HIF-miR21在急性缺血性肾损伤发病中的作用及防治新靶点的研究

Acute kidney injury (AKI) is a common clinical critical disease, the main cause of which is renal ischemia/hypoxia. Uncontrolled immune-inflammatory response is an important mechanism in the progression of ischemic AKI. The core regulatory factor of hypoxic response is hypoxia inducible factor (HIF) and important hypoxia-related miRNA miR-21 is possibly regulated by HIF. Both of them were confirmed to participate in immune-inflammatory response. On the basis of our previous studies of the mechanism of ischemic kidney injury, we found that short period of ischemic preconditioning ameliorated kidney injury and immune-inflammatory responses after the subsequent prolonged renal ishcemia, which accompanied with activation of HIF-miR21 system. Therefore, it is speculated that this signaling system may be involved in the regulation of immune-inflammatory response during ischemic AKI. Building upon the mutual regulatory mechanism between HIF and miR-21, we want to further clear the role of HIF-miR21-target gene signaling pathway in the modulation of the immune-inflammatory responses and the interaction between tubular epithelial cells and dendritic cells in the process of ischemic AKI through gene modification methods, as well as the estiblishment of molecular, cellular and animal models. Finally, we try to search new technologies to intervene AKI targeted as miR-21 in order to provide new ideas and new strategies for the early diagnosis and prevention of AKI clinically.

急性肾损伤（AKI）是临床常见危重症，缺血/缺氧是AKI的主要致病因素，免疫炎症反应失控则是推动缺血性AKI发生发展的重要机制。低氧反应调节的核心低氧诱导因子（HIF）及其可能调控的重要低氧相关miRNA miR-21均被证实参与免疫炎症反应过程。我们前期工作发现短时间缺血预处理可以减轻长时间肾脏缺血所致的肾损伤与免疫炎症反应，并活化HIF-miR21信号系统，推测该信号系统可能参与调控缺血性肾损伤的免疫炎症反应。本项目拟在阐明肾脏缺血/缺氧时HIF与miR-21相互调控机制的基础上，分别从分子、细胞、动物模型三个层面，采用基因修饰等方法，以HIF-miR21-靶基因为主线，研究缺血性AKI过程中免疫炎症的信号机制及肾小管上皮细胞与树突状细胞的交互作用，并进一步以miR-21为靶点寻找干预AKI的新技术，从而为AKI临床早期诊断和防治提供新思路和新策略。

急性肾损伤（AKI）是临床常见危重症，缺血/缺氧是AKI的主要致病因素，免疫炎症反应失控则是推动缺血性AKI发生发展的重要机制。低氧反应调节的核心低氧诱导因子（HIF）及其可能调控的重要低氧相关miRNA miR-21均被证实参与免疫炎症反应过程。本项目拟在阐明肾脏缺血/缺氧时HIF与miR-21相互调控机制的基础上，分别从分子、细胞、动物模型三个层面，采用基因修饰等方法，以HIF-miR21-靶基因为主线，研究缺血性AKI过程中免疫炎症的信号机制及肾小管上皮细胞与树突状细胞（DC）的交互作用。研究中首先我们通过荧光素酶报告基因检测和定点突变实验发现，低氧培养条件下，肾小管上皮细胞HIF-1α可以增强miR-21的活性，再通过染色质免疫沉淀实验，证实HIF-1α可以与miR-21结合，并且证实HIF-1α或HIF-2α对缺氧情况下miR-21的调控作用。此外，我们通过体内外研究明确miR-21通过PTEN/AKT/mTOR信号途径间接上调HIF-1α。我们观察到低氧抑制DC成熟并促进凋亡，与低氧上调miR-21和抑制其靶基因CCR7有关。低氧条件下，与肾小管上皮细胞共培养可影响DC成熟和免疫活性，与低氧持续时间有关，进一步我们研究证实共培养的肾小管上皮细胞可能通过上调miR-21抑制CCR7表达，从而减少DC的凋亡、成熟和迁移，与miR-21/PDCD4/NF-κB通路无关。低氧诱导的DC中miR-21对共培养肾小管上皮细胞内PDCD4-NFκB通路具有一定的调控作用，对肾小管上皮细胞CCR7表达无影响。在前面研究的基础上，我们以miR-21为靶点寻找干预AKI的新技术。首先我们发现晚期缺血预适应诱导的miR-21通过抑制PDCD4-NFκB通路和CCR7的表达，减少缺血再灌注（I/R）后肾脏DC的成熟分化，抑制免疫炎症反应。此外，肾脏或远端肢体预缺血可通过分泌含miR-21的外泌体，抑制PDCD4-NFκB通路，减少肾脏的炎症反应，减轻脓毒症肾损伤。同时，我们输注miR-21 mimic修饰DC对缺血性AKI具有保护作用，可能与miR-21抑制PDCD4-NFκB通路，减轻肾脏I/R后炎症反应有关。本项研究不仅有望在AKI发病机制的理论方面获得创新和突破，并有可能为AKI临床早期诊断和防治提供新思路和新策略。