MEDIATION OF ANTIBODY-INDUCED GLOMERULAR INJURY

该项目的长期目标是确定体液机制 肾小球损伤 目前的注意力集中在内脏 以肾小球上皮细胞（GEC）为靶点， 补体介导的损伤。 该提案利用被动海曼 肾炎（PHN）的大鼠，一个模型，忠实地再现了许多特点 人类膜性肾病 具体目标1基于两项观察。 鱼油喂养 改善PHN肾小球损伤，补体引起膜 磷脂水解和细胞内钙动员 培养的GECs。提出了三组实验来检查 鱼油的好处 大鼠GECs的细胞膜磷脂 将富含ω-3（n-3）或ω-6（n-6） 脂肪酸和对补体攻击的反应将在 术语：对亚致死细胞毒性的敏感性; 磷脂水解和细胞内钙水平的变化; 细胞膜流动性 n-3脂肪酸的功能作用 目标GEC的替代，独立于系统性影响，将是 在离体灌流的大鼠肾脏中检查。 肾小球血流动力学 将通过显微穿刺和大分子生物学方法在体内研究其作用。 筛分技术 具体目标2旨在确定GEC细胞骨架是否发挥作用， 在伴随的形态和功能变化中的作用 补体介导的PHN损伤。 形态学、标记和 功能性技术，以及破坏或稳定 微丝和微管将被用来研究的影响， 补体在细胞培养物中和分离的GEC细胞骨架中的作用 肾 特异性目的3检查抗体和补体在 PHN中的细胞外基质产生。 这项工作将初步扩大 发现细胞占据PHN肾小球周围 与探针原位杂交的选择性表达的mRNA转录物 I型胶原蛋白的α 1链。 计划进行两组研究。 首先，将在体内进行研究，以确定I型的时间过程 胶原mRNA表达及其与IV型胶原表达的关系 胶原和层粘连蛋白的原位杂交。 来自PHN和对照的RNA 肾小球将通过北方印迹和核糖核酸酶保护进行分析 测定并与来自大鼠成纤维细胞的RNA进行比较，以确定 与真正的I型胶原mRNA杂交。 免疫检测 技术将用于确定I型胶原是否嵌入 进入基底膜或分泌到泌尿空间。 第二、 培养的大鼠GEC将用于开发体外模型以研究 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.