黏膜源性IgA肾病的发病机制研究

IgA nephropathy (IgAN) is a group of glomerular diseases, which has a high incidence in our country. Although its etiology and pathogenesis is still unclear, a lot of information shows its correlation with mucosal infection. Our previous studies have confirmed that after administration of BSA + LPS + CCL4, experimental mice developed intestinal mucosal injuries, increased intestinal permeability and typical pathological changes of IgAN in kidneys, which demonstrated a correlation between intestinal injuries and occurrence of IgAN, and confirmed the protective repairing role of rhein on intestinal mucosal injuries and renal damage of IgAN. This project intends to establish a rat model of IgAN using the same method, to detect cytokines including BAFF, APRIL, TGF-β1, TLR4, myd88, NF-κB, etc. by using immunological, molecular biological, stereological and gene knockout methods in order to analyze the role of innate immunity in the pathogenesis of IgAN and the role of TLR-mediated signaling pathway in the pathogenesis of such mucosa-derived IgAN from three levels of intestinal mucosal injuries, lymphocyte transformation and dislocation migration, and renal damage and to further confirm the role of rhein in it and its mechanism, and to identify functional differential proteins and demonstrate their role in the pathogenesis of IgAN by using proteomic methods, providing a theoretical basis for prevention and treatment of IgAN in clinical setting.

IgA肾病（IgAN）是一组在我国发病率较高的肾小球疾病，病因和发病机制至今不甚清楚，大量资料显示与黏膜感染有关。我们前期的研究已经证实给予BSA+LPS+CCL4灌服后，实验鼠出现肠粘膜损伤、肠通透性增加和肾脏IgAN病理变化，证明了肠粘膜损伤与IgAN发病相关关系，并证实了大黄酸保护和修复肠粘膜损伤及IgAN肾脏损伤的作用。本项目拟通过同样方法建立大鼠IgAN模型，采用免疫学、分子生物学、体视学和基因敲除等方法检测细胞因子BAFF、APRIL、TGF-β1、TLR4、myd 88、NF-κB 等，分析粘膜免疫在IgAN发病过程中的作用及TLR介导的天然免疫信号通路在此类粘膜源性IgAN肠黏膜损伤、淋巴细胞转换与错位迁移及肾脏损伤三个层面的发病机制，并进一步证实大黄酸的作用及机理，用蛋白组学方法找出肠粘膜功能性差异蛋白并进一步论证其在IgAN发病中的作用，为临床IgAN的防治提供理论依据

IgA肾病是一组在我国发病率较高的肾小球疾病，病因和发病机制尚不完全明了，本项目通过建立黏膜源性IgA肾病大鼠和小鼠模型，并通过敲除基因分析TLR4在IgA 肾病发病过程中的作用，通过蛋白组学分析进一步查找差异蛋白并验证其在IgA 肾病发病过程中的作用，观察了大黄酸防治IgA肾病的效果并进一步验证其作用是否通过TLR4信号通路产生作用。.实验结果显示，BSA+LPS+CCL4联合法构建IgA肾病大鼠模型和口服BSA联合尾静脉注射ESB构建小鼠IgA肾病模型均可出现典型的血尿生化变化和肾脏IgA免疫荧光沉积，方法稳定可靠；在IgA肾病鼠肠Peyer结中Th数量增多、Ts数量减少以及Th2免疫反应增强，肠Peyer结和骨髓IgA+B淋巴细胞都增加，肠Peyer结T细胞亚群的变化及细胞因子的改变促进IgA+B细胞的转化；在IgA肾病鼠肠Peyer结TLR4mRNA和蛋白的表达升高，NF-κB、TGF-β1升高，TLR4基因敲除后得到缓解，表明TLR4参与介导黏膜源性IgA肾病模型炎症级联反应参与肾脏损伤及慢性纤维化的进程；蛋白组学分析发现，在模型组显著降低而在TLR4敲除组升高的蛋白有PHF7、ACTN2、DAPK3、KIF4A、ABHD1等，在模型组显著升高而在TLR4敲除组降低的有PEA15、TSPAN1、RAP1A、AKAP7、BABAM1等。KEGG通路分析结果显示在TLR4敲除组上调的蛋白主要参与紧密连接的形成，而下调的蛋白主要参与白细胞跨内皮迁移过程；在大黄酸预防组和治疗组血尿生化指标和肾IgA免疫荧光沉淀减轻，IgA+B细胞转化减少，TLR4、NF-κB、TGF-β1、Smad3、α-SMA、FN的升高得到缓解。.这些结果表明，黏膜源性IgA肾病的发病机制是通过激活TLR4信号通路，影响细胞连接蛋白的表达和增加肠黏膜通透性，诱导BAFF的产生，促进非T细胞依赖性IgA+B细胞的类别转换，从而启动IgA肾病发病过程；TLR4还参与介导黏膜源性IgA肾病模型炎症级联反应参与肾脏损伤及慢性纤维化的进程。大黄酸能有效防治黏膜源性IgA肾病，大黄酸防治IgA肾病的机制是通过影响TLR4通路而产生的，而大黄酸防治IgA肾病肾纤维化是通过调控TGF-β1/Smad通路实现。本项目的完成为阐明黏膜源性IgA肾病的发病机制和阐明大黄酸作用机制并促进大黄的临床应用产生积极作用。