The long-term goal of the proposed research is to elucidate the molecular mechanisms of renal interstitial fibrosis, the final common pathway leading to end-stage renal disease (ESRD). The two major extracellular pathways present in the tubulointerstitial renal compartment will be investigated: the urokinase-plasmin cascade and the metalloproteinase (MMP) family. Preliminary data suggests that interstitial fibrosis is characterized by inactivation of both proteolytic pathways due to up-regulated expression of protease inhibitors: plasminogen activator inhibitors (PAI) and tissue inhibitors of metalloproteinases (TIMP). Using a combination of in vivo and in vitro approaches, three specific aims will be investigated.
Aim #1 : To test the hypothesis that down-regulation of the intrarenal urokinase- plasmin cascade plays a role in renal interstitial fibrosis and to elucidate the cellular origin and mechanistic pathways involved.
Aim #2 : To test the hypothesis that down-regulation of intrarenal metalloproteinase activity plays a role in renal interstitial fibrosis and to determine the cellular origin and mechanistic pathways involved.
Aim #3 : To produce, characterize and test two recombinant proteins, auto-activating stromelysin-Ig and PAI-l-inhibitor-Ig, for their ability to increase renal proteolytic activity and to attenuate renal interstitial fibrosis. Models of experimental renal disease (protein- overlord proteinuria and adriamycin nephropathy) will be studied in normal mice and in mice that have genetically-engineered manipulations in renal matrix-degrading proteolytic activity: PAI-l over-expressing mice, PAI-l deficient mice, uPA-receptor deficient mice. TIMP-1, TIMP-1- 2 and TIMP-1-3 deficient mice and TIMP-1 over-expressing mice. Recombinant stromelysin and a PAI-l inhibiting peptide will be generated fused to an immunoglobulin chain and tested for their ability to attenuate renal fibrosis. The primary outcome will be the severity of interstitial fibrosis based on quantification of the interstitial area occupied by matrix proteins, tubular area and peritubular capillary surface area. Intrarenal proteolytic activity will be evaluated by measuring mRNA levels for genes encoding the proteases and their inhibitors. Changes in renal collagenolytic activity, transforming growth factor-beta (TGF-Beta) activation, fibrin deposition, leukocyte migration and collagen fibril cross-link formation. In vitro studies will investigate the ability of the uPA-receptor to modulate the production of the fibrogenic molecules (TGF-beta, angiotensin II, endothelin-l) and matrix proteins. The results of these studies should clearly establish the role and importance of these intrarenal proteolytic pathways in renal fibrosis.
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