CYTOCHROME P-450 AND GLOMERULAR PROTEIN PERMEABILITY

描述（由申请人提供）：局灶节段性肾小球硬化症（FSGS）和微小病变（MCD）是人类特发性肾病综合征的最常见原因。其病因尚不清楚，治疗是经验性的，包括皮质类固醇和环孢素 A (CSA) 等药物。人们越来越认识到细胞色素 P450 (CYP450) 花生四烯酸代谢产物在肾脏生理学中发挥着重要作用。 CYP450 4A 产品对肾小球功能的影响尚未描述。在初步研究中，我们已经表明，CYP450 4A 的主要产物外源性 20-羟基二十碳四烯酸 (20-HETE) 可防止肾小球损伤模型中体外白蛋白通透性 (P(alb)) 的增加，并且用已知诱导肾 CYP450 4A 表达的药物治疗的大鼠的肾小球也可免受 P(alb) 增加的影响。我们假设 20-HETE（CYP450 4A 的花生四烯酸代谢物）可以保护肾小球滤过屏障免受非炎症损伤，例如导致 FSGS 和 MCD 的损伤。我们将使用 FSGS 因子（一种来自 FSGS 患者血浆的物质）或嘌呤霉素氨基核苷 (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）或基因操作改变 CYP450 4A 表达和/或活性的大鼠。我们将追求以下具体目标：1）确定肾小球损伤模型中CYP450 4A的表达； 2) 测定肾小球损伤模型中CYP450 4A的活性； 3) 记录20-HETE对损伤模型中肾小球蛋白通透性的影响； 4) 明确药物对肾小球CYP450 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。我们还将对保护机制进行初步研究。我们将采用蛋白质印迹、实时 PCK、液相色谱/质谱 (LC/MS)、微穿刺和尿清除等成熟技术。我们还将使用独特的方法、试剂和实验动物。这些包括测量分离肾小球的 P(alb)、使用活体双光子显微镜观察肾小球白蛋白过滤、使用 20-HETE 的独特类似物和阻断剂以及具有不同 CYP450 4A 表达的同源大鼠。拟议研究的结果将深入了解肾小球对损伤的反应以及治疗药物提供肾小球保护的机制。这些见解将有助于开发新疗法来治疗人类蛋白尿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.