GLOMERULAR MATRIX DEGRADING METALLOPROTEINASES

我们对孤立肾小球的研究发现了一种独特的 金属蛋白酶--一种与其他经典的 基质降解金属蛋白酶的几个方面，包括缺乏 金属蛋白酶组织抑制剂的抑制作用。肾小球 金属蛋白酶是肾小球基底膜的主要成分。 中性pH下的降解活性表明其在 肾小球病理生理学。具体目标：A.净化和 描述肾小球金属蛋白酶的特征。因为肾小球 酶是膜结合的，它会被溶解然后纯化 使用包括离子在内的各种层析技术的组合 交换、羟基磷灰石、凝集素亲和力和明胶底物亲和力 层析法。纯化的肾小球酶将被鉴定 生物化学；用于获得N-末端的氨基酸序列和一些 内胰蛋白酶多肽(特异靶C)，用于培养多克隆 抗体(特异性靶标B)。B.免疫定位和免疫筛选 用纯化的肾小球酶的多克隆抗体。 针对纯化的酶产生的多克隆抗体将用于 用免疫细胞化学方法确定酶在细胞中的定位 亚细胞器的类型和定位/S。多克隆 抗体还将用于a)免疫筛查大鼠肾脏和/或 肾小球lambda/gt11 cDNA文库(特异靶C)和，b)至 用酶联免疫吸附试验测定疾病模型中的酶蛋白含量 比色法和Western blotting(特异靶D)。C.为了获得DNA探针和 分离肾小球酶的全长c DNA。DNA探针基于 纯化产物的部分N-末端和内部氨基酸序列 肾小球酶，将被开发并用于筛查lambda/gt10大鼠 肾和/或肾小球cDNA库，以分离出一个cDNA克隆。 此外，本研究中开发的探针，包括全长 Cdna，将成为检测疾病中mrna水平的有价值的工具。 模型以及未来有关调节的研究 酵素。D.疾病模型研究。这些研究的活体研究 两种非炎症性肾小球疾病模型 氨基核苷(PAN)模型，一种微小病变疾病的模型 人和被动型Heymann肾炎模型、膜性肾炎模型 肾病，将诱导和在不同时间点的mRNA在 肾小球将使用Northern印迹和原位检测 杂交。此外，肾小球酶蛋白(使用 用多克隆抗体开发的化验)以及酶 活跃度，将在这些模型中进行衡量。肾小球的体外研究 上皮细胞(GEC)似乎是血管内皮细胞损伤的重要部位。 泛型和被动型Heymann肾炎模型。腐霉素类药物的抗炎作用 抗Fx1a抗体(含和不含补体)对细胞分泌的影响 培养基中酶活性和蛋白质含量，以及培养基中酶活性 将测定细胞液和细胞膜的部分。此外，在选定的位置 时间点将检测对GEC基因表达的影响。

Our studies in isolated glomeruli have identified a distinct metalloproteinase which is strikingly different from other classical matrix degrading metalloproteinases in several respects including lack of inhibition by tissue inhibitor of metalloproteinase. The glomerular metalloproteinase accounts for most of the glomerular basement membrane degrading activity at neutral pH suggesting its potential importance in glomerular pathophysiology. Specific Aims: A. To purify and characterize the glomerular metalloproteinase. Since the glomerular enzyme is membrane associated, it will be solubilized and then purified using a combination of various chromatographic techniques including ion exchange, hydroxylapatite, lectin affinity and gelatin substrate affinity chromatography. The purified glomerular enzyme will be characterized biochemically; used to obtain amino acid sequence of N-terminal and some internal tryptic peptides (specific aim C), and used to raise polyclonal antibody (specific aim B). B. Immunolocalization and immunoscreening using polyclonal antibody against the purified glomerular enzyme. Polyclonal antibody raised against the purified enzyme will be used to determine by immunocytochemistry the localization of the enzyme by cell type and localization in subcellular organelle/s. The polyclonal antibody will also be used a) for immunoscreening rat kidney and/or glomerular lambda/gt11 cDNA libraries (specific aim C) and, b) to determine the enzyme protein content in the disease models by an ELISA assay and Western blotting (specific aim D). C. To obtain DNA probes and isolate full length cDNA of the glomerular enzyme. DNA probes based on partial N-terminal and internal amino acid sequences of the purified glomerular enzyme, will be developed and used to screen lambda/gt10 rat kidney and/or glomerular cDNA libraries in order to isolate a cDNA clone. In addition, probes developed in this study, including the full length cDNA, will be valuable tools in determining the mRNA levels in disease models as well as in future studies related to the regulation of the enzyme. D. Studies in disease models. In vivo studies For these studies two non-inflammatory glomerular disease models, the puromycin aminonucleoside (PAN) model, a model for minimal change disease in humans, and the passive Heymann nephritis model, a model for membranous nephropathy, will be induced and at various time points the mRNA in glomeruli will be examined using Northern blotting and in situ hybridization. In addition, the glomerular enzyme protein (using the assay developed with the polyclonal antibodies), as well as the enzyme activity, will be measured in these models. In vitro studies Glomerular epithelial cells (GEC) appear to be an important site of injury in the PAN and passive Heymann nephritis models. The effect of puromycin and anti-Fx1A antibody (with and without complement) on secretion of the enzyme activity and protein in the medium, and the enzyme activity in cytosolic and membrane fractions will be determined. Also, at selected time points the effect on mRNA in GEC will be examined.