MEDIATION OF ANTIBODY INDUCED GLOMERULAR INJURY

描述(改编自《调查人员摘要》)：此应用程序 建议研究抗体诱导的肾小球损伤现象 在没有补体结合的情况下可能会发生这种情况。调查员已经 先前确定的绵羊肾毒性血清诱导蛋白尿 大鼠和小鼠在肾小球中没有补体结合，以及 进一步证明了这种多克隆抗体的一个主要靶点是 肾小球上皮细胞中的β1整合素。当前的目标是 研究目的是确定该化合物的抗β1整合素活性 肾毒性血清是产生所观察到的 蛋白尿，或是否有额外或替代的抗原 对这一影响负有责任。第一个具体目标将检查是否 抗β1整合素抗体活性是必要且充分的 诱导肾小球损伤。这将使用免疫吸收进行提纯 此外，还将产生针对融合蛋白的抗体。 β1整合素和α3整合素的外部结构域(可能是 与肾小球上皮细胞中的β1整合素配对)，以及 这些抗体的肾脏毒性将在体内进行测试，并将 在初步尝试绘制该病毒的致病表位图时很重要 肾毒性血清，如果β1整合素确实被确定为 主要或唯一的抗原性部位。第二个具体目标是检查 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。 这些实验的目的是确定其他尚未确定的 肾小球抗原参与了肾脏的发生作用。 免疫提纯和分子生物学技术将用于 确定其他可能的肾小球细胞膜蛋白 为效果做出贡献。此外，有可能 IV型胶原的α3链的非胶原区将是 被检查为可能的抗原。第三个具体目标将审查 肾毒性血清与肾小球上皮细胞结合的假说 细胞通过改变肾小球通透性来激活破坏肾小球通透性的事件 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 肾毒性血清的抗整合素作用是主要原因 对于肾小球的改变。在这一修订的具体目标中，研究将 在培养的大鼠肾小球上皮细胞培养系统中进行 在可渗透支撑物上。暴露在抗体中的细胞将被检查 蛋白质的分布和磷酸化状态的变化 参与整合素介导的细胞和细胞外形成 基质粘附性。细胞骨架元素和蛋白质的形态 它们与整合素的联系也将被检查与改变的 肾毒性血清诱导的大分子通透性。整合素相关 信号将通过磷脂水解和第二信使进行分析 制作。最后，调查员将检查是否存在抗原 暴露于抗体后的脱落，或抗原是否内吞 对抗体的反应。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在糖尿病中的作用 蛋白尿性肾脏疾病，并确定整合素在 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.