中性粒细胞明胶酶相关脂质运载蛋白在缺血再灌注急性肾损伤中对自噬的调控作用和机制研究

Acute kidney injury (AKI) is a common clinical complication. Its pathogenesis is unclear, and the treatments of AKI are limited. The role of neutrophil gelatinase-associated lipocalin (NGAL) as a biomarker of human AKI has been established through a variety of clinical trials. But the role and mechanism of NGAL in renal ischemia-reperfusion injury (IRI) were unknown. Our previous study found that a certain concentration of NGAL acted as a protective factor against hypoxia-reoxygenation injury by regulating apoptosis-related genes. While NGAL increased to a certain extent, its anti-apoptotic protective effect weakened. We also found autophagy involved in IRI, and the intensity of autophagy in reperfusion stage was higher than ischemia stage, which was positively correlated with NGAL level. This study intends to establish renal IRI cell and animal models, to explore the function and regulation mechanism of autophagy in different stages of IRI, and to clarify the role and targets of NGAL in this process. It will provide new idea for clinical effective treatment. Meanwhile, we focus on the comprehensive judgement and evaluation of early biomarkers of AKI, to improve the clinical diagnosis ability ,and to provide basis on the determination and establishement of new diagnostic criteria and definition for AKI.

急性肾损伤（AKI）是临床上常见的并发症，发病机制不明确，治疗手段有限。临床试验证实AKI早期中性粒细胞明胶酶相关脂质运载蛋白（NGAL）水平升高，但其升高对肾缺血再灌注损伤（IRI）的作用和机制不详。前期研究发现一定浓度的NGAL通过调节凋亡相关基因的表达发挥对肾小管上皮细胞的保护作用，但NGAL水平升高到一定程度后其抗凋亡的保护作用相对减弱，同时发现自噬参与了IRI，且再灌注阶段自噬的强度高于缺血阶段，与NGAL水平正相关。本项目拟制备肾IRI细胞模型和动物模型，深入研究自噬在肾脏IRI不同阶段中的作用和调控机制，明确NGAL在其中发挥的作用和靶点，为临床治疗效果提供新思路。此研究拟对AKI的早期标志物进行综合判断和评价，提高临床对AKI的诊断能力，为AKI新定义和诊断标准的制定提供依据。

急性肾损伤（AKI）是临床上常见的严重并发症，肾脏缺血/再灌注（I/R）损伤是导致AKI的主要原因之一。中性粒细胞明胶酶相关脂质运载蛋白（NGAL）是AKI的重要标志物，但其在I/R损伤中发挥的作用及机制尚缺乏系统研究。本课题重点探究NGAL表达水平与肾脏I/R损伤过程中自噬状态和损伤程度的关系，以明确NGAL能否对肾脏I/R损伤发挥保护作用，并探讨相关机制。本研究以人肾小管上皮细胞（HK-2）为研究对象构建I/R损伤的细胞模型，明确了在缺氧1h并复氧24h后，NGAL水平增加，自噬水平增强。通过转染NGAL高表达质粒、敲减质粒，加入NGAL蛋白处理，证实NGAL在HK-2细胞缺氧/复氧过程中起保护作用，NGAL高表达后细胞中自噬相关蛋白表达增高，敲减后则降低，明确NGAL可诱发自噬。构建I/R损伤大鼠模型，明确大鼠血清和尿液中NGAL可作为良好的AKI早期诊断的标志物，同时自噬相关蛋白表达上升。应用3-MA抑制自噬后，大鼠肾脏自噬减轻而肾损伤加重，应用自噬诱导剂则自噬增强、肾损伤减轻。肾脏缺血前、后分别应用外源性NGAL蛋白后，自噬增加，凋亡减少，肾损伤改善，且缺血前应用效果更为显著。对NGAL调控自噬的分子机制研究发现NGAL敲减后p-AMPK 、p-TSC2水平明显降低，p-mTOR、p-P70s6k水平升高，提示NGAL通过增强AMPK和TSC2磷酸化，抑制mTOR及其下游底物P70s6k的磷酸化，诱导缺氧/复氧后自噬发生。. 通过对肾移植术后不同时间点患者尿液标本检测分析，发现NGAL有助于移植肾功能延迟恢复（DGF）的诊断。联合检测NGAL、IL-18和血清肌酐可使DGF诊断的阳性预测值达到90.9%，阴性预测值达到100%，明显优于其他联合方案。. 综上，体外细胞和动物实验均证实I/R损伤过程中，自噬处于激活状态，NGAL可通过AMPK-TSC2-mTOR-P70S6K通路诱发自噬，发挥保护作用，提示NGAL有望成为预防或治疗肾脏I/R损伤的一种新的治疗思路。通过多指标的综合判断和评价，提高了临床对AKI、DGF的诊断能力，为AKI新定义和诊断标准的制定提供依据。