MEDIATION OF ANTIBODY INDUCED GLOMERULAR INJURY

描述（改编自研究者摘要）：本申请 建议检查抗体诱导的肾小球损伤现象 在缺乏补体结合的情况下会发生。 研究者已 先前确定绵羊肾毒性血清诱导蛋白尿， 大鼠和小鼠在肾小球中不存在补体结合的情况下，以及 进一步证明了这种多克隆抗体的主要靶点是 肾小球上皮细胞中的β 1整合素。 目前的目标是 研究的目的是确定这种抗β 1整合素活性是否 肾毒血清是必要的，足以产生所观察到的 蛋白尿，或是否有其他或替代抗原 对这种影响负责。 第一个具体目标将审查是否 抗β 1整联蛋白抗体活性是必需的且足以 导致肾小球损伤。 这将使用免疫吸附进行纯化 此外，将产生融合蛋白的抗体 β 1整合素和α 3整合素的外部结构域（可能的α 肾小球上皮细胞中β 1整联蛋白的伴侣）， 将在体内测试这些抗体的肾毒性，并且还将在体内测试这些抗体的肾毒性。 重要的是，在最初尝试映射的致病性表位， 肾毒性血清，如果β 1整合素确实被确定为 主要或唯一的抗原位点。 第二个具体目标是审查 肾毒性血清的抗整合素特性可能是 必要但不足以引起肾损伤，或者是一种附带现象。 这些实验旨在确定其他尚未确定的 肾小球抗原有助于肾发生效应。 免疫纯化和分子生物学技术将用于 鉴定其他可能的肾小球细胞膜蛋白， 有助于效果。 此外， IV型胶原的α 3链的非胶原结构域将被 作为一种可能的抗原。 第三个具体目标将审查 肾毒血清与肾小球上皮细胞结合的假说 细胞激活事件，通过改变肾小球的通透性选择性， 细胞与基底膜的关系。 这一具体目标 如果第一个特定目标的实验确定了 肾毒血清的抗整合素作用是 肾小球的改变 在这一修订后的具体目标中，研究将 在大鼠肾小球上皮细胞的培养系统中进行， 在可渗透的支持物上。 将检查暴露于抗体的细胞， 蛋白质的分布和磷酸化状态的改变， 参与整合素介导的细胞和细胞外 基体附着力 细胞骨架元件和蛋白质的形态 将它们与整合素联系起来的蛋白质也将被检查， 肾毒血清诱导的大分子通透性。 整合素相关 将通过磷脂水解和第二信使分析信号传导 生产 最后，研究人员将检查是否有抗原 暴露于抗体后脱落，或抗原是否被内吞 对抗体作出反应。 这些研究旨在进一步调查， 肾小球上皮细胞作为损伤的主要靶点的作用 蛋白尿性肾病，并确定整合素的作用， 维持正常的肾小球结构和功能。

DESCRIPTION (Adapted from Investigators Abstract): This application proposes to examine the phenomenon of antibody-induced glomerular injury that can occur in the absence of complement fixation. The investigator has previously determined that sheep nephrotoxic serum induces proteinuria in rats and mice in the absence of complement fixation in the glomerulus, and has further demonstrated that a major target of this polyclonal antibody is beta1 integrins in glomerular epithelial cells. The goal of the present studies is to determine whether the anti-beta1 integrin activity of this nephrotoxic serum is necessary and sufficient to produce the observed proteinuria, or whether there are additional or alternative antigens responsible for this effect. The first specific aim will examine whether the anti beta1 integrin antibody activity is necessary and sufficient to induce the glomerular injury. This will be purified using immunoabsorption techniques and in addition, antibodies will be produced to fusion proteins of external domains of beta1 integrin and alpha3 integrin (the likely alpha partner to the beta1 integrin in glomerular epithelial cells), and the nephrotoxicity of these antibodies will be tested in vivo, and will also be important in initial attempts to map the pathogenic epitope of the nephrotoxic serum, if indeed the beta1 integrin is determined to be the major or only antigenic site. The second specific aim examines the possibility that anti-integrin properties of nephrotoxic serum may be necessary but insufficient to cause renal injury, or is an epiphenomenon. These experiments are designed to determine how other as yet undefined glomerular antigens contribute to the nephrogenic effect. Immunopurification and molecular biological techniques will be used to identify other possible glomerular cell membrane proteins that may contribute to the effects. In addition, the possibility that the noncollagenous domain of the alpha3 chain of type IV collagen will be examined as a possible antigen. The third specific aim will examine the hypothesis that the binding of nephrotoxic serum to glomerular epithelial cells activates events that disrupt glomerular permselectivity by altering the relationship between the cells and basement membrane. This specific aim will be important if the experiments in the first specific aim determine that the anti-integrin effect of nephrotoxic serum is primarily responsible for the glomerular alterations. In this revised specific aim, studies will be performed in a cultured system of rat glomerular epithelial cells grown on permeable supports. Cells exposed to antibodies will be examined for alterations in distribution and phosphorylation status of proteins that are involved in integrin mediated formation of cell and cell extracellular matrix adhesion. The morphology of cytoskeletal elements and the proteins that link them to integrins will also be examined in relation to the altered macromolecular permeability induced by nephrotoxic serum. Integrin-related signaling will be analyzed by phospholipid hydrolysis and second messenger production. Finally, the investigator will examine whether there is antigen shedding after exposure to antibody, or whether the antigen is endocytosed in response to antibody. These studies are designed to investigate further, the role of the glomerular epithelial cell as a primary target of injury in proteinuric renal diseases, and to define the role of integrins in the maintenance of normal glomerular structure and function.