功能化肾靶向MSCs胞外囊泡通过保护线粒体治疗AKI-CKD转归的作用及机制研究

Mitochondrial dysfunction plays a key role in the acute kidney injury (AKI) to chronic kidney disease (CKD) transition (AKI-CKD Transition). Mesenchymal stem cells (MSCs)-derived extracellular vesicles (EVs) exert multiple biological properties, such as transport of exogenous bioactive substances, inhibition of inflammatory and fibrotic injuries. In this study, MSC-EVs are re-engineered to generate multi-function bionanomaterials that can target renal lesions to deliver mitochondria-target antioxidants and MRI imaging at the same time, and thus improve their diagnostic and therapeutic potential..This research project intends to: 1) Establishment of the labeling method for lipid-PEG-KIM-1 affibody complex and SPIO nanoparticles (NPs) in MSCs, which enables MSCs to secrete functionalized EVs containing SPIO and surface modification of KIM-1 affibody ; 2) In vitro investigation of the cellular uptake rates of functionalized MSC-EVs in human proximal tubular cell lines (HK2), and their effects on hypoxia/reoxygenation (H/R)-induced oxidative stress, mitochondrial dysfunction and inflammation in HK2 cells are also assayed; 3) Drug-loaded functionalized MSC-EVs were injected into the mice model of AKI-CKD transition, and the in vivo distribution, MRI imaging and drug release of functionalized MSC-EVs are measured. In addition, the protective effect of MSC-EVs on renal function, histopathology and mitochondrial function are evaluated; 4) In order to clarify the molecular mechanism of functionalized MSC-EVs on AKI-CKD, the changes of key signaling pathways respected to renal mitochondria, apoptosis, inflammation and fibrosis are analyzed. This study will decrease the risk of AKI-CKD transition, and highlights that re-engineering of MSC-EVs is a promising method for the renal translational medicine.

线粒体功能紊乱是急性肾损伤（AKI）向慢性肾病（CKD）转归的关键因素。MSCs分泌的胞外囊泡（EVs）具有运输内外源活性物质，降低炎症/纤维化损伤等作用。功能化修饰MSC-EVs，构建能靶向受损肾脏递送线粒体抗氧化剂及MRI成像的多能纳米材料，将提升其诊疗潜能。.本项目拟：1）建立脂质-PEG-KIM-1亲合体和SPIO标记MSCs方法，由MSCs代谢产生含SPIO及表面修饰亲合体的功能化EVs；2）体外研究功能化EVs的肾小管细胞摄取，及其对H/R所致肾小管氧化应激、线粒体和炎症损伤的作用；3）注射载药功能化EVs治疗小鼠AKI-CKD模型，研究其体内分布、MRI成像和药物释放情况，评价其对肾功能、组织病理和线粒体功能等的保护作用；4）研究功能化EVs对肾脏线粒体、凋亡、炎症和纤维化信号通路的影响，阐明其分子机制。本研究有助于降低AKI-CKD转归风险，为EVs的临床转化提供新的思路。

急性肾损伤（AKI）是一种临床常见危重症，具有较高的发病率及死亡率。AKI后肾脏不良修复还将诱导肾间质纤维化，最终导致慢性肾脏病（AKI-CKD转归）和肾功能衰竭，给患者家庭及国家公共健康资源带来沉重负担。本项目研究计划针对AKI后肾脏不良修复这一临床重要问题，围绕氧化应激所致线粒体损伤及炎症反应等导致肾脏损伤的关键病因，提出构建能靶向受损肾脏的工程化细胞外囊泡（EVs），递送干预线粒体氧化应激和炎症反应等的外源性或内源性治疗分子、促进肾损伤修复的新型治疗策略，从而降低AKI后慢性肾脏纤维化及长期CKD风险。我们主要开展了以下几个方面的工作：1）阐明了线粒体氧化应激所致线粒体转录因子A（TFAM）信号受损是AKI炎症反应及肾功能损伤的重要因素，提示其可能作为治疗AKI肾损伤的潜在靶点，为后续研究建立理论基础；2）采用基因工程技术构建了表面展示白蛋白结合肽段的工程化EVs（ABP-EVs），其通过与内源性血白蛋白结合提升EVs的体内循环时间及肾脏驻留率，可用于递送小分子化合物如姜黄素等和大分子蛋白质药物如HGF等并增强其对肾脏线粒体氧化应激、炎症损伤和肾小管坏死等的治疗效果，促进肾小管再生；3）揭示了调节性免疫细胞如M2巨噬细胞来源的EVs（M2-EVs）含有多种免疫调节分子包括miRNAs和蛋白如IL-10、TGF-β等，可通过同时抑制多条关键促炎通路如NF-κB、p38 MAPK、JAK/STAT等降低促炎型巨噬细胞激活，减轻系统性炎症所致的多器官如肾脏氧化应激和细胞死亡等。这些研究结果提示，线粒体氧化应激是参与AKI后肾脏炎症及功能损伤的关键因素；采用基因工程化技术可构建长循环肾靶向EVs，用于递送多种类型药物并提升其肾脏治疗指数；M2巨噬细胞来源的EVs可减轻多器官如肾脏炎症损伤，促进损伤修复。在进一步改进的基础上，工程化EVs有潜能应用于不同病因所致的肾损伤修复，改善肾脏纤维化及长期预后，为未来临床治疗肾脏疾病提供新思路。