探讨C3a受体信号在慢性感染引起的肾脏纤维化过程中的保护作用及其机制

Renal tubulointerstitial fibrosis is the common final outcome of almost all progressive chronic kidney diseases. It causes significant morbidity and mortality. There are currently no specific therapies for renal fibrosis. Therefore, more studies are needed to better understand the molecular and cellular mechanisms of renal fibrosis and develop new treatment strategies. As we known, the complement activation contributes to the pathogenesis of many renal diseases (etc. infection, ischemia-reperfusion injury and transplant rejection). However, our recent study has demonstrated a protective role of C3a/C3aR axis in uropathogenic E.coli-induced renal injury, conferred by the suppression of proinflammatory responses and enhanced phagocytosis by macrophages. In the proposed research, we hypothesise that C3a/C3aR axis has a protect role against the development of tubulointerstitial fibrosis following chronic renal infection through influencing macrophage phenotype and modulating local inflammatory environment. To this end, we will employ a well-established murine chronic renal infection model and using C3aR knockout (KO) mice as well as C3aR agonist to determine the protective roles of C3a/C3aR in the process of renal fibrosis. We will generate mice with macrophage-specific C3aR deficiency (Lyz2-C3aR-/-) and mice with kidney-specific C3aR deficiency (Six2-C3aR-/-) to evaluate the importance of C3aR on macrophages and renal cells in the development of tubulointerstitial fibrosis following chronic infection. Furthermore, we will use in vitro cell culture or co-culture models to further examine the cellular mechanisms by which contributes to the pathogensis of renal fibrosis. Results from these studies will provide new insight into the mechanisms underlying renal fibrosis and the development of novel therapies for prevention and treatment of this disease.

肾间质纤维化是各种肾脏疾病进展到终末期肾衰竭共有的病理损伤过程，目前尚无特异性治疗方法，临床预后差。因此，深入认识纤维化的发病机制并找出对其进程起调节作用的关键途径将具有重要意义。补体系统激活与各种肾脏疾病的发生、转归关系密切。申请人前期工作发现,补体活化小片段C3a与其受体C3aR作用在细菌感染引起的急性肾损伤过程中发挥重要保护作用，其主要机制是抑制炎症反应以及促进巨噬细胞吞噬细菌功能。本课题我们提出，C3a受体信号同样在感染引起的肾脏慢性炎症以及间质纤维化过程中发挥保护作用。我们将使用受体敲除小鼠、受体激动剂以及慢性肾脏感染模型来明确C3aR的保护作用；将分别构建肾脏组织特异性或髓系细胞特异性C3aR敲除小鼠以及体外细胞实验来探讨可能的细胞分子机制。我们的工作将有助于进一步认识肾脏慢性炎症以及间质纤维化的病理机制，可能为临床治疗开辟新靶点提供理论依据。

肾间质纤维化是各种肾脏疾病进展到终末期阶段所共有的病理损伤过程，目前尚无特异性治疗方法，临床预后差。泌尿系感染是最常见的感染性疾病之一，肾脏感染的反复发作或持续性感染可引起肾间质纤维化。因此，深入认识肾间质纤维化的发病机制并找出对其进程起调节作用的关键途径将具有重要意义。补体作为机体炎症及免疫反应的重要调节者，能够参与影响多种炎症及免疫相关疾病的进程。C3aR为补体活化小片段C3a的受体，广泛表达于髓系细胞和非髓系细胞，其与配体结合后可以发挥多种生物学活性。因此，本研究拟探讨C3aR在慢性感染引起的肾间质纤维化过程中的作用及其机制。.采用上行性感染慢性致病性大肠杆菌（IH11128）建立慢性肾脏感染模型，并通过构建髓系细胞特异性C3aR缺失小鼠进一步探究其机制。我们的研究发现，C3aR-/-组肾脏组织和膀胱组织的细菌负荷量显著高于WT组，提示C3aR-/-组肾脏组织和膀胱组织细菌负荷量显著高于WT组；病理结果提示C3aR-/-组肾小管基底膜损伤明显较WT组严重；C3aR缺失加重了肾脏组织炎症细胞的浸润；C3aR缺失小鼠的炎症因子Tnfa、IL1b、TGF-b、CXCL1表达量显著高于WT组，提示C3aR缺失加重了肾脏组织炎症反应；SR染色结果发现C3aR-/-组肾脏组织纤维化评分明显高于WT组，WB和IF表明C3aR-/-组FN和COL1表达量显著高于WT组；提示C3aR缺失加重了肾脏组织纤维化；表达C3aR的髓系细胞（尤其是巨噬细胞）为慢性肾脏感染中发挥保护作用的关键细胞，C3aR主要通过影响巨噬细胞，从而在慢性肾脏感染中发挥保护性作用。巨噬细胞吞噬细菌体内/外实验证实，C3aR通过上调巨噬细胞吞噬受体的表达，增强巨噬细胞吞噬能力，促进巨噬细胞对UPEC的清除，但C3aR对巨噬细胞吞噬凋亡细胞的能力无明显影响，因此C3aR缺失削弱了巨噬细胞吞噬病原菌能力，影响其在UTI中对病原菌的杀伤及清除。.我们的研究提示，C3aR依赖的巨噬细胞吞噬UPEC的上调和局部炎症反应的调节有助于抵御持续感染、慢性炎症和肾纤维化。C3aR激动剂对小鼠慢性肾脏感染具有治疗作用，因此，C3aR激动剂可能具有临床应用价值。