Role of Cytochrome P450 Eichosanoids in Pressure-Natriuresis

以前的研究表明，肾脏在长期控制动脉压方面起着主导作用。 在每一种基因和实验中，压力-尿钠关系都转移到更高的压力 到目前为止已经研究过的高血压模型。然而，改变肾功能的因素和 涉及的基因和途径仍有待确定。这个节目之前做的工作显示， 压力-钠尿与肾髓质血流量和间质流体静力升高相关 压力(RIHP)和抑制近端小管的Na+转运。在上一个资助期内，我们发现 RIHP升高刺激20-HETE的肾脏形成，20-HETE参与 通过抑制近端小管钠转运的压力-利钠反应。我们进一步展示了 阻断20-HETE的形成促进盐敏感型高血压的发展和 获得了令人振奋的新证据，即5号染色体5厘米区域内含有CYP4504A的转移 从Lewis大鼠到Dahl盐敏感(SS)遗传背景的等位基因增加了肾脏的表达 细胞色素P4A蛋白，延缓高血压的发展。我们现在有初步数据表明 从棕色Norwary(BN)大鼠到SS遗传背景的5号染色体也反对 SS.5BN经济株高血压的发生。这些发现支持我们的工作假说 肾脏20-HETE缺乏导致高血压的发生。 SS大鼠。这个项目的目标是确定是否存在降低活性的序列变体或 一种细胞色素P4A基因在SS大鼠肾脏中的表达及其在糖尿病发病中的作用 如果这一途径可能是继发性参与的，因为CYP4A亚型的表达是 由5号染色体上的其他基因调节。其具体目的是：1)确定是否 BN大鼠5号染色体转入SS基因的降压和肾保护作用 背景依赖于肾脏细胞色素P4A蛋白表达的增加和20-羟色胺的产生 HETE；2)确定四个CYP4A基因中的一个是否存在序列变异，从而降低 这些酶在SS大鼠肾脏中的表达或活性；以及3)检测是否有任何序列变体 发现细胞色素P4A基因与SS大鼠高血压和肾脏疾病的发生有关 使用转基因技术。这些研究的新颖之处在于，它们将采用独特的 我们在过去5年中培育的SS大鼠的5号染色体同源和共体品系，一种新的 LC/MS/MS法测定20-HETE的合成及其作用的新型抑制剂20-HETE，REAL 时间-聚合酶链式反应检测细胞色素P4A亚型的表达及慢病毒新策略的建立 转基因大鼠品系的拟议研究，以确定肾脏形成中的遗传异常 20-HETE通过重置血压-钠尿剂参与SS大鼠高血压的发展 这种关系是特别独特和相关的，因为CYP4A11基因的一个多态性会降低 20-HETE的形成最近与三名独立的人类血压升高有关 这些研究将提供一种新的同源动物模型系统供探索 解释20-HETE肾脏形成缺陷如何导致 盐敏感型高血压在男性的发展。所获得的结果也可能刺激 为治疗高血压和肾脏疾病而开发上调这一途径的药物。

Previous studies have indicated the kidney plays a dominant role in the long-tern control of arterial pressure and that the pressure-natriuresis relationship is shifted to higher pressures in every genetic and experimental model of hypertension that has been studied to date. However, the factors that alter renal function and the genes and pathways involved remain to be determined. Previous work done in this program revealed that pressure-natriuresis is associated with elevations in renal medullary blood flow and interstitial hydrostatic pressure (RIHP) and inhibition of Na+ transport in the proximal tubule. During the last funding period, we found that elevations in RIHP stimulate the renal formation of 20-HETE and that 20-HETE contributes to the pressure-natriuretic response by inhibiting sodium transport in the proximal tubule. We further demonstrated that blockade of the formation of 20-HETE promotes the development of salt-sensitive hypertension and obtained exciting new evidence that transfer of a 5 cM region of chromosome 5 containing the CYP4504A alleles from Lewis rats onto the Dahl salt-sensitive (SS) genetic background increases the renal expression of CYP4A protein and attenuates the development of hypertension. We now have preliminary data that transfer of chromosome 5 from the Brown Norwary (BN) rat onto the SS genetic background also opposes the development of hypertension in a SS.5BN consomic strain. These findings support our working hypothesis that a deficiency in the renal formation of 20-HETE contributes to the development of hypertension in SS rats. The goal of this project is to determine whether there is a sequence variant that reduces the activity or expression of one of the CYP4A genes in the kidney of SS rats and plays a causal role in the development of hypertension or if this pathway maybe secondarily involved because the expression of CYP4A isoforms are regulated by some other gene on chromosome 5. The Specific Aims are: 1) to determine whether the antihypertensive and renoprotective effects of transfer of chromosome 5 from the BN rat into the SS genetic background is dependent on an increase in the renal expression of CYP4A protein and the production of 20- HETE; 2) to determine if there is a sequence variant in one of the four CYP4A genes that reduces the expression or activity of these enzymes in the kidney of SS rats; and 3) to test if any of the sequence variants identified in the CYP4A genes contribute to the development of hypertension and renal disease in SS rats using transgenic techniques. The novel aspects of these studies are that they will employ unique chromosome 5 congenic and consomic strains of SS rats that we developed over the last 5 years, a new LC/MS/MS assay for measurement of 20-HETE, novel inhibitors of the synthesis and actions of 20-HETE, real time-PCR assays for measuring the expression of CYP4A isoforms and a new lentiviral strategy for creating transgenic strains of rats The proposed studies to determine if a genetic abnormality in the renal formation of 20-HETE contributes to the development of hypertension in SS rats by resetting the pressure-natriuretic relationship are especially unique and relevant, since a polymorphism in the CYP4A11 gene that reduces the formation of 20-HETE has recently been associated with elevated blood pressure in three independent human populations.77'183'184 These studies will provide a new homologous animal model system to explore mechanisms to explain how a deficiency in the renal formation of 20-HETE could contribute to the development of salt-sensitive forms of hypertension in man. The results obtained may also spur the development of drugs that upregulate this pathway for the treatment of hypertension and renal disease.