GLOMERULAR MATRIX DEGRADING METALLOPROTEINASES

我们在离体肾小球中的研究已经确定了一种独特的 金属蛋白酶，这是显着不同于其他经典的 基质降解金属蛋白酶在几个方面，包括缺乏 金属蛋白酶组织抑制剂的抑制作用。 肾小球 金属蛋白酶占肾小球基底膜的大部分 在中性pH下的降解活性表明其在 肾小球病理生理学 具体目标：A。纯化和 表征肾小球金属蛋白酶。 由于肾小球 酶是膜结合的，它将被溶解，然后被纯化 使用各种色谱技术的组合， 交换、羟基磷灰石、凝集素亲和性和明胶基质亲和性 层析 纯化的肾小球酶将被表征 生物化学;用于获得N-末端的氨基酸序列和一些 内部胰蛋白酶肽（特异性目标C），并用于提高多克隆 抗体（特异性目标B）。 B。免疫定位和免疫筛选 使用针对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 通过免疫细胞化学确定酶在细胞中的定位 亚细胞器的类型和定位。 多克隆 a）用于免疫筛选大鼠肾脏和/或 肾小球λ/gt 11 cDNA文库（特异性目标C）和，B）， 通过ELISA测定疾病模型中的酶蛋白含量 测定和蛋白质印迹（特异性目的D）。 C.为了获得DNA探针， 分离肾小球酶的全长cDNA。 DNA探针基于 部分N-末端和内部氨基酸序列的纯化 肾小球酶，将开发和用于筛选λ/gt 10大鼠 肾和/或肾小球cDNA文库，以分离cDNA克隆。 此外，本研究中开发的探针，包括全长 cDNA将是确定疾病中mRNA水平的有价值的工具 模型，以及在未来的研究有关的监管， 酵素 D.疾病模型研究。 体内研究对于这些研究 两种非炎症性肾小球疾病模型，嘌呤霉素 氨基糖苷（PAN）模型，一种用于微小病变疾病的模型， 被动Heymann肾炎模型，一种膜性肾炎模型， 肾病，将被诱导，并且在不同的时间点， 将使用北方印迹法和原位法检查肾小球 杂交方法 此外，肾小球酶蛋白（使用 用多克隆抗体开发的测定），以及酶 活动，将在这些模型中进行测量。 体外研究肾小球 上皮细胞（GEC）似乎是一个重要的部位损伤， PAN和被动Heymann肾炎模型。 嘌呤霉素和 抗Fx 1A抗体（含和不含补体）对 培养基中的酶活性和蛋白质， 将测定胞质和膜级分。 此外，在选定的 将检查时间点对GEC中mRNA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.