细胞内补体激活介导的足细胞损伤在三氯乙烯免疫性肾损害中作用

Immune renal damage of Occupational medicamentosa like dermatitis induced by trichloroethylene (ODMLT) contains glomerular injury and renal tubular injury, in which complement activation plays a vital role, while the mechanisms of complement activation between glomerular injury and renal tubular injury may be diffenrent. Our previous study showed anaphylatoxin C5a and membrane attack complex C5b-9 formed by complement activation induced renal tubular injury, while the mechanism of glomerular injury remains unknow. The earlier studies suggested that consistent with the distribution of podocytes which is an important component of the glomerular filtration membrane, the large number of complement C3 deposited in glomeruli of sensitive mice caused by TCE treatment. Combined with the latest advancements of complement, this program puts forward hypothesis that "intracellular complement activation mediated podocytes injury, which promote the TCE immune renal injury". Therefore, in this study, we observe complement activation and podocytes injury in ODMLT patients through occupational population study. We also investigate the mechanism that intracellular complement activation mediates podocytes damage, then damaged podocytes would promote the inflammatory reaction and crosstalk with endothelial cell, aggravating immune renal injury induced by trichloroethylene Sensitization，with CTSL-/- mice and inflammatory cell migration assay in vitro. The results will provide a new theoretical basis for elucidating the mechanism of renal damage in ODMLT.

职业性三氯乙烯药疹样皮炎（ODMLT）患者出现的免疫性肾损害既有肾小管损伤也有肾小球损害，补体激活在此损害中发挥重要作用，但肾小球和肾小管损伤机制可能各异。课题组前期研究显示补体激活形成的过敏毒素C5a和膜攻击复合物C5b-9介导了肾小管损伤，而补体如何介导肾小球损伤，机制不清。课题组前期研究显示：TCE致敏小鼠肾小球内有大量补体C3沉积且C3分布与肾小球滤过膜重要组成细胞—足细胞形态一致，结合补体研究最新进展，本课题提出“足细胞内补体激活介导的足细胞损伤，参与了TCE免疫性肾损害”的假设。为此，本课题采用人群研究观察ODMLT患者足细胞损伤与肾功能受损关系，用CTSL-/-小鼠和体外细胞迁移实验，探讨TCE免疫性肾损害中足细胞内补体激活及其介导的足细胞损伤，以及足细胞受损后促进炎症反应并与内皮细胞交互作用，参与免疫性肾损害的机制。研究结果将为阐明ODMLT患者肾损害机制提供新的理论依据。

职业性三氯乙烯药疹样皮炎（ODMLT）是由于职业活动中暴露于三氯乙烯（TCE）后所引起的一种职业性重症皮肤病。肾脏损伤是其常见的并发症，不仅影响临床的治疗难度，同时加重患者的疾病负担。因此，深入探讨ODMLT肾损伤的发病机制对于TCE的防治意义重大。细胞内补体激活是一种由组织蛋白酶L（CTSL）在胞内切割补体C3所引发的补体激活路径，普遍发生于多种免疫细胞和组织细胞内，发挥调控固有免疫和适应性免疫的重要作用。本研究通过收集ODMLT患者临床资料和外周血、尿液等生物样品，检测肾脏损伤相关指标BUN、Cre、PCX、Nephrin、内皮素-1（ET-1），发现ODMLT患者肾脏滤过功能出现明显障碍、肾小球足细胞标志蛋白Nephrin丢失明显增加，说明肾小球及其足细胞损害是ODMLT肾损害的重要组成部分。接下来，我们将足细胞作为肾小球损害研究的靶细胞，建立TCE致敏BALB/c小鼠模型，在用或不用CTSL抑制剂的情况下，检测TCE致敏阳性小鼠肾小球结构和功能的损伤情况、CTSL的表达情况和补体C3a的沉积情况，发现CTSL抑制剂预处理后的TCE致敏阳性小鼠肾小球损害有所缓解，足细胞C3a的沉积减少、炎症细胞因子表达减少，说明CTSL介导的足细胞内补体C3激活是肾小球炎性损伤的关键分子机制。不仅如此，我们还发现CTSL抑制剂可以减少内皮素-1（ET-1）的表达，而ET-1在肾脏中主要表达于肾小球内皮细胞，其受体ETAR则主要表达于足细胞，所以我们进一步聚焦ET-1/ETAR通路，研究细胞内补体激活介导的足细胞损伤与肾小球内皮细胞损伤之间的关系，发现ET-1/ETAR路径激活可能是受损足细胞诱导肾小球内皮细胞损伤的关键分子机制，阻断ET-1/ETAR路径不仅可以有效减少内皮细胞凋亡，还可以减轻足细胞结构损伤，从而有效改善肾小球滤过功能损伤。本项目研究成果表明细胞内补体激活及其介导的足细胞损伤，通过ET-1/ETAR交联肾小球内皮细胞损伤是TCE免疫性肾小球炎性损伤的内在发病机理，从而为ODMLT免疫性肾损伤的防治提供新视角。