cPLA2 derived Eicosanoids modulate Renal Fibrosis

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DESCRIPTION (provided by applicant): Renal fibrosis is the final common pathologic finding in chronic kidney disease. Recent research efforts have uncovered a vast network of inflammatory signals and cell types responsible for generating renal myofibroblasts that are instrumental in creating and propagating fibrosis. An emerging literature base supports that eicosanoids, broadly characterized as biologically active lipid products of enzymatic cleavage, may play a major role in renal disease, in particular renal fibrosis. Our project seeks to characterize the contribution of eicosanoids in propagating renal fibrosis using an animal model of chronic kidney disease as well cell culture techniques. Specifically, we are using genetic knockout mice to elucidate the role that cytosolic phospholipase A2 (cPLA2) derived eicosanoids have in creating fibrosis. Specific aim 1 will investigate the role that eicosanoids play in propagating fibrosis in an animal model of chronic kidney disease. We will be using unilateral ureteral obstruction (UUO) in wild type (WT) and cPLA2 genetic knockout (KO) mice. Using techniques such as liquid chromatography and mass spectrometry we will be able to profile tissue levels of cPLA2 derived eicosanoid products in these mouse populations after UUO, look at degree of fibrosis and inflammation, and perform bone marrow transplants to delineate the contribution from circulating inflammatory cells. Our preliminary data shows upregulated tissue expression of prostanoid, lipoxygenase, and cytochrome P450 products after UUO. These are potential unexplored pathways to target with existing drug therapies. Specific aim 2 will attempt to establish the cell type responsible for the phenotypes observed above using a co-culture system. Specifically, we plan to use a mouse immortalized cell line, MCT cells in co-culture with macrophages isolated from our experimental mice. By cell specific genetic knockout we can observe the molecular cross-talk between these cell lines to determine the major cell type contributing to injury relating this to the studies done in animals. Our preliminary data in human immortalized epithelial cells demonstrates that cPLA2 gene silencing induces phenotypic changes that may indicate an altered resident cell response to injurious stimuli.