Redox control of medullary function and blood pressure

这个项目的总体目标是促进我们对活性氧簇(ROS)和活性氮簇(RNS)在确定肾功能中所起作用的理解，特别是肾髓质，这是一个容易产生过量O2-的区域。我们先前已经证实，肾髓质血流量的变化在钠稳态和动脉压的长期调节中起着重要作用，并显示 一氧化氮(NO)的产生在调节流向该区域的血液中起着关键作用。我们假设，肾髓质中ROS的过量产生降低了NO的生物利用度，降低了髓质的血流量，增加了肾小管对钠的重吸收，并导致了盐敏感型高血压。这个项目，就像这个PPG中的所有其他项目一样，利用了遗传定义的Dahl盐敏感大鼠(SS/MCW)和来自近交系SS/MCW品系的新开发的共体对照品系，在该品系中，来自盐不敏感的BN/MCW品系的13号染色体已经导入SS/MCW大鼠的基因组背景(SS.BN13共体品系)。该共体菌株与SS/MCW品系有98%的同源性，但对盐相对不敏感。目标1将确定Dahl盐敏感大鼠(SS/MCW)的肾髓质是否产生过量的ROS(O2，H2O2，ONOO)，从而反馈到 解偶联一氧化氮合酶(NOS)，降低四氢生物蝶呤(BH4)/二氢生物蝶呤(BH2)的重做比(即O2的恶性循环)。生产)。新开发的荧光微透析和高效液相分析技术将能够比较盐敏感(SS/MCW)和盐不敏感的共体体SS.BN13大鼠的ROS和NO途径。我们的初步数据表明，这种对照菌株表现出明显较低的髓质ROS水平。研究还将确定SS/MCW延髓中ROS的降低 大鼠长期延髓注射抗氧化剂可增加延髓血流量，降低盐性高血压。目的2确定盐不敏感的SS.BN13染色体组大鼠髓质内ROS水平升高的影响。髓血流量、肾间质静水压力(RIHP)、动脉压(AP)和血压-尿钠关系将根据O2-、H2O2和ONOO-的慢性升高而确定。一氧化氮合酶解偶联的概念将通过测量确定的骨髓组织BH4/BH2比率来检验 用高效液相色谱法测定，用微渗析技术测得NO、O2和H2O2的值。目标3将使用薄的、超融合的髓质组织条来确定肾髓质血管和管段内以及管段之间O2和NO的动态相互关系。在对照SS.BN13和盐敏感的SS/MCW大鼠中，不会比较刺激和抑制ROS的药物的反应。新型荧光染料，结合显微图像的高速捕捉，将使细胞内NO的测量独立于间质中的NO，从而确定从小管释放的NO是否可以作为旁分泌物质来控制髓内血流。综上所述，这些研究将提供新的和临床相关的数据，确定ROS在调节肾脏髓质功能和盐敏感型高血压的发展中所起的作用，这些信息有望导致人类高血压治疗的新的治疗方式。

The overall goal of this project is to advance our understanding of the role that reactive oxygen species (ROS) and reactive nitrogen species (RNS) play in determining renal function, especially that of the renal medulla, a region that is vulnerable to excess O2- production. We have previously established that changes in renal medullary blood flow play an important role in sodium homeostasis and the long-term regulation of arterial pressure and shown that nitric oxide (NO) production plays a key role in the regulation of blood flow to this region. We hypothesize that excess production of ROS in the renal medulla reduces NO bioavailability, lowers medullary blood flow, increases tubular sodium reabsorption and results in a salt-sensitive form of hypertension. This project, as all others in this PPG, utilizes Dahl salt-sensitive rats that are genetically defined (SS/Mcw) and a newly developed consomic control strain derived from the inbred SS/Mcw strain in which chromosome 13 from the salt-insensitive BN/Mcw strain has been introgressed into the genomic background of the SS/Mcw rat (SS.BN13 consomic strain). This consomic SS.BN13 strain is 98% identical to the SS/Mcw strain but is relatively salt-insensitive. Aim 1 will determine if the renal medulla of Dahl salt-sensitive rats (SS/Mcw) produces excess ROS (O2, H2O2, ONOO) that feeds back to uncouple nitric oxide syntheses enzymes (NOS) and reduce the redo ratio of tetrahydrobiopterin (BH4)/dihydrobiopterin (BH2) (i.e., a vicious cycle of O2. production). Newly developed fluorescent microdialysis and HPLC analytical techniques will enable comparison of ROS and NO pathways in salt-sensitive (SS/Mcw) and in salt-insensitive consomic SS.BN13 rats. Our preliminary data indicate that this control strain exhibits significantly lower levels of medullary ROS. Studies will also determine whether lowering of ROS in the medulla of SS/Mcw rats by chronic medullary infusion of antioxidants increases medullary blood flow and reduces salt-induced hypertension. Aim 2 will determine the effects of induced medullary elevations of ROS in salt-insensitive SS.BN13 consomic rats. Medullary blood flow, renal interstitial hydrostatic pressure (RIHP), arterial pressure (AP) and the pressure-natriuresis relationships will be determined in response to chronic elevations of O2-, H2O2, and ONOO-. The concept of NOS uncoupling will be examined by measurement of medullary tissue BH4/BH2 ratios determined by HPLC, and NO, O2 and H2O2 values obtained by microdialysis techniques. Aim 3 will determine the dynamic inter-relationships of O2 and NO within and between vascular and tubular segments of the renal medulla using thin, superfused medullary tissue stripes. NO responses to agents that stimulate and inhibit ROS will be compared in control SS.BN13 and salt-sensitive SS/Mcw rats. Novel fluorescent dyes, coupled with high speed capture of microscopic images will enable the measurement of intracellular NO independent from NO in the interstitial space thereby determining whether NO released from tubules could act as a paracrine substance to control medullary blood flow. Taken together, these studies will provide novel and clinically relevant data defining the role that ROS play in the regulation of renal medullary function and the development of salt-sensitive hypertension, information that would be expected to lead to new therapeutic modalities in the treatment of human hypertension.