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A study on the mechanism of IgA-deposition on renal glomerular mesangium in IgA nephropathy -the role of circulating complex of IgA-fibronectin fragment in the interaction between IgA and mesangial cell matrix-

IgA肾病肾小球系膜IgA沉积机制研究-IgA-纤连蛋白片段循环复合物在IgA与系膜细胞基质相互作用中的作用-

基本信息

批准号：
11670733
负责人：
WAGA Shinobu
金额：
$ 1.54万
依托单位：
Hirosaki University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

项目摘要

Introduction : It is generally believed that IgA nephropathy is a consecquence of mesangial trapping of circulating macromolecular IgA.We have demonstrated that fibronectin fragment was found in patient's serum and was capable of binding IgA in vitro (J Am Soc Nephrol, 10 : 256, 1999). Therefore, we studied here if serum IgA from patient exists in association with fibronectin fragment, thereby forming macromolecular IgA, and if it deposits on the extracellular matrix in a fibronectin-dependent manner. Methods : Serum IgA from 10 patients with IgA nephropathy and 10 normal controls were obtained by jacalin-immobilized affinity chromatography. They were reacted with the confluent cell layer of cultured human fibroblasts for 3 hr and were cultured with medium for an additional 24 hr. IgA was detected by immunofluorescence microscory. Each three samples from both groups were applied to native SDS-PAGE (3.5%), and IgA and fibronectin fragments were detected by western blotting. Results : In … More all patients, but not in controls, the coarse and punctate clusters of IgA staining were demonstrated on the cell layer. The deposition was abolished by pretreatment of samples with anti-human fibronectin monoclonal antibodies, and was augmented with an addition of cathepsin D-digested fibronectin. Western blot analysis showed that major reactivities of patient's IgA were at 380 kDa and 200 kDa, shifting to higher molecular mass than an expected size from multiples of monomeric IgA (160 kDa). In a parallel experiment, a panel of anit-human fibronectin monoclonal antibodies reacted mainly with 440 kDa molecule presumably representing intact fibronectin in both patients and controls. However, broad and intense reactivity from 480-to 330-kDa was demonstrated by a monoclonal antibody to C-terminal region of fibornectin only in patients. These reactivities were also confirmed by using ELISA experiment, in which fibronectin fragment was detected in serum IgA captured by anti-human IgA coated plate. Thus serum IgA co-existed with fibronectin (fragment) in patient. Conclusion : Serum IgA from patients with IgA nephropathy is associated with fibronectin fragments to form a macromolecular IgA, thereby contributing to IgA depostion on extracellular matrix of mesangium through the mechanism of assembly of exogenous fibronectin into extracellular matrix. Less

前言：一般认为，IgA肾病是一种系膜捕获循环大分子IgA的过程。我们已经证明，在患者血清中发现了纤维连接蛋白片段，并在体外能与IgA结合(J Am Soc Nephrol，10：256,1999)。因此，我们在这里研究了患者的血清IgA是否与纤维连接蛋白片段结合，从而形成大分子IgA，以及它是否以纤维连接蛋白依赖的方式沉积在细胞外基质上。方法：采用印迹亲和层析法测定10例IgA肾病患者和10例正常对照血清中的IgA。将它们与培养的人成纤维细胞融合细胞层反应3小时，并与培养液一起培养24小时。免疫荧光显微镜检测免疫球蛋白。两组各取3个样本，用天然的SDS-PAGE(3.5%)，用免疫印迹法检测IgA和纤维连接蛋白片段。结果：在…更多的是ALL患者，而不是对照组，在细胞层上可见粗大的斑点状的IgA染色。用抗人纤维连接蛋白单抗对样本进行预处理，可消除这种沉积，而加入组织蛋白酶D消化的纤维连接蛋白则可增强这种沉积。Western印迹分析表明，患者的免疫球蛋白A的主要反应性在380 kDa和200 kDa，从单体免疫球蛋白A的倍数(160 KDa)转移到比预期更高的分子质量。在一项平行实验中，一组抗人纤维连接蛋白单抗主要与440 kDa分子反应，推测在患者和对照组中代表完整的纤维连接蛋白。然而，仅在患者中，抗纤维粘连蛋白C末端区域的单抗才显示出从480 kDa到330 kDa的广泛和强烈的反应。用抗人IgA包被板捕获的血清中检测到纤维连接蛋白片段的ELISA实验也证实了这些反应性。因此，患者血清中的IgA与纤维连接蛋白(片段)共存。结论：IgA肾病患者血清中的IgA与纤维连接蛋白片段结合形成大分子的IgA，通过外源性纤维连接蛋白组装成细胞外基质的机制促进IgA沉积在系膜细胞外基质上。较少