深低温停循环致急性肾损伤过程中miRNA对细胞自噬的调节机制

Deep hypothermic circulatory arrest (DHCA) induced acute kidney injury (AKI) is one of the major complications after aortic surgery. The pathogenesis of AKI remains unknown. Possible molecular mechanism is still under investigation. Our previous work demonstrated association between DHCA induced AKI and endoplasmic reticulum stress (ERS). This process is partially regulated by miR-205. It is known that autophagy is one important link between ERS to apoptosis in renal tubular epithelial cells, we postulated that miRNAs may participate in regulation of cell autophagy and thereby influence the process of DHCA induced AKI. First of all, we plan to observe the difference of the protein biomarker of autophagy body and ERS before and after cell autophagy is inhibited. The observation will be done under different level of hypothermia and hypoxemia. Through results derived from this observation, we hope to elucidate the relation of ERS and cell autophagy and the effect of cell autophagy on cell apoptosis. Secondly, on the cellular level, we will search for key regulatory miRNAs which control ERS, cell autophagy and apoptosis in the condition of different level of hypothermia and hypoxemia. Finally, in the animal model, we will verify the regulation effect of the target miRNA on cell autophagy of renal tubular epithelial cells to further investigate role of DHCA induced AKI. Through the purposed study, we try to provide a new insight into the mechanism of DHCA induced AKI in cardiovascular surgery and find new targets for the prevention and treatment of the AKI.

深低温停循环后急性肾损伤（AKI）是主动脉弓等心血管手术的严重并发症，其发生机制的基础研究匮乏，分子水平调控机制不明。我们前期研究发现深低温停循环后AKI与内质网应激相关，miR-205参与了调控。由于细胞自噬是肾小管上皮细胞内质网应激到细胞凋亡过程中的必经环节，所以我们推测miRNAs可能通过对细胞自噬的调控，影响深低温停循环后AKI过程。本项目拟先在细胞水平，通过观察细胞自噬被抑制前后，内质网应激标志蛋白和自噬体在不同低温缺氧状态下的形成情况，明确该过程中内质网应激与细胞自噬的关系和对细胞凋亡的影响；然后在细胞水平进一步寻找、筛选，发现在低温缺氧条件下调控内质网应激、细胞自噬和细胞凋亡的关键miRNAs；最后在动物水平，验证目标miRNA对细胞自噬的调控和在术后AKI过程中的作用。本研究将为阐明深低温停循环后AKI发生机制提供新思路，为AKI的防治找到新靶点。

深低温停循环过程中的全身停循环和降温、复温过程是引起心脏手术术后急性肾损伤（AKI）的重要原因，AKI仍然是影响此类重大手术临床疗效的重要制约因素。细胞自噬在肾小管上皮细胞损伤中扮演着重要的角色，但其作用和调控机制仍未阐明。细胞自噬产生的物质和能量用于细胞更新和稳态维持，在细胞代谢适应、胞内质量控制、细胞更新乃至疾病发生发展中发挥重要作用。细胞自噬功能障碍可以引起细胞能量代谢功能障碍、触发细胞凋亡甚至坏死的发生。我们在构建的人肾小管上皮细胞和GFP-LC3以及GFP-LC3+mCherry-siBeclin 1稳转细胞系上通过多种手段平行检测了细胞内质网应激、自噬和凋亡，以此提取RNA建立了microRNA 片段文库，进行了microRNA生信分析，随后在大鼠体外循环动物模型上进行了动物实验。以期探明细胞自噬在肾小管上皮细胞应激反应过程中的作用，初步探索其调控机制。结果发现：1、低温暴露和复温可以诱导肾小管上皮细胞温度相关性的内质网应激、细胞自噬和进行性的细胞凋亡；2、自噬抑制剂3-MA和Beclin 1基因沉默都能明显抑制肾小管上皮细胞低温暴露和复温诱导的自噬，促进细胞凋亡；3、microRNA可能通过信号通路参与了对肾小管上皮细胞自噬的调节作用。研究结果提示，自噬在肾小管上皮细胞中深低温暴露中发挥保护作用。本研究有望阐明microRNA调控自噬信号通路基因表达，参与肾小管上皮细胞自噬调节的信号转导机制，为心脏体外循环围术期肾保护保护提供新的思路和治疗靶点。