肾损伤分子1启动子调控的miR-181d对缺血性急性肾损伤的保护作用及机制研究

Inflammation is one of the most important pathophysiological changes in acute ischemic renal injury, its main immune cell responses include neutrophil chemotaxis, dendritic cell activation and regulatory T cells dysfunction. The miR-181 family has a number of functions such as inhibition of cell proliferation, promotion of cell apoptosis, regulation of T lymphocyte proliferation and differentiation, as well as inhibition of inflammation. The expression of miR-181d decreased significantly in the early stage of renal hypoxia-ischemia. Therefore, we hypothesized that the high expression of miR-181d may attenuate early inflammatory processes in renal injury by inhibiting neutrophil aggregation, reducing dendritic cell activation, and improving regulatory T cell function. To test this hypothesis, we will construct the miR-181d expression vector regulated by the kidney injury molecule 1 promoter, using several methods such as lentiviral vector transfection, in situ hybridization, and Western blot, to explore the role of miR-181d on ischemic injury and inflammation in renal tissue of AKI, discuss whether miR-181d could alleviate the inflammatory reaction and protect the renal tissue after renal injury by inhibiting the aggregation of neutrophils, reducing the activation of dendritic cells and improving the function of regulatory T cells. This study will investigate the ameliorative effect of miR-181d in inflammation after acute ischemic renal injury.

炎症是缺血性急性肾损伤中重要的病理生理改变，其主要免疫细胞反应包括：中性粒细胞趋化聚集、树突状细胞活化、调节性T细胞功能减退。miR-181家族具有抑制多种细胞增殖、促进细胞凋亡、调节T淋巴细胞增生分化与发育、抑制炎症等功能。在肾脏缺氧缺血早期，miR-181d表达显著下降。为此，我们提出假说：高表达miR-181d可能通过抑制中性粒细胞的聚集、减轻树突状细胞的活化、改善调节性T细胞功能，减轻肾损伤早期炎症进程。为了验证这一假说，我们构建肾损伤分子1启动子调控的miR-181d表达载体，采用慢病毒载体转染、原位杂交等手段，探讨miR-181d在缺血性AKI肾组织损伤和炎症反应中的作用，明确miR-181d通过抑制中性粒细胞聚集、减轻树突细胞活化、改善调节性T细胞功能，减轻肾损伤后炎症反应，保护肾组织的潜在机制。本研究将探讨miR-181d 对缺血性急性肾损伤后炎症的改善作用。

肾小管上皮细胞（RTEC）凋亡和肾间质炎症是缺血再灌注性急性肾损伤（I/R-AKI）最重要的病理生理改变。本课题组发现在缺氧/复氧损伤的人近端RTEC模型和I/R-AKI小鼠模型中miR-181d-5p表达减少而Krueppel样因子6（KLF6）表达增加，且主要分布于肾小管。miR-181d-5p过表达明显抑制I/R-AKI后炎症介质IL-6和TNF-α等促炎细胞因子的表达，减少细胞凋亡，改善肾功能。KLF6可加重RTEC的损伤，并作为核因子κB（NF-κB）共激活剂加重I/R-AKI的炎症反应。通过生物信息学分析和荧光素酶报告基因证实，KLF6是miR-181d-5p新的潜在靶基因，过表达miR-181d-5p和抑制KLF6后miR-181d-5p改善肾损伤的作用减弱，证明miR-181d-5p可能通过靶向抑制KLF6抗炎和抗凋亡，对I/R-AKI发挥保护作用。因此，本课题组相关结果提供了与I/R损伤相关的分子机制的新见解和潜在的新的治疗靶点。以此为基础，课题组对AKI的分子病理学机制、新型潜在生物学标志物、靶向治疗、临床风险评估进行了一系列的探索和研究，并明确了Resolvin D1（RvD1）通过LXA4受体（ALX/FPR2）途径上调调节性T细胞百分比减轻AKI；通过高通量分子对接技术及基础实验验证蒙花苷、烟花苷、野黄芩苷、柚皮素等通过抑制炎症、凋亡和ROS等过程对AKI具有靶向治疗作用；血清反应因子在尿酸诱导的RTEC中高表达，并通过上调slug促进上皮间质转化，加重高尿酸肾损伤，并首次提出尿液血清反应因子可作为AKI的早期诊断指标，其敏感性和特异性均优于血肌酐；基于临床数据分析，创新性构建Nomogram风险评估模型实现心脏手术、肝切除术、肾切除术、颅内动脉瘤夹闭术患者继发AKI风险个体化预测，筛选高危人群与危险因素，指导早期临床干预，改善患者预后。