RIP3激酶/TFEB介导肾小管上皮细胞necroptosis自噬流障碍的机制

Unregulated necrosis in renal tubular epithelial cells is a therapeutic challenge in severe acute kidney injury. We observed recently that necroptosis, a novel proposed regulated necrosis, played a role in renal tubular cells. Activation of RIP3 and autophagy are symbolic characters of necroptosis. TFEB is a key transcription factor of autophagy. We found that TFEB inactivation mediated impaired autophagy promoted necroptosis. Because the TFEB inactivation is modulated by serine phosphorylation and RIP3 is a serine kinase, we speculated that RIP3 might block TFEB through kinase phosphorylation. We confirmed the interaction between RIP3 and TFEB using CoIP, and observed that RIP3 kinase inhibitor was able to activate TFEB. Based on these results above, this program proposes that RIP3 blocks TFEB activation through a kinase pathway. To confirm this hypothesis, we plan to explore the mechanism of interaction between the two molecules, using pull down, HPLC/MS, in vitro phosphorylation assay and point mutant technique. Finally, the therapeutic value of RIP3 kinase inhibitor will be testified in mRFP-GFP-LC3 transgene mouse. These works will facilitate to translate the RIP3 kinase inhibitor into clinical practice and drug development, and provide new ideas for therapy of severe acute kidney injury.

肾小管坏死不受调控是治疗重症急性肾损伤的难点。我们最近观察到肾小管necroptosis参与急性肾损伤。这是一种可被调控的细胞坏死，RIP3活化和自噬是其标志性特征。TFEB是自噬的关键转录因子，我们发现由它失活所介导的自噬障碍会促进necroptosis。TFEB失活受丝氨酸磷酸化调控，由于RIP3是丝氨酸激酶，我们推测RIP3可能会调控TFEB。我们用CoIP发现它们在necroptosis时相互结合，并观察到RIP3激酶抑制剂可活化TFEB，因此提出“RIP3可能通过激酶作用阻抑TFEB活化”。为了阐明这一推测，课题拟用pull down、液相质谱、磷酸化实验和点突变等技术来研究两者的作用，并利用mRFP-GFP-LC3转基因小鼠来验证抑制RIP3激酶对急性肾损伤的治疗价值。这些工作将为应用和研发RIP3激酶抑制剂提供理论基础，为治疗重症急性肾损伤提供新思路。

本项目通过体内外实验研究，探索了RIP3在内毒素血症AKI发病中的作用及具体机制，本研究主要从以下几方面进行研究：.1）发现在脓毒血症AKI患者近端肾小管上皮细胞中，RIP3表达上调，核内TFEB表达减少，同时观察到RIP3与TFEB存在共定位。.2）LPS腹腔注射建立小鼠内毒素血症AKI模型，利用免疫印迹法检测内毒素血症小鼠模型肾组织中RIP3 、p62、LC3蛋白表达上调。并利用LPS诱导的mRFP-GFP-LC3双荧光转基因小鼠，动态观察到内毒素血症AKI时RTECs自噬流障碍。通过免疫荧观察到LPS处理下核内TFEB水平明显下降。使用RIP3激酶抑制剂GSK’872预处理LPS诱导的内毒素血症AKI小鼠模型，可增加TFEB入核、减轻内毒素诱导的RTECs自噬流障碍，并改善肾脏功能。.3）在体外实验中，进一步验证了动物实验相关结果。应用RIP3激酶抑制剂或沉默RIP3，可增加LPS刺激的肾小管上皮细胞中TFEB表达水平明显增加，而过表达RIP3可下调肾小管上皮细胞中TFEB表达水平。进一步通过沉默或过表达细胞中TFEB，检测到沉默细胞中TFEB后，肾小管上皮细胞自噬相关蛋白P62及LC3表达水平明显增加，而过表达TFEB可下调肾小管上皮细胞中P62及LC3表达水平。此外，免疫共沉淀结果显示，LPS刺激下RIP3与TFEB存在结合。.4）同时发现了通过体内外实验证实RIP3介导了内毒素血症AKI肾小管上皮细胞凋亡。.5）同时验证了达拉菲尼、necrostatin-1作为RIP3激酶抑制剂或活化抑制剂对内毒素血症AKI的保护性作用。.上述结果显示内毒素血症时，肾小管上皮细胞RIP3表达水平上调，减少了TFEB入核，介导肾小管上皮细胞自噬潮障碍，引起内毒素血症AKI；而抑制RIP3激酶活性可增加TFEB入核，改善肾小管上皮细胞自噬潮，减轻内毒素血症AKI。该研究阐明了内毒素血症AKI新的发病机制，同时为内毒素血症AKI防治提供新的靶点。