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New Animal Model for Studies of mucosal Immunity and IgA Nephropathy

研究粘膜免疫和 IgA 肾病的新动物模型

基本信息

批准号：
7787214
负责人：
JAN NOVAK
金额：
$ 18.31万
依托单位：
UNIVERSITY OF ALABAMA AT BIRMINGHAM
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-07-01 至 2012-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7787214
关键词：
Acetylgalactosamine Animal Model Antibodies Antigen-Antibody Complex Binding Biochemical Blood Circulation Bone Marrow Carbohydrates Cell Line Cells Characteristics Clinical Complex Defect Deposition Development Diagnosis End stage renal failure Enzyme-Linked Immunosorbent Assay Enzymes Epitopes Exoglycosidases Exons Experimental Animal Model Exposure to Foundations Future Galactose Galactosyltransferases Gas Chromatography Gene Targeting Generations Genes Genetic screening method Glomerular Mesangial Cell Glomerulonephritis Glycopeptides Hematuria Human IgA1 Immune Immunoglobulin A Immunoglobulin G In Vitro Injection of therapeutic agent Injury Kidney Kidney Diseases Knock-in Mouse Laboratories Link Modeling Molecular Mouse Strains Mucosal Immunity Mucous Membrane Mus Nude Mice Pathogenesis Patients Physiological Play Polysaccharides Positioning Attribute Preparation Primates Production Proteinuria Renal Tissue Research Design Role Serum Site Small Interfering RNA Splenocyte Structure of glomerular mesangium Study models Testing Transgenic Mice Transgenic Organisms Urine Variant Western Blotting Wild Type Mouse base embryonic stem cell glycosylated IgA glycosylation glycosyltransferase human disease in vivo inhibitor/antagonist interest lymph nodes molecular mass novel public health relevance research study tool

项目摘要

DESCRIPTION (provided by applicant): is characterized by mesangial IgA1 immune deposits that originate from circulating immune complexes containing aberrantly-glycosylated IgA1, i.e., IgA1 with galactose (Gal)-deficient O-glycans. Several lines of evidence suggest a direct causal relationship between aberrant glycosylation, the formation of immune complexes containing aberrantly glycosylated IgA1, their deposition in the mesangium, and renal injury in IgAN. We determined that IgA1-producing cells of IgAN patients secrete IgA1 with Gal-deficient O- glycans and that this aberrancy is due to dysregulation in expression/activity of specific glycosyltransferases. These findings indicate that Gal-deficient IgA1 plays a pivotal role in the pathogenesis of IgAN. An understanding of the molecular basis for the variations in the carbohydrate content of IgA1 in IgAN is essential for the definition of the fundamental defects that result in the synthesis of the aberrant glycans in IgAN. Such studies have been frustrated, however, by the fact that it is not feasible to obtain sufficient cells of interest from human mucosal tissues, lymph nodes, or bone marrow and by the lack of animal models, as IgA1 is present exclusively in humans and hominoid primates. As a first step toward developing a murine model, we have now shown that immune complexes, prepared in vitro between human Gal-deficient IgA1 and anti-glycan IgG and i.v.-injected to nude mice, deposit in the mesangium and induce hematuria and proteinuria. Because mice do not have IgA with hinge-region O-glycans, we hypothesized that generating transgenic mice with IgA containing the human hinge region would result in murine IgA containing O-linked glycans that can be manipulated to resemble the aberrant human IgA1. Such a transgenic mouse strain would represent a new tool for studies of the role of the O-glycans in IgAN and in mucosal immunity. We propose to generate a knock-in transgenic mouse strain producing IgA with hinge region from human IgA1, assess its O-glycosylation, and, by inhibiting the key enzyme - 21,3-galactosyltransferase, to generate Gal-deficient O-glycans on the murine transgenic IgA. Furthermore, we will determine whether the immune complexes composed of murine transgenic Gal-deficient IgA and glycan-specific IgG deposit in renal mesangium of mice causing nephropathy. We will further develop and validate this first animal model truly reflecting the human disease by performing histological analysis of renal tissue and laboratory analysis of urine and serum. This transgenic mouse will open new possibilities for testing genetic and biochemical mechanisms involved in production of aberrantly O- glycosylated IgA and its mesangial deposition and clearance. Relevance: IgAN is the most common primary glomerulonephritis and leads to end-stage renal failure in 20% to 40% of patients. The current gaps in the understanding of the pathogenesis of IgAN represent a major barrier to the development of IgAN-specific treatments. The proposed studies will provide the foundation for the future development of a relevant physiological animal model of IgAN that will advance the understanding of the pathogenesis of human IgAN. PUBLIC HEALTH RELEVANCE: We propose to develop a murine model of IgAN. We will generate transgenic mice in which IgA will include the human hinge region containing O-linked glycans. We will further generate Gal-deficient O-glycans on the murine transgenic IgA and use anti-glycan IgG to form pathogenic complexes that will deposit in the kidneys.

描述(由申请人提供)：以系膜免疫沉积为特征，其起源于含有异常糖基化的IgA1的循环免疫复合体，即具有半乳糖(Gal)缺陷的O-糖链的IgA1。多条证据表明，异常糖基化、含有异常糖化免疫球蛋白A1的免疫复合体的形成、它们在系膜中的沉积与IgAN的肾脏损伤之间存在直接的因果关系。我们确定，IgAN患者的IgA1产生细胞分泌Gal缺乏O-糖链的IgA1，这种异常是由于特定糖基转移酶的表达/活性失调所致。这些发现表明Gal缺乏的IgA1在IgAN的发病机制中起着关键作用。了解IgAN中IgA1糖类含量变化的分子基础，对于确定导致IgAN中异常糖链合成的基本缺陷是至关重要的。然而，这些研究一直受挫，因为从人类粘膜组织、淋巴结或骨髓中获取足够的感兴趣细胞是不可行的，而且缺乏动物模型，因为IgA1仅存在于人类和类人灵长类动物中。作为建立小鼠模型的第一步，我们现在已经证明，在体外制备的人半乳糖缺乏的IgA1和抗多糖Ig G之间的免疫复合物，以及静脉注射到裸鼠身上，会沉积在系膜上，并诱导血尿和蛋白尿。由于小鼠没有带有铰链区O-糖链的IgA，我们假设，产生含有人铰链区的IgA转基因小鼠将导致含有O-连接糖链的小鼠IgA，可以被操纵成类似于异常的人类IgA1。这样的转基因小鼠品系将为研究O-糖链在IgAN和粘膜免疫中的作用提供新的工具。我们建议从人的IgA1中产生带有铰链区的敲入转基因小鼠株系，评估其O-糖基化，并通过抑制关键酶-21，3-半乳糖基转移酶，在转基因小鼠的IgA上产生缺乏半乳糖的O-糖链。此外，我们还将确定转基因小鼠Gal缺陷IgA和葡聚糖特异性IgG组成的免疫复合体是否沉积在导致肾病的小鼠的肾小球系膜上。我们将通过对肾脏组织进行组织学分析以及对尿液和血清进行实验室分析，进一步开发和验证这第一个真实反映人类疾病的动物模型。这一转基因小鼠将为测试异常O-糖基化IgA的产生及其系膜沉积和清除所涉及的遗传和生化机制开辟新的可能性。相关：IGAN是最常见的原发性肾小球肾炎，在20%到40%的患者中会导致终末期肾功能衰竭。目前对IgAN发病机制的认识存在差距，这是发展针对IgAN的治疗的主要障碍。这些研究将为今后建立相关的免疫球蛋白肾病动物模型奠定基础，从而促进对人类免疫球蛋白肾病发病机制的认识。公共卫生相关性：我们建议建立一种免疫球蛋白肾病的小鼠模型。我们将产生转基因小鼠，其中IgA将包括含有O-连接糖链的人类铰链区。我们将在转基因小鼠的IgA上进一步产生缺乏半乳糖的O-糖链，并使用抗葡聚糖免疫球蛋白形成致病复合体，这些复合体将沉积在肾脏中。