New Animal Model for Studies of mucosal Immunity and IgA Nephropathy

描述（由申请人提供）：其特征在于来源于含有异常糖基化的IgA 1的循环免疫复合物的系膜IgA 1免疫沉积，即，具有半乳糖（Gal）缺陷型O-聚糖的IgA 1。一些证据表明异常糖基化，形成含有异常糖基化的IgA 1的免疫复合物，它们在系膜中的沉积和IgAN的肾损伤之间存在直接的因果关系。我们确定，IgA肾病患者的IgA 1产生细胞分泌的IgA 1与半乳糖缺乏的O-聚糖，这种异常是由于在特定的糖基转移酶的表达/活性失调。这些结果表明，缺乏半乳糖的IgA 1在IgA肾病的发病机制中起着关键作用。了解IgAN中IgA 1碳水化合物含量变化的分子基础对于定义导致IgAN中异常聚糖合成的基本缺陷至关重要。然而，由于从人粘膜组织、淋巴结或骨髓中获得足够的感兴趣的细胞是不可行的，并且由于缺乏动物模型，因为IgA 1仅存在于人类和类人猿灵长类动物中，因此这些研究受到了挫折。作为开发鼠模型的第一步，我们现在已经表明，在体外制备的人Gal缺陷型IgA 1和抗聚糖IgG之间的免疫复合物和i. v.注射到裸鼠体内，存款在系膜中并引起血尿和蛋白尿。因为小鼠不具有具有铰链区O-聚糖的伊加，所以我们假设产生具有含有人铰链区的伊加的转基因小鼠将导致含有O-连接聚糖的鼠伊加，其可以被操纵以类似于异常的人IgA 1。这种转基因小鼠品系将代表一种新的工具，研究的作用，O-聚糖在IgAN和粘膜免疫。我们建议建立一个敲入转基因小鼠品系，生产伊加与铰链区从人IgA 1，评估其O-糖基化，并通过抑制关键酶-21，3-半乳糖基转移酶，产生的Gal缺陷的O-聚糖的小鼠转基因伊加。此外，我们将确定是否由小鼠转基因Gal缺陷型伊加和聚糖特异性IgG组成的免疫复合物存款在小鼠肾系膜中引起肾病。我们将通过对肾组织进行组织学分析和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正反映人类疾病的动物模型。这种转基因小鼠将为检测参与异常O-糖基化伊加及其系膜沉积和清除的遗传和生化机制开辟新的可能性。相关性：IgAN是最常见的原发性肾小球肾炎，导致20%至40%的患者发生终末期肾衰竭。目前对IgAN发病机制的理解存在差距，这是发展IgAN特异性治疗的主要障碍。拟议的研究将提供基础，为未来发展的相关生理动物模型的IgAN，将促进人类IgAN的发病机制的理解。 公共卫生相关性： 我们建议建立IgAN的小鼠模型。我们将产生转基因小鼠，其中伊加将包括含有O-连接聚糖的人铰链区。我们将进一步在鼠转基因伊加上产生缺乏Gal的O-聚糖，并使用抗聚糖IgG形成致病复合物，其将存款在肾脏中。

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.