The mechanisms responsible for the degradation of the mesangial matrix by mesangial cells are unknown. The overall objective of this application is to delineate the biochemical mechanisms by which glomerular mesangial cells degrade mesangial matrix, including identification of the specific proteinases involved and the factors and interactions that regulate their activity and expression. Mesangial cells produce several agents potentially involved in matrix degradation including plasminogen activator(s), latent gelatinase and the gelatinase inhibitors, TIMP-1 and TIMP-2 (tissue inhibitor of metalloproteinase). Mesangial cells also produce and respond to polypeptide growth factors and reactive oxygen metabolites, agents which in other tissues modulate the activities of matrix degrading proteinases and their inhibitors. The applicant's preliminary studies indicate that extracellular matrix degradation by cultured mesangial cells involves cooperative interactions between plasmin (generated from plasminogen by MC plasminogen activator) and mesangial cell gelatinase. His hypothesis is that in vivo, degradation of the mesangial matrix is controlled by interactions among matrix degrading proteinases (gelatinase, plasmin), their inhibitors (TIMPs and alpha-2-antiplasmin), and specific modulators (polypeptide growth factors, reactive oxygen metabolites) produced by MC and/or leukocytes. Using mesangial cells cultured on thin films of radiolabeled ECM (Matrigel) or purified matrix components (type IV collagen, laminin, fibronectin, heparan sulfate proteoglycan), he will: 1. Confirm and extend his preliminary observations that ECM degradation by cultured mesangial cells is plasmin(ogen) dependent but also involves mesangial cell gelatinase. 2. Determine the relative contribution of plasmin and mesangial cell gelatinase to ECM degradation by examining: a) the effect of increasing concentrations of plasminogen; b) the effect of exogenously added specific inhibitors of gelatinase (eg TIMP, antigelatinase antibodies) and plasmin (eg alpha-2-antiplasmin, aprotinin); c) the correlations among ECM degradation and gelatinase, plasmin, TIMP, and mesangial cell plasminogen activator activities. 3. Determine the role of reactive oxygen metabolites in ECM degradation by cultured mesangial cells by examining the effects of reactive oxygen metabolite scavengers (including SOD, catalase, DMTU, methionine, and deferroxamine) on ECM degradation by cultured mesangial cells. 4. Examine potential interactions among gelatinase, plasmin, TIMPs, alpha-2- antiplasmin, and reactive oxygen metabolites in ECM degradation by cultured mesangial cells including a) the ability of plasmin and reactive oxygen metabolites to activate latent mesangial cell gelatinase and inactivate TIMPs; and b) the ability of mesangial cell gelatinase to inactivate alpha-2 antiplasmin; 5. Examine the effects of TGFbeta, TNFalpha, and IL-1 (each known to alter the expression of ECM degrading proteinases and/or their inhibitors) on: a) the degradation of intact ECM and purified ECM components by cultured mesangial cells; and b) the activities of plasmin, gelatinase, and TIMPs in medium and plasminogen activator in mesangial cells.
