Role of Cytochrome P450 Eichosanoids in Pressure-Natriuresis

以往的研究表明，肾脏在动脉血压的长期控制中起着主导作用 压力-尿钠排泄关系在每个遗传和实验中都向更高的压力转移， 高血压模型，迄今已研究。然而，改变肾功能的因素和 涉及的基因和途径仍有待确定。该计划的前期工作显示， 压力性尿钠排泄与肾髓质血流量和间质流体静力学的升高有关 压力（RIHP）和近端小管中Na+转运的抑制。在上一次融资期间，我们发现 RIHP升高刺激肾脏形成20-HETE，20-HETE有助于 通过抑制近端小管中的钠转运来抑制压力-利钠反应。我们进一步表明 阻断20-HETE的形成促进盐敏感性高血压的发展， 获得了令人兴奋的新证据，转移5号染色体上含有CYP 4504 A的5 cM区域， 将来自刘易斯大鼠的等位基因转移到达尔盐敏感（SS）遗传背景上， CYP 4A蛋白和减弱高血压的发展。我们现在有初步的数据， 来自布朗诺维里（BN）大鼠的5号染色体到SS遗传背景上也反对 在SS.5BN同源株中发生高血压。这些发现支持了我们的工作假设 肾内20-HETE形成的缺乏会导致高血压的发生， 党卫军老鼠。该项目的目标是确定是否存在降低活性的序列变体， SS大鼠肾脏中CYP 4A基因的表达，并在SS的发生中起因果作用。 高血压或如果该途径可能继发性参与，因为CYP 4A亚型的表达是 由5号染色体上的其他基因调控。具体目标是：1）确定 BN大鼠5号染色体转入SS基因的抗高血压和肾脏保护作用 背景依赖于肾脏CYP 4A蛋白表达的增加和20- HETE; 2）确定在四个CYP 4A基因之一中是否存在降低CYP 4A表达的序列变异。 这些酶在SS大鼠肾脏中的表达或活性;和3）测试是否有任何序列变体 在SS大鼠中，CYP 4A基因中发现的一个突变导致高血压和肾脏疾病的发生 使用转基因技术。这些研究的新颖之处在于，他们将采用独特的 我们在过去5年中开发的SS大鼠的5号染色体同源和同源品系， 用于测量20-HETE、20-HETE合成和作用的新型抑制剂、真实的的LC/MS/MS分析 用于测量CYP 4A亚型表达的时间PCR测定法和用于产生CYP 4A亚型的新的慢病毒策略 大鼠的转基因品系的拟议研究，以确定是否在肾脏形成的遗传异常， 20-HETE通过重置血压-利钠肽参与SS大鼠高血压的发生 这种关系是特别独特和相关的，因为CYP 4A 11基因的多态性降低了 最近在三个独立的人中发现20-HETE的形成与血压升高有关 77 '183'184这些研究将提供一种新的同源动物模型系统， 机制来解释如何在肾形成的20-HETE的不足可能有助于 盐敏感型高血压的发展。所获得的结果也可能刺激 开发上调这一途径的药物，用于治疗高血压和肾脏疾病。

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.