MESANGIAL REGULATION OF GLOMERULAR LEUKOCYTE TRAFFIC

该项目的长期目标是制定一个明确的 了解肾小球单核细胞浸润的过程， 被认为是急性肾小球功能障碍的重要原因， 慢性肾小球硬化 该实验室表明， 单核细胞趋化蛋白-1（MCP-1）的产生， 特异性趋化肽，可在肾小球系膜中诱导 细胞 因此推测肾小球系膜细胞 通过产生MCP-1调节单核细胞运输。 这一假设 最初将使用肾小球损伤的体外模型进行研究， 通过将培养的人肾小球系膜细胞暴露于补体 成分，免疫复合物和脂蛋白，刺激物， 显示在体内引发肾小球损伤。 系膜MCP-1表达 将通过系膜mRNA的北方分析来评估。 mcp-1分泌 将通过免疫吸附确认进入培养物上清液， 特异性抗人MCP-1 β抗体，其功能活性将 可以使用单核细胞趋化性测定来测量。 为了了解这种 不同的刺激可以激活系膜MCP-1的合成，一种常见的 将寻求行动机制。 可能会是 研究包括：1）刺激诱导另一个系膜 最终调节MCP-1基因的细胞因子（如IL-1、PDGF） 表达，以及2）通过共同第二信使的信号传导 系统（例如蛋白激酶C）。 该模型还将用于 研究趋化性抑制剂的系膜加工，提供 进一步的证据表明，系膜细胞在 肾小球炎症 抗炎药在这方面的作用 系统将被检查，以测试MCP-1的敏感性 表达到药物干预。 进一步的研究将 使用从人肾活检标本获得的组织进行， 啮齿动物的肾脏 MCP-1在人肾小球中存在的证据 疾病将寻求使用免疫组织化学分析的肾 活检材料; MCP-1 mRNA的原位杂交也将被用于 将MCP-1的肾小球来源鉴定为系膜细胞， cell. 肾小球MCP-1 mRNA表达的时间过程， 将评估肾小球肾炎的诱导（通过北方分析 和原位杂交）在单核细胞依赖性 肾小球损伤，并与不断发展的白细胞浸润。 最后，MCP-1的中和抗体将被给予啮齿动物， 诱导肾小球肾炎，试图消除 单核细胞浸润和随后的蛋白尿的发展。 因此，这些研究将确定系膜细胞在肾小球疾病中的核心作用。 肾小球损伤的初始阶段的调节，以及文献 新的促炎细胞因子MCP-1在招募 单核细胞到肾小球。 了解系膜细胞的机制 MCP-1的表达可以为治疗性调节 这种细胞因子的释放，从而预防或改善急性 肾小球损伤和进行性肾功能不全 浸润单核细胞。

The long term objective of this project is to develop a clear understanding of the process of glomerular monocyte infiltration, which is believed to be an important cause of acute glomerular dysfunction and chronic glomerular sclerosis. This laboratory has shown that the production of monocyte chemoattractant protein-1 (MCP-1), a monocyte specific chemotactic peptide, may be induced in glomerular mesangial cells. It was thus postulated that the glomerular mesangial cell regulates monocyte traffic via the production of MCP-1. This hypothesis will initially be studied using an in vitro model of glomerular injury, created by exposing cultured human mesangial cells to complement components, immune complexes, and lipoproteins, stimuli which have been shown to initiate glomerular injury in vivo. Mesangial MCP-1 expression will be assessed by Northern analysis of mesangial mRNA. MCP-1 secretion into the culture supernatants will be confirmed by immunoadsorption with a specific anti-human MCP-1beta antibody, and functional activity will be measured using a monocyte chemotaxis assay. To understand how such diverse stimuli may activate mesangial MCP-1 synthesis, a common mechanism of action will be sought. Possibilities which will be investigated include: 1) stimulus induction of another mesangial cytokine (e.g. IL-1, PDGF) which ultimately regulates MCP-1 gene expression, and 2) signal transduction through a common second messenger system (e.g. protein kinase C). this model will also be used to investigate mesangial elaboration of chemotaxis inhibitors, providing further evidence for a regulatory role of the mesangial cell in glomerular inflammation. The effect of anti-inflammatory agents in this system will be examined in order to test the susceptibility of MCP-1 expression to pharmacologic interventions. Further studies will be conducted using tissue obtained from human renal biopsy specimens and rodent kidneys. Evidence for the presence of MCP-1 in human glomerular disease will be sought using immunohistochemical analysis of the renal biopsy material; in situ hybridization for MCP-1 mRNA will also be performed to identify the glomerular source of MCP-1 as the mesangial cell. The time course of glomerular MCP-1 mRNA expression following induction of glomerulonephritis will be assessed (by Northern analysis and in situ hybridization) in a rodent model of monocyte-dependent glomerular injury, and correlated to the evolving leukocyte infiltration. Finally, neutralizing antibodies to MCP-1 will be given to rodents during the induction of glomerulonephritis in an attempt to abrogate the monocyte infiltration and the subsequent development of proteinuria. These studies will thus define a central role for the mesangial cell in the regulation of the initial phase of glomerular injury, and document the importance of the novel pro-inflammatory cytokine MCP-1 in recruiting monocytes to the glomerulus. Understanding the mechanisms of mesangial expression of MCP-1 could provide a basis for therapeutically modulating the release of this cytokine, thus preventing or ameliorating the acute glomerular damage and progressive renal insufficiency caused by infiltrating monocytes.