GLOMERULAR MATRIX DEGRADING METALLOPROTEINASES

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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.