α-防御素基因在中国人群IgA肾病中的功能研究

IgA nephropathy (IgAN) is the most common form of idiopathic glomerulonephritis (GN) in China. However，the exact pathogenic mechanism of IgAN remains unclear. The result published on "Nature Genetics" from our lab showed that single nucleotide polymorphrism of α-defensin gene DEFAs (rs2738048) was significantly related with IgAN, and α-defensin gene maybe a susceptibility gene for IgAN in Chinese Han patients. In order to characterize the expression pattern of α-defensin in IgAN patients, we plan to detect all types of α-defensins expression level in serum, urine and renal tissues of IgA nephropathy patients and normal control by ELISA,real-time PCR and immunofluorescence. Our previous results revealed that HNP1-4 was significantly increased in serum and renal tissue of IgAN patients, but no change in urine. It is reported that some types of α-defensins (HNP1-4 and HD5) have lectin-like activity. Whether α-defensins have ability to bind galactosylated IgA1 or not, and if it done, what are their roles in IgAN pathogenesis? In order to answer these questions, we have isolated IgA1 from serum of IgAN patients and normal control by affinity purification and gal filtration, and applied surface plasmon resonance (SPR) techniques to gain insights into this property. The results of SPR revealed that α-defesin (HNP1-3) have affinity to bind galactosylated IgA1 from IgAN patients or from normal control in a concentration-dependent manner, while the kinetics of interaction were significantly different. Based on these, we plan to further study the interaction pattern and mechanism of α-defensin with galactosylated IgA1, and make clearly the mechanisms of α-defensin and/or α-defensin-galactosylated IgA1 interaction on cell proliferation, collagen, Fn and cytokines production in human glomerular mesangial cells, hoping to find their potential roles as new therapeutic targets or biomarkers. In brief, we hope that this research will shed some new light on the mechanism study of IgAN pathogenesis and offer new strategies for the clinical treatment.

IgA肾病(IgAN)是我国最常见的原发性肾小球疾病，其发病机理尚未完全清楚。本实验室已完成的IgAN全基因组关联分析提示α-防御素单核苷酸多态性与IgAN显著相关，α-防御素可能参与IgAN的发生(Nature Genetics,2011)。项目组前期从mRNA和蛋白质表达水平分析发现IgAN患者外周血、尿液和肾组织中α-防御素某些类型的表达水平显著升高；表面等离子共振结果显示α-防御素能与异常糖基化IgA1结合，但其动力学与正常对照来源的IgA1差异显著。为进一步阐明α-防御素与疾病的关系，我们拟探讨其与异常糖基化IgA1相互作用的模型与机制，通过体外实验研究其对肾小球系膜细胞的影响和效应机制，如α-防御素和α-防御素-异常糖基化IgA1对细胞增殖、外基质形成及细胞因子表达的作用效应和信号调控机制。通过上述研究，旨在阐明α-防御素在IgAN发生中的作用及机理，为疾病治疗提供新的思路。

IgA肾病(IgAN)是我国最常见的原发性肾小球疾病，其病理特征是以IgA1为主的免疫复合物沉积于肾小球系膜区，IgA1的异常糖基化是导致IgAN发病的关键因素。但其中有关IgA1如何形成多聚体，如何沉积在肾小球系膜区还不十分清楚。IgAN是多基因、多因素参与的复杂性疾病。本实验室已完成的IgAN全基因组关联分析提示DEFA基因单核苷酸多态性与IgAN显著相关，提示DEFA基因编码蛋白α-防御素可能参与IgAN的发生。本项目探讨了α-防御素在IgAN患者外周血、尿液和肾脏中的表达水平及其与临床病理特征的相关性，α-防御素与异常糖基化IgA1相互作用的模式和机制，以及和α-防御素与IgA1系膜沉积的关系。研究表明IgAN患者外周血中α-防御素HNP1-3 (461.37 (224.10, 1340.74) ng/ml vs. 242.45 (92.41, 547.52) ng/ml)和HNP4 (118.73 (93.84, 149.18) ng/ml vs. 102.98 (87.00, 127.57) ng/ml)的表达水平显著升高，与外周血淋巴细胞数、IL-8和IgA水平显著正相关。体外细胞分离培养实验表明，IL-8能浓度依赖的提高中性粒细胞分泌HNP1-3和HNP4的水平。组织免疫荧光结果显示HNP1-3和HNP4在IgAN患者肾脏组织中性粒细胞中高表达。体外细胞实验表明HNP1-3和HNP4能浓度依赖的提高系膜细胞的增殖活性和外基质蛋白Fn的表达。而肠道相关的HD5血清水平显著升高(1.65 (1.05, 2.00) ng/ml vs. 1.18 (0.74, 1.45) ng/ml)，而且与肾小球率过滤显著负相关，与肌酐显著正相关，但HD5表达仅限于小肠，在IgAN患者和正常对照肾组织中都没有检测到荧光信号。表面等离子共振和体外细胞实验结果表明，HD5可能通过与异常糖基化IgA1结合加速IgA1多聚体形成来提高IgAN患者来源的IgA1沉积在肾小球系膜细胞表面。HNP1-3和HNP4虽然也能与IgA1结合，但并没有显著提高IgA1沉积在肾小球系膜细胞表面。通过上述研究，阐明了各类型α-防御素在我国IgAN患者中的表达水平及其对系膜增生和IgA1沉积中的作用，α-防御素可能是我国IgAN发生发展的潜在生物标记物，为阐明IgAN的发病机理提供了新的实验依据。