C型凝集素受体Clec7a介导免疫炎症参与急性肾损伤的功能和机制研究

Mounting evidence suggested that macrophage inflammation and immune response play an important role in acute kidney injury (AKI). Our previous studies have showed that: Clec7a, one of C-type lectin receptor, was highly expressed on pro-inflammatory M1 macrophages after acute renal ischemia reperfusion (I/R) injury; We further demonstrated that kidney injury was significantly improved in Clec7a knockout mice after kidney I/R injury, compared with wild type mice; We identified a DAMPs-peroxiredoxin 6 (Prdx6) as an endogenous, activating ligand for Clec7a, which was highly expressed after kidney I/R injury. Based on these findings, we proposed that Prdx6 released from damaged kidney, mediates macrophage polarization towards M1 phenotype through binding its receptor Clec7a, which contributes to kidney injury. Thus, the present study aimed to further explored: ①the mechanism and functional significance of Clec7a mediated macrophage inflammation in acute kidney I/R injury; ②Prdx6 contributes to kidney ischemic injury through binding its receptor Clec7a; ③Lastly, we will examine circulating levels of Prdx6 and circulating Clec7a+ monocyte in patients with acute kidney injury, and further explore the relationship between prdx6/Clec7a axis and the severity of AKI. Our findings will provide new therapeutic target for acute kidney disease.

巨噬细胞免疫炎症在急性肾损伤（AKI）发病中的作用日益受到关注。我们前期研究发现C型凝集素受体Clec7a在肾脏缺血再灌注（I/R）损伤早期促炎性M1巨噬细胞表达显著升高；Clec7a敲除小鼠肾损伤显著减轻；过氧化物酶Prdx6可与Clec7a结合，在I/R早期即升高，提示Prdx6可能为Clec7a的内源性配体。在此基础上我们提出科学假设：AKI时组织损伤释放Prdx6，结合巨噬细胞表面Clec7a，促进M1巨噬细胞免疫炎症反应参与AKI发病。为此，本课题将继续探索：①Clec7a介导M1巨噬细胞活化参与AKI的免疫炎症机制；②Prdx6与Clec7a结合介导巨噬细胞免疫炎症反应及其在AKI中的作用；③临床验证循环Prdx6水平、Clec7a＋单核细胞与AKI的相关性，从而为阻抑AKI提供新的治疗策略和干预靶点。

【背景】急性肾损伤（acute kidney injury, AKI）是临床常见的危重疾病，不仅严重影响患者的生存质量，也给全球带来了沉重的经济负担。巨噬细胞介导的炎症免疫反应在AKI肾脏缺血再灌注（renal ischemia reperfusion, RIR）损伤中发挥重要作用。模式识别受体Clec7a在活化巨噬细胞上表达，参与调节机体免疫炎症反应。然而，Clec7a在RIR中的具体作用及其机制尚不明确。本研究旨在探索模式识别C型凝集素受体Clec7a在肾脏IR损伤中的作用和机制。.【方法】研究采用质粒、腺相关病毒转染技术、骨髓基质干细胞的分离和分化，通过流式细胞学、ELISA、免疫印迹及免疫荧光染色等方法探究AKI-IRI后肾脏巨噬细胞中Clec7a的表达，以及clec7a对免疫细胞及机体炎症状态的影响。.【结果】研究证实，AKI后肾脏中表达CD68+CD11b+巨噬细胞以及Clec7a+CD163-M1巨噬细胞和Clec7a-CD163+M2巨噬细胞比例均显著增加。诱导型一氧化氮合酶（iNOS）、肿瘤坏死因子α（TNFα）和IL-1β在Clec7a+CD163-M1巨噬细胞中表达较高，精氨酸酶1（ARG1）、TGFβ1和VEGF-A在Clec7a+CD163+M2巨噬细胞中的水平显著降低，表明Clec7a可能增加M1巨噬细胞促炎和吞噬功能，降低M2巨噬细胞的增殖和迁移能力。同时证实Clec7a不改变M1或M2巨噬细胞的极化。在此基础上，我们分别利用Clec7a或si-Clec7a 转染LPS/IFN-γ诱导的M1巨噬细胞和IL-4诱导的M2巨噬细胞，检测M1巨噬细胞的吞噬功能和M2巨噬细胞的增殖和迁移能力。结果显示Clec7a增加M1巨噬细胞的吞噬能力，M2巨噬细胞中Clec7a的耗竭增加了巨噬细胞的增殖和迁移。此外，我们进一步利用缺血再灌注小鼠模型证实M1巨噬细胞中Clec7a和M2巨噬细胞中Clec7a耗竭的联合表达可显著改善AKI后肾功能。.【结论】Clec7a对AKI中巨噬细胞表型的精细调节至关重要，可调节巨噬细胞的吞噬作用和增殖迁移能力，是临床干预急性肾损伤免疫反应的新靶点，具有潜在的转化医学价值。

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