C3a/C3aR轴过度活化在糖尿病肾病中的作用及机制研究

Studies in recent years have demonstrated that diabetic nephropathy (DN) is a chronic inflammatory renal disease. The complement system has been found involved in the pathologic process of DN. However, the inflammatory mechanisms of DN and the mechanisms by which the complement system is involved in the disease are not clear. In preliminary studies, we found that C3aR, the receptor for C3a, was upregulated in both the kidney of patients with DN and the kidney of model animals, indicating that a situation of C3a/C3aR axis over-activation exists in DN. The increased C3aR was found distributed mainly in the tubular epithelial cells, glomerular parietal epithelial cells and podocytes. Significantly increased C3aR was often observed in the injured tubulus, detached podocytes, proliferating podocytes and parietal epithelial cells. Also, C3aR was found in the renal infiltrating cells. The increased expression of C3aR was found correlated with clinical index reflecting renal injury. These findings suggested that over-activation of C3a/C3aR axis might have important roles in DN, especially in tubular injury, podocytes injury and renal inflammation. The present items aim to clarity the pathological roles of C3a/C3aR in DN and to determine whether C3a/C3aR can be used as a target for treatment of DN. It will provide a new insight in renal inflammation mechanisms under DN condition and the mechanisms by which the complement system is involved in the disease. Also, the items will be helpful in developing new strategy for treatment of DN. To achieve these goals, patients at different stages of DN will be recruited, the expression changes of C3a and C3aR will be examined, and the correlation between C3a/C3aR axis and DN will be evaluated. In the mean while, db/db mice were used as a model and the influences of SB290157 (an antagonist for C3aR) and WWGKKYRASKLGLAR (an agonist for C3aR) on the initiation and development of DN will be evaluated. Further more, transgenic mice specifically express C3aR miRNA in tubular epithelial cells or in glomerular podocytes will be constructed and the difference between these mice and wild mice in the renal injury under diabetic conditions will be examined. In the in vitro studies, cell line HK-2 and HPC will be used as models to investigate the possible roles of C3a/C3aR axis in the injury of tubular epithelial cells and podocytes respectively. Also, these cell lines will be used to elucidate the signal pathway involved.

越来越多的证据表明：糖尿病肾病（DN）是一种慢性炎症疾病，补体活化参与了DN病理过程。但有关DN炎症机制和补体系统参与DN的具体机制仍不是很清楚。在前期研究中，我们发现：补体活化产物C3a的受体C3aR在DN患者和模型动物肾组织中的表达显著上调，DN肾组织存在C3a/C3aR过度活化的情况；从C3aR的分布及相关性分析来看，C3a/C3aR过度活化很可能在DN炎症、肾小管和足细胞损伤中有重要作用。本项目拟在已有研究基础上，利用临床观察、动物实验和体外细胞实验相结合，采用C3aR激动剂和拮抗剂干预，以及转基因动物等手段对C3a/C3aR过度活化在DN中的具体作用（特别是在DN炎症、小管和足细胞损伤中的作用）和可能机制进行研究，以阐明C3a/C3aR在DN中的病理意义，增加对DN炎症机制和补体系统参与DN机制的认识，了解C3a/C3aR能否作为DN防治的新靶标，最终为提高DN诊疗水平创造条件。

已有研究表明，补体和C3a/C3aR可能在糖尿病肾病（DN）中有作用，但具体作用和有关机制有待阐明。本项目围绕探讨C3a/C3aR在DN中的作用和机制而展开：1）临床研究部分，首先分析了补体活化产物在DN患者肾组织中的沉积、C3aR在患者肾组织中的表达、补体活化产物在患者尿液和血浆中的变化情况。发现：患者肾组织和尿液中的补体活化产物水平，以及肾组织C3aR水平随DN的进展而升高，与反映肾损伤的指标具有相关性。在此基础上，又对DN下补体活化的机制进行分析，发现替代途径和甘露糖结合凝集素途径可能是DN下肾组织补体活化的主要原因。2）体外细胞实验部分，首先分析了激活C3aR对小管细胞和足细胞的影响情况。观察到：激活C3aR可诱使小鼠小管细胞发生细胞骨架重排和上皮间充质转分化；激活人足细胞可引起足细胞细胞骨架重排、标记分子表达下降；而在小鼠足细胞，高糖环境中激活C3aR还可引起足细胞凋亡。此外，利用分泌性表达C3a以持续激活C3aR可严重影响足细胞的分化功能。接着，对DN下致C3aR表达上调的机制进行了探讨。发现：缺氧和炎症因子IFN-γ都可诱使小管上皮细胞C3aR表达增加，而缺氧对C3aR的这种诱导作用与HIF-1α和NF-κB的活化有关。3）动物实验部分，首先是进行了C3aR激动剂和拮抗剂干预DN模型小鼠的研究。初步的结果发现，C3aR激动剂可增加而C3aR激抗剂可减轻模型小鼠肾组织损伤。此外，本项目还进行了可诱导的肾小管上皮细胞和足细胞特异性的C3a分泌性转基因小鼠的构建。现已初步鉴定出了可诱导表达转基因的小鼠。.本项目研究证实DN肾组织确实存在C3aR过度活化现象，C3aR过度活化可能通过包括诱使肾脏细胞发生结构重塑、细胞凋亡、足细胞分化受阻等方式对肾脏造成损伤，成为DN的致病因素。本项研究还提示C3a/C3aR可能是糖尿病肾病防治的重要靶标，而尿液补体活化产物水平检测可望成为新的反映DN肾组织损伤的无创性指标。构建的肾小管上皮细胞和足细胞特异性的C3a分泌性转基因小鼠将是一个很有希望的可用于揭示肾组织C3a/C3aR功能的重要工具。