RIP3/MLKL/GSDMD程序性调控坏死与炎症正反馈环路反应在急性肾损伤慢性化中的作用机制研究

Acute kidney injury (AKI) confers a greater risk of progression to chronic kidney disease (CKD). Renal tubular necrosis and inflammation plays a crucial role in the progression of AKI to CKD, but the underlying mechanisms are still not clear. 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 ischemia/reperfusion (IR), cisplatin and a combination of cytokines-induced necroptosis of renal proximal tubular cells (PTCs). The dying PTCs released danger associated molecular patterns (DAMPs) and induced NLRP3 inflammasome activation and Gasdermin D (GSDMD) cleavage, resulting in macrophage pyroptosis to produce more inflammatory cytokines, which in turn triggered more necrosis to enhance inflammation. This positive feedback loop of necrosis and inflammation in AKI, termed as necroinflammation, may eventually lead to chronic renal failure. In this study, we will establish AKI-CKD model in RIP3/GSDMD-dKO and MLKL/GSDMD-dKO mice to investigate the contribution of cross-talk between PTC necroptosis and macrophage pyroptosis in the progression of AKI to CKD. Furthermore, we will do clinical observational studies in AKI patients with histologic evidence of tubular necrotosis to assess the role of RIP3/MLKL/GSDMD in prediction and evaluation of post-AKI renal function recovery on long-term renal outcomes. Our findings in future will provide evidences that blockade of the cross-talk between PTC necroptosis and macrophage pyroptosis by inhibiting RIP3/MLKL/GSDMD signaling represents a promising strategy for clinical therapy of AKI progression to CKD.

目前急性肾损伤(AKI)仍有相当比例的患者进展为慢性肾脏病，肾小管坏死及炎症细胞浸润是驱使AKI向慢性化进展的重要病理改变，但其调控机制尚不清楚。我们近期研究发现，缺血再灌注、顺铂及炎症因子诱导的近端肾小管上皮细胞坏死受程序性坏死（Necroptosis）核心信号通路RIP1/RIP3/MLKL有序调控，而其坏死产物能激活巨噬细胞炎症小体、剪切GSDMD、导致巨噬细胞焦亡（Pyroptosis）、促进炎症反应、并进一步加重肾小管坏死，形成坏死与炎症正反馈恶性循环反应。本研究拟采用双基因敲除RIP3/GSDMD和MLKL/GSDMD、以阻断肾小管上皮细胞Necroptosis及其激活的巨噬细胞Pyroptosis，探究RIP3/MLKL/GSDMD程序性调控肾小管坏死与巨噬细胞炎症反应间的Cross-talk在AKI向慢性化进展中的作用机制，探索临床预测和评估AKI慢性化的重要生物学标志物。

通过建立缺血再灌注肾损伤（IRI）诱导的AKI慢性化模型，采用RIP3-KO、MLKL-KO单基因敲除小鼠、RIP3/MLKL-dKO双基因敲除小鼠及骨髓移植实验，明确在AKI中首先启动肾小管程序性坏死（Necroptosis）核心信号通路RIP3/MLKL、进而激活巨噬细胞焦亡（Pyroptosis）信号通路Nlrp3/ASC/Caspase1/GSDMD、最后肾小管上皮细胞的Necroptosis及巨噬细胞的Pyroptosis 之间的Cross-talk促进AKI慢性化的过程。 我们还建立盲肠结扎穿刺术引起的脓毒症-AKI模型，由于单纯基因敲除RIP3或MLKL不能完全抑制脓毒症休克和AKI，因此我们还同时敲除了细胞焦亡的关键蛋白GSDMD，以探究双重阻断程序性坏死（Necroptosis）和细胞焦亡（Pyroptosis）能否更好保护脓毒症AKI。

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