MEDIATION OF ANTIBODY-INDUCED GLOMERULAR INJURY

这个项目的长期目标是定义体液机制 肾小球损伤。目前的注意力集中在内脏上 肾小球上皮细胞(GEC)作为抗体靶点， 补体介导的损伤。这项提议利用了被动的海曼 大鼠肾炎(PHN)，一种忠实地复制许多特征的模型 人类膜性肾病。 具体目标1基于两个观察结果。鱼油饲料 改善PHN肾小球损伤，补体致膜 磷脂的降解与细胞内钙的动员 培养的血管内皮细胞。提出了三组实验来检验 鱼油的有益效果。大鼠肾小球内皮细胞的细胞膜磷脂 在培养中将富含omega-3(n-3)或omega-6(n-6) 脂肪酸和对补体攻击的反应将在 条款：它们对亚致死性细胞毒性的敏感性； 磷脂水解和细胞内钙水平；以及 细胞膜流动性。N-3脂肪酸的功能作用 不受系统性影响的目标GEC的替代将是 在分离的灌流大鼠肾脏中进行检查。肾小球血流动力学 将通过微穿刺法和大分子法在体内研究效果。 筛分技术。 特殊目的2旨在确定GEC细胞骨架是否发挥作用 在伴随而来的形态和功能变化中的作用 补体介导的PHN损伤。形态、标签和 功能技术，以及破坏或稳定的试剂 微丝和微管将被用来研究 细胞培养和分离培养中对GEC细胞骨架的补充 肾脏。 特异性目标3检查抗体和补体在 PHN细胞外基质的产生。这项工作将初步延长 肾小球周围细胞占据PHN的表现 选择性表达的与探针原位杂交的mRNA转录本 对于I型胶原蛋白的A1链。计划进行两组研究。 首先，将在体内进行研究，以确定I型的时间进程 胶原蛋白mRNA表达及其与IV型胶原表达的关系 胶原蛋白和层粘连蛋白的原位杂交。来自PHN的RNA和对照 肾小球将通过Northern杂交和核糖核酸酶保护进行分析 检测并与大鼠成纤维细胞的RNA进行比较，以确定 杂交是为了获得真正的I型胶原mRNA。免疫检测 将使用技术来确定I型胶原是否被嵌入 进入基底膜或分泌到尿腔。第二, 将培养的大鼠肾小管上皮细胞用于建立体外模型进行研究 补体可能影响基质合成的信号通路。

The long-term goals of this program are to define the humoral mechanisms of glomerular damage. Attention is presently focused on the visceral glomerular epithelial cell (GEC) as the target of antibody-directed, complement-mediated injury. The proposal utilizes passive Heymann nephritis (PHN) in rats, a model that faithfully reproduces many features of human membranous nephropathy. Specific aim 1 is based on two observations. Fish oil feeding ameliorates glomerular injury in PHN, and complement causes membrane phospholipid hydrolysis and intracellular calcium mobilization in cultured GECs . Three groups of experiments are proposed to examine the beneficial effects of fish oil. Cell membrane phospholipids of rat GECs in culture will be enriched with either omega-3 (n-3) or omega-6 (n-6) fatty acids and the response to complement attack will be compared in terms of: their susceptibility to sublethal cytotoxicity; alterations in phospholipid hydrolysis and intracellular calcium levels; and changes in cell membrane fluidity. The functional effects of n-3 fatty acid substitution of target GECs, independent of systemic influences, will be examined in the isolated perfused rat kidney. Glomerular hemodynamic effects will be studied in vivo by micropuncture and macromolecular sieving techniques. Specific aim 2 is designed to determine if the GEC cytoskeleton plays a role in the morphological and functional changes that accompany complement-mediated injury in PHN. Morphological, labelling and functional techniques, and agents that either disrupt or stabilize microfilaments and microtubules will be used to study the effect of complement on the GEC cytoskeleton in cell culture and in the isolated kidney. Specific aim 3 examines the role of antibody and complement on extracellular matrix production in PHN. The work will extend preliminary findings in which cells occupying the periphery of PHN glomeruli selectively expressed mRNA transcripts that hybridize in situ to a probe for the a1 chain of type I collagen. Two groups of studies are planned. First, studies will be done in vivo to define the time course of type I collagen mRNA expression and relate it to the expression of type IV collagen and laminin by in situ hybridization. RNA from PHN and control glomeruli will be analyzed by Northern blot and ribonuclease protection assays and compared to RNA from rat fibroblasts to determine if hybridization is to genuine type I collagen mRNA. Immunodetection techniques will be used to determine if type I collagen is intercalated into the basement membrane or secreted into the urinary space. Second, rat GECs in culture will be used to develop an in vitro model to study signalling pathways by which complement may influence matrix synthesis.