The long-term goal of the applicant is to explore a paradigm of disease, hypothesizing that in the nephrotic syndrome, both proteinuria and hypercholesterolemia have a causative role in the development of interstitial inflammation and fibrosis. The applicant proposes that these two components of the nephrotic syndrome contribute separately but synergistically to this process, which inevitably leads to end-stage renal disease when proteinuria is unremitting. In this project, the applicant will focus on the role of the interstitial macrophage, and on the macrophage product, TGF-beta, in the generation of interstitial fibrosis. The TGF-beta family is a group of multifunctional peptides which influence growth, differentiation and morphogenesis. It is proposed that TGF-beta plays a pivotal profibrotic role in the interstitium in proteinuric renal disease. The effects of hypercholesterolemia on macrophage infiltration and activation will be addressed. Two animal models of proteinuria (one normocholesterolemic and one hypercholesterolemic) will be used; complementary experiments in cell culture systems will also be performed. In vivo interventional studies in animal models will be utilized to identify the infiltrating macrophage as the origin of TGF-beta upregulation, as well as to explore the role of hypercholesterolemia in macrophage activation. Proof of TGF-beta activation in the interstitium will be obtained via bioassay for the peptide, and will also be demonstrated indirectly by identifying """"""""downstream"""""""" effect of TGF-beta upregulation in the interstitium. These processes will be documented with molecular biological and immunohistochemical methods in whole animal and cell culture models. Particular focus will be placed on the role of proteases in this system, both on the exploration of proteolytic steps necessary for TGF-beta activation, and on the effect of TGF-beta upregulation on matrix metalloproteinase production in the interstitium.
