Focal segmental glomerulosclerosis (FSGS) and minimal change disease (MCD) are the most common causes of idiopathic nephrotic syndrome in humans. Their etiologies are unknown and treatment is empirical, consisting of drugs such as corticosteroids and cyclosporine A (CSA). Products of cytochrome P450 (CYP450) arachidonic acid metabolism are increasingly recognized as playing important roles in renal physiology. The effect of CYP450 4A products on glomerular function has not been described. In preliminary studies, we have shown that exogenous 20- hydroxyeicosatetraenoic acid (20-HETE), the major product of CYP450 4A, prevents increased in vitro albumin permeability (P(alb)) in models of glomerular injury, and that glomeruli from rats treated with agents known to induce renal CYP450 4A expression are also protected from increased P(alb). We hypothesize that 20-HETE, an arachidonic acid metabolite of CYP450 4A, protects the glomerular filtration barrier from noninflammatory injury such as that responsible for FSGS and MCD. We will induce glomerular injury using the FSGS factor, a substance from the plasma of patients with FSGS, or puromycin aminonucleoside (PAN), a glomerular epithelial cell toxin. We will also study rats in which expression and/or activity of CYP450 4A has been altered by pharmacological agents (CSA, corticosteroid or fibrate) or genetic manipulations. We will pursue the following Specific Aims: 1) To determine expression of CYP450 4A in models of glomerular injury;2) To determine activity of CYP450 4A in models of glomerular injury;3) To document the effect of 20-HETE on glomerular protein permeability in models of injury;4) To define the effect of pharmacological agents on glomerular CYP450 4A expression and activity;5) To define the effect of pharmacological agents on glomerular protein permeability in models of injury. We will also perform initial studies on mechanism of protection. We will employ established techniques of Western blotting, real-time PCK, liquid chromatography/mass spectrometry (LC/MS), micropuncture and urinary clearance. We will also use unique methods, reagents and experimental animals. These include measurement of P(alb) of isolated glomerular, observation of glomerular albumin filtration using intravital two-photon microscopy, use of unique analogs and blockers of 20-HETE and of congenic rats with varying expression of CYP450 4A. Results of the proposed studies will provide insights into glomerular responses to injury and the mechanisms by which therapeutic agents provide glomerular protection. These insights will permit development of new therapies to treat human proteinuric renal diseases and arrest the progression of glomerular injury.
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