MESANGIAL REGULATION OF GLOMERULAR LEUKOCYTE TRAFFIC

这个项目的长期目标是制定一个明确的 对肾小球单核细胞浸润过程的认识 被认为是急性肾小球功能障碍的重要原因 慢性肾小球硬化症。这个实验室已经证明了 单核细胞单核细胞趋化蛋白-1的产生 肾小球系膜细胞可诱导特异性趋化性多肽 细胞。由此推测，肾小球系膜细胞 通过产生单核细胞趋化蛋白-1调节单核细胞的运输。这一假设 最初将使用体外肾小球损伤模型进行研究， 通过暴露培养的人肾小球系膜细胞来补充 成分，免疫复合体和脂蛋白，刺激物 被证明在活体内引发肾小球损伤。系膜MCP-1的表达 将通过系膜mRNA的Northern分析进行评估。单核细胞趋化蛋白-1分泌 进入培养上清液将通过免疫吸附确认与 一种特异性的抗人MCP-1β抗体，并具有功能活性 用单核细胞趋化试验测定。为了理解为什么会这样 不同的刺激可以激活系膜MCP-1的合成，这是一种常见的 将寻求行动机制。这些可能性将是 研究内容包括：1)刺激诱导另一系膜细胞 最终调控MCP-1基因的细胞因子(如IL-1、PDGF) 表达，以及2)通过共同的第二信使的信号转导 系统(如蛋白激酶C)。该模型还将用于 研究趋化抑制物的系膜精细加工，提供 肾小球系膜细胞调控作用的进一步证据 肾小球炎症。抗炎药在本病中的作用 将对系统进行检查，以测试MCP-1的敏感性 对药物干预的表达。进一步的研究将是 使用从人肾活检标本中获得的组织进行 啮齿动物的肾脏。人肾小球中存在单核细胞趋化蛋白-1的证据 将使用肾脏的免疫组织化学分析来寻找疾病 活组织检查材料；MCP-1mRNA的原位杂交也将 用于确定MCP-1的肾小球来源为系膜 手机。肾小球单核细胞趋化蛋白-1表达的时程变化 将评估肾小球肾炎的诱因(通过Northern分析 和原位杂交)在单核细胞依赖的啮齿动物模型中 肾小球损伤，并与不断演变的白细胞浸润相关。 最后，将给啮齿动物注射MCP-1的中和抗体 诱导肾小球肾炎试图消除肾小球疾病 单核细胞的渗透和随后的蛋白尿的发展。 因此，这些研究将确定肾小球系膜细胞在 肾小球损伤初期的调节，以及文献 新的促炎细胞因子MCP-1在新兵招募中的重要性 单核细胞进入肾小球。了解肾小球系膜病的机制 单核细胞趋化蛋白-1的表达可为治疗调控提供依据 这种细胞因子的释放，从而防止或改善急性 肾小球损害和进展性肾功能不全 渗入单核细胞。

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.