现，线粒体自噬通过清除受损线粒体抑制NLRP3炎症体；而NLRP3炎症体激活后可抑制线粒体自噬的发生。NLRP3炎症体与线粒体自噬cross-talk在CI-AKI中的作用尚未明确。.本研究拟使用碘海醇刺激小鼠原代肾小管上皮细胞或诱发小鼠AKI建立CI-AKI实验模型，通过抑制NLRP3炎症体，探讨其对线粒体自噬及细胞损伤、肾损伤的影响；通过多种途径抑制或促进线粒体自噬，分析其对NLRP3炎症体和细胞损伤、肾损伤的影响；同时抑制NLRP3炎症体并促进线粒体自噬，进一步明确NLRP3炎症体与线粒体自噬cross-talk在CI-AKI中的作用。本研究可为干预CI-AKI发病及转归提供新的理论依据。

【背景】含碘对比剂在临床疾病诊断和治疗中广泛应用导致对比剂所致急性肾损伤（CI-AKI）发病率逐年升高，已成为住院患者新发急性肾损伤的第三大原因，其发病机制不完全明确，因此缺乏有效治疗手段，导致预后不良。进一步明确CI-AKI发病机制及发现潜在治疗靶点至关重要。线粒体自噬是特异性吞噬并降解受损线粒体的损伤后修复机制，课题组前期研究证实NLRP3炎症体活化加重CI-AKI，但是线粒体自噬在CI-AKI中的作用以及NLRP3炎症体是否与线粒体自噬存在cross-talk仍然未知。故本课题探讨：1. 线粒体自噬在对比剂所致急性肾损伤中的作用和机理；2. 线粒体自噬是否参与调控CI-AKI中的NLRP3炎症体活化及具体信号通路；3. NLRP3炎症体是否调控线粒体自噬水平及其在CI-AKI转归中的作用。.【主要研究内容】实验通过尾静脉注射碘海醇诱导CI-AKI小鼠模型及碘海醇干预HK-2细胞模型探讨NLRP3炎症体与线粒体自噬cross-talk在CI-AKI中的作用。通过Western blot、免疫荧光染色、TUNEL染色、流式细胞术、电镜等手段，评估线粒体自噬、mtROS、NLRP3炎症体、细胞凋亡通路的调控作用。.【重要结果及关键数据】对比剂上调线粒体自噬水平，并促进NLRP3炎症体活化。抑制PINK1-PARK2介导的线粒体自噬促进NLRP3炎症体活化及肾脏损伤。MitoTEMPO通过清除mtROS减少NLRP3炎症体活化。抑制NLRP3炎症体促进HIF1A-BNIP3介导的线粒体自噬。HIF脯氨酰羟化酶抑制剂罗沙司他通过稳定HIF1A促进BNIP3介导的线粒体自噬。BNIP3介导线粒体自噬减轻对比剂所致线粒体损伤及细胞凋亡。.【结论及科学意义】1. 对比剂通过释放mtROS激活NLRP3炎症体，加重肾脏损伤。2. 线粒体自噬水平在CI-AKI肾小管上皮细胞中保护性升高。3. PINK1-PARK2介导线粒体自噬通过清除mtROS释放，抑制NLRP3炎症体及细胞凋亡。4. 抑制NLRP3炎症体同时上调HIF1A-BNIP3介导线粒体自噬，减轻肾小管上皮细胞凋亡。5. CIAKI中NLRP3炎症体与线粒体自噬存在cross-talk，促进线粒体自噬、线粒体靶向抗氧化、抑制NLRP3炎症体、稳定HIF1A表达可能成为预防和改善CI-AKI预后的重要靶点。