程序性坏死信号通路RIP1/RIP3/MLKL调控脓毒症急性肾损伤的机制研究

As the morbidity and mortality of septic AKI remains very high, it is of great significance to explore the mechanisms of AKI in sepsis. Inflammation triggered by LPS from gram-negative bacteria plays an important role in septic AKI, but the underlying mechanisms are still largely unknown. Macrophages activated by LPS accumulate in the renal interstitial compartment, promoting the production of proinflammatory cytokines and chemokines. As a consequence, increased cytokines, such as TNFα, IFN-γ, Tweak, IL-1β and IL-6, induce renal injury, which is a predominant pathogenic factor in early sepsis-related AKI. Therefore, the proinflammatory macrophages could largely account for the extent of tubular necrosis and thereby determine the extent of AKI. Evidences have already revealed that necrosis is a tightly regulated rather than an unregulated and uncontrollable process. In our previous study, we found that inhibition of core components of the necroptotic pathway, receptor-interacting protein 1 (RIP1), RIP3, or mixed lineage kinase domain-like protein (MLKL) by gene knockout or a chemical inhibitor diminished a combination of cytokines-induced necroptosis of renal proximal tubular cells (PTCs). In this study, we will establish cecal ligation and puncture (CLP) septic model using genetically modified mice to investigate the contribution of inflammation-induced necroptosis in sepsis-induced AKI. To explore the role of inflammation in triggering necroptosis of tubular cells, PTCs will be treated with a combination of cytokines or co-cultured with proinflammatory macrophages-activated by LPS. After that, cell survival, RIP1-RIP3-MLKL kinase activity and their subcellular localization will be detected. The function and interaction of these three proteins will be further evaluated during inflammation-induced necroptosis and necroinflammation. In summary, our study are aiming to delineate the molecular mechanisms underlying septic AKI. Inflammation triggers RIP1/RP3/MLKL-dependent necrotic cell death in renal tubules, which drives inflammation by forming an autoamplification loop, and eventually contributes to renal dysfunction. Our findings in future will provide evidences that blockade of RIP1/RIP3/MLKL signaling represents a promising strategy for clinical therapy of septic-AKI.

脓毒症相关的急性肾损伤 (acute kidney injury，AKI) 的发病率和病死率仍居高不下，探究脓毒症-AKI隐藏的机制具有重要的临床意义。脓毒症中，细菌脂多糖（LPS）诱发的肾内炎症反应引起肾小管坏死是脓毒症-AKI的重要原因，但其调控机制尚不清楚。近年研究证实许多细胞坏死并不是不可调控的。我们既往研究发现炎症因子联合诱导的近端肾小管上皮细胞坏死是受程序性坏死核心信号通路RIP1/RIP3/MLKL所调控的。因此，本研究拟在不同基因型小鼠建立盲肠结扎穿刺术引起的脓毒症-AKI模型，模拟脓毒症体内炎症反应环境、采用LPS诱导活化的巨噬细胞和炎症因子诱导近端肾小管上皮细胞坏死，探究RIP1/RIP3/MLKL通过分子间的相互作和激酶活性调控细胞程序性坏死及坏死性炎症反应、从而导致AKI进展的机制，阐明干预RIP1/RIP3/MLKL信号通路是抑制脓毒症-AKI进展的重要手段。

急性肾损伤(acute kidney injury，AKI)是ICU脓毒症患者最常见的并发症，也是重症患者最主要的引起AKI的原因，目前脓毒症相关的AKI发生率和死亡率仍然居高不下，临床的干预治疗手段仍然有限。脓毒症肾损伤的病理生理机制十分复杂，包含了肾内血流动力学改变引起的肾脏缺血、肾内毛细血管内皮细胞功能障碍、炎症细胞浸润、大量炎症因子的释放、进而导致肾小管损伤、AKI发生发展，然而肾小管损伤在脓毒症AKI进展中所隐藏的细胞分子调控机制尚不明。LPS刺激下的具有促炎症表型的巨噬细胞和其分泌的炎症因子是如何诱导肾小管上皮细胞坏死、从而促进脓毒症-AKI的进展机制尚不清楚，因此研究脓毒症中调控肾小管上皮细胞坏死的机制对干预脓毒症-AKI的进展具有重要的意义。. 通过建立盲肠结扎穿刺术引起的脓毒症-AKI小鼠模型，采用RIP3/MLKL/GSDMD单基因敲除鼠、RIP3/GSDMD和MLKL/GSDMD双基因敲除鼠，明确程序性坏死核心信号通路RIP3/MLKL与细胞焦亡执行蛋白GSDMD在脓毒症-AKI中的作用及其与近端肾小管上皮细胞坏死和肾内炎症反应之间的关系。 通过原代分离骨髓源性巨噬细胞，模拟体内脓毒症炎症反应微环境，观察RIP3/MLKL和GSDMD如何协同调控炎症反应，从而导致脓毒症AKI进展的机制。通过原代内皮细胞，模拟体内脓毒症炎症反应微环境，观察RIP3/MLKL和GSDMD如何协同调控内皮细胞功能，从而使得炎症细胞迁移到肾组织导致脓毒症AKI进展的机制。 通过原代内皮细胞，模拟体内脓毒症炎症反应微环境（LPS及缺血缺氧环境），观察对肾小管上皮细胞坏死的影响。. 本研究阐明了脓毒症中炎症反应所导致的近端肾小管上皮细程序性坏死依赖于RIP3/MLKL的相互作用、RIP3的激酶活性、MLKL的多聚化和向细胞膜转位。我还发现双基因敲除RIP3/GSDMD或者MLKL/GSDMD（即同时抑制Necroptosis和Pyroptosis能更有效抑制脓毒症休克和肾损伤。在细胞水平上，我们模拟脓毒症微环境采用不同刺激物干预巨噬细胞、内皮细胞、肾小管上皮细胞，细胞的死亡和坏死性炎症反应能被双基因敲除RIP3/GSDMD或者MLKLMLKL/GSDMD能更有效抑制。. 因此，同时阻断Necroptosis和Pyroptosis能更有效保护脓毒症肾损伤。