Complosome与传统补体旁路途径过度活化在恶性高血压肾损害中的作用机制研究

Malignant nephrosclerosis is a critical renal emergency caused by malignant hypertension. Despite well-established roles for mechanical stretch and angiotensin in malignant nephrosclerosis, mechanisms on the onset and development of the condition is not yet fully understood. The pathologic change of malignant nephrosclerosis is characterized by the injury of endothelium, which is almost identical to those of atypical hemolytic uremic syndrome (aHUS). One of the key mechanisms of aHUS is the activation of complements, while the role for complements in malignant nephrosclerosis remains largely unknown. Recently, complement activation via the alternative pathway has been observed in patients with malignant nephrosclerosis. Our previous work found marked deposition of C3, C5a and C5b-9 along the arteriolar walls in renal biopsies of patients with malignant nephrosclerosis. Whole-genome exon sequencing highlighted significant enrichment of a missense mutation of membrane cofactor protein, a regulatory protein of alternative complement pathway, in the genome of patients with malignant nephrosclerosis. These pieces of evidence strongly suggest a close association between alternative pathway of complement activation and malignant nephrosclerosis. Furthermore, it was recently reported that complement activation also occurs inside cells, a phenomenon known as the ‘complosome’. However, the role for the ‘complosome’ in the pathogenesis of malignant nephrosclerosis has not been well studied. In this proposal, we hypothesize that intracellular and extracellular alternative pathways of complement activation plays causal roles in the pathogenesis of malignant nephrosclerosis. To test this hypothesis, we will test a series of key questions by examining biological specimens of patients, animal models induced in wild-type and gene-knockout mice in which specific regulators of complement activation are deleted, and in-vitro models in cultured primary human glomerular microvascular endothelial cells. It is expected that this study will lay a more solid foundation for understanding the mechanisms of malignant nephrosclerosis and may lead to novel therapeutic strategies for the disease.

恶性高血压肾损害是肾脏病的急危重症，发病机制仍不清楚。现有观点聚焦在机械压力和血管紧张素的作用，但未完全解释恶高肾损害的发生发展。恶高肾损害以血管内皮细胞损伤为核心的病理特征与不典型溶血尿毒综合征（aHUS）基本一致，aHUS关键发病机制是补体旁路过度活化，最新研究显示恶高肾损害患者存在补体过度激活，我们前期工作也发现恶高肾损害患者肾组织C3、C5a和C5b-9小动脉壁大量沉积，全外显子组测序显示补体旁路途径膜辅蛋白基因突变显著聚集，提示恶高肾损害与补体旁路过度活化密切相关。近年发现细胞内存在补体系统（complosome），但其在恶高肾损害发病中的作用研究甚少。本课题将从细胞内/外补体旁路途径的新视角，利用恶高肾损害患者生物标本、整体/细胞特异性基因敲除小鼠恶高肾损害模型及肾小球内皮细胞，从不同层面揭示细胞内/外补体旁路途径过度活化在恶高肾损害中的作用机制，为恶高肾损害的治疗提供新靶点。

项目背景：恶性高血压肾损害是肾脏病的急危重症，发病机制仍不清楚。现有观点聚焦在机械压力和血管紧张素的作用，但未完全解释恶性高血压肾损害的发生发展。恶性高血压肾损害以血管内皮细胞损伤为核心的病理特征与不典型溶血尿毒综合征（aHUS）基本一致，aHUS关键发病机制是补体旁路过度活化，提示补体旁路过度活化可能在恶高肾损害发生发展中发挥重要作用。主要研究内容：本研究利用恶性高血压肾损害患者生物标本、补体旁路CFB基因敲除小鼠恶性高血压肾损害模型及肾小球微血管内皮细胞进行了体内、外实验，用激光微切割联合质谱分析、全外显子测序、激光共聚焦等新技术，从不同层面阐明补体旁路途径过度活化在恶性高血压肾损害发病机制中的作用，为寻找恶性高血压肾损害治疗新靶点奠定理论基础。研究结果：免疫组化及激光共聚焦显示恶性高血压肾损害患者肾组织中C3、C3a、C5a、C5b-9沿小动脉壁及肾小球毛细血管壁沉积，激光微切割恶性高血压肾损害患者肾小球和小动脉，靶向质谱定量分析显示补体旁路D因子和活化产物MAC（C9，C8a，C8b）的独有肽段的表达丰度明显增高，ELISA结果显示恶性高血压肾损害患者较正常对照血浆/尿液中补体B因子、P因子、D因子、C3a、C5a显著升高（P＜0.05），而补体H因子显著降低（P＜0.05）。全外显子组基因测序发现恶性高血压肾损害患者补体旁路相关MCP、B因子、H因子基因变异。利用Ang-II泵入建立恶性高血压肾损害小鼠模型，C3及B因子基因敲除小鼠较野生型小鼠肾功能损伤减轻（P<0.05），光镜及电镜下内皮细胞损伤减轻。在培养的人肾小球内皮细胞，Ang-II刺激后内皮细胞胞内补体旁路途径D因子表达增高，内皮细胞损伤标志蛋白ICAM-1、VCAM-1、vWF和ET-1的表达增高，特异性干扰D因子表达可以减少内皮细胞表面MAC沉淀及Ang-II诱导下的ICAM-1、VCAM-1、vWF和ET-1的表达上调。科学意义：本项目发现了补体旁路过度活化在恶性高血压肾损害发病中的重要作用及其机制，探讨了补体旁路敲除/拮抗对恶性高血压肾损害的保护作用，为寻找恶性高血压肾损害治疗的新靶点开拓了思路。