CYTOCHROME P-450 AND GLOMERULAR PROTEIN PERMEABILITY

描述（由申请方提供）：局灶节段性肾小球硬化（FSGS）和微小病变疾病（MCD）是人类特发性肾病综合征的最常见病因。其病因不明，治疗是经验性的，包括药物，如皮质类固醇和环孢素A（CSA）。细胞色素P450（CYP 450）花生四烯酸代谢产物在肾脏生理学中的作用日益受到重视。尚未描述CYP 450 4A产物对肾小球功能的影响。在初步研究中，我们已经表明，外源性20-羟基二十碳四烯酸（20-HETE），CYP 450 4A的主要产物，防止肾小球损伤模型中的体外白蛋白渗透性（P（alb））增加，并且用已知诱导肾脏CYP 450 4A表达的药物治疗的大鼠肾小球也受到保护，免于P（alb）增加。我们假设20-HETE，一种CYP 450 4A的花生四烯酸代谢产物，保护肾小球滤过屏障免受非炎症性损伤，如FSGS和MCD。我们将使用FSGS因子（一种来自FSGS患者血浆的物质）或嘌呤霉素氨基糖苷（PAN）（一种肾小球上皮细胞毒素）诱导肾小球损伤。我们还将研究通过药物（CSA、皮质类固醇或贝特类药物）或遗传操作改变CYP 450 4A表达和/或活性的大鼠。本研究的主要目的是：1）检测肾小球损伤模型中CYP 450 4A的表达; 2）检测肾小球损伤模型中CYP 450 4A的活性; 3）观察20-HETE对肾小球蛋白通透性的影响; 4）观察药物对肾小球CYP 450 4A表达和活性的影响; 5）确定药物对损伤模型中肾小球蛋白通透性的影响。我们亦会就保护机制进行初步研究。我们将采用Western印迹、实时PCK、液相色谱/质谱（LC/MS）、显微穿刺和尿液清除等已建立的技术。我们还将使用独特的方法、试剂和实验动物。这些方法包括测量离体肾小球P（alb）、用活体双光子显微镜观察肾小球白蛋白滤过、使用独特的20-HETE类似物和阻断剂以及具有不同表达CYP 450 4A的同类大鼠。拟议的研究结果将提供深入了解肾小球损伤的反应和治疗药物提供肾小球保护的机制。这些见解将允许开发新的疗法来治疗人类蛋白尿性肾病并阻止肾小球损伤的进展。

DESCRIPTION (provided by applicant): 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.