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（FSGS）和微小改变病（MCD）是人类特发性肾病综合征最常见的原因。其病因不明，治疗是经验性的，包括皮质类固醇和环孢素A （CSA）等药物。细胞色素P450 （CYP450）花生四烯酸代谢产物在肾脏生理中起着重要作用。CYP450 4A产物对肾小球功能的影响尚未见报道。在初步研究中，我们已经证明，CYP450 4A的主要产物外源性20-羟基二糖四烯酸（20- hete）可以防止肾小球损伤模型中体外白蛋白通透性（P(alb)）的增加，并且用已知的诱导肾CYP450 4A表达的药物治疗的大鼠肾小球也可以防止P（alb）的增加。我们假设20-HETE是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CYP450 4A的表达和/或活性被药理学药物（CSA、皮质类固醇或贝特）或基因操作改变的大鼠。我们将追求以下具体目标：1)确定CYP450 4A在肾小球损伤模型中的表达；2)测定肾小球损伤模型中CYP450 4A的活性；3)观察20-HETE对大鼠损伤模型肾小球蛋白通透性的影响；4)明确药物对肾小球CYP450 4A表达及活性的影响；5)明确药物对损伤模型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。我们将采用已建立的Western blotting、实时PCK、液相色谱/质谱（LC/MS）、微穿刺和尿液清除技术。我们还将使用独特的方法、试剂和实验动物。这些方法包括测定离体肾小球的P(alb)，使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类似物和阻滞剂以及不同CYP450 4A表达的同源大鼠。拟议研究的结果将提供肾小球对损伤的反应和治疗药物提供肾小球保护的机制的见解。这些见解将允许开发新的疗法来治疗人类蛋白尿肾病和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.