Renal Vascular Reactivity in Hypertension

描述(申请人提供)：CD38ADP核糖(ADPR)环化酶是一种膜结合酶，能产生已知促进小动脉平滑肌细胞兰尼定受体(RyR)介导的钙动员的代谢物。我们推测肾脏CD38在血管紧张素II(Ang II)诱导的高血压(AIH)的发生发展中起中心作用，CD38缺陷小鼠比野生型(WT)小鼠表现出更少的明显的肾血管收缩、Na+滞留和AIH。目的1验证CD38 ADPR环化酶参与AIH发生的假说，使Ang II在CD38-/-(全球遗传缺陷)和WT小鼠中产生较少明显的高血压。通过siRNA诱导的CD38的靶向性、肾脏特异性的部分敲除，WT小鼠也可以预测不那么严重的高血压。AIH的严重程度是通过在慢性注射Ang II之前和期间测量清醒的无拘束小鼠的24小时动脉压(遥测)来确定的。还测量了24小时尿中亚硝酸盐/硝酸盐和8-iso-PGF21的排泄量，以评估一氧化氮(NO)的产生和氧化应激。肾小球前血管的CD38mRNA和ADPR周期酶活性将被定量。目的2探讨CD38ADPR环化酶在AIH肾血管收缩中的作用及压力-钠尿关系的右移。我们预测，在AIH的发育过程中，CD38缺陷小鼠的肾脏对急性盐负荷的反应比WT小鼠更快地排泄Na+，而在已建立的AIH中排泄率变得相似。清醒和麻醉的小鼠具有CD38的全局敲除和肾脏特异性敲除，将在这两个阶段进行评估。目的3评价CD38是介导G蛋白偶联受体介导的钙信号转导通路的假说，涉及RyR的钙信号通路和钙离子诱导的肾小动脉内钙释放和肾血管收缩。我们预测，在对照和AIH条件下，CD38缺陷小鼠与WT小鼠相比，钙信号和血管收缩药物(Ang II，ET-1，TXA2)的反应性减弱。放射配基结合的Scatchard分析将表征肾微血管中Ang II、ET-1和TP受体的亲和力和/或密度。我们的目标是确定在高血压发展之前或发展早期发生的一系列事件，因此比起继发性的、压力依赖的后果，这些事件更有可能是因果关系。结合CD38基因的全局性和肾脏特异性的基因靶向缺失，结合药物抑制ADPR周期酶和RyR介导的钙释放，将为CD38是介导肾微循环中钙信号转导的主要周期酶家族成员及其在肾血管收缩、Na+滞留和AIH发生发展的长期调节中的功能意义提供重要的新信息。我们对这一被低估的途径的新研究的成功完成将极大地促进我们对钙信号在肾脏微循环中的细胞/分子机制的理解，以及对健康和疾病中肾脏血管反应性的调节，对该领域产生重大影响。 公共卫生相关性：超过7000万美国人患有高血压，这是一个主要的心血管风险因素，高度预测其他血管疾病，如动脉粥样硬化、糖尿病、肾功能衰竭和中风。有人建议进行研究，以探讨收缩血管的平滑肌细胞钙代谢的调节机制。异常的细胞信号可导致动脉对血管活性激素高度反应，并导致肾脏保留不适当数量的盐和水，导致血容量扩大和高血压的发生。

DESCRIPTION (provided by applicant): CD38 ADP ribosyl (ADPR) cyclase is a membrane-bound enzyme that produces metabolites known to promote Ca2+ mobilization mediated by ryanodine receptors (RyR) in arteriolar smooth muscle cells. We posit that renal CD38 is central to the development of angiotensin II (Ang II)-induced hypertension (AIH) and that CD38-deficient mice exhibit less pronounced renal vasoconstriction, Na+ retention and AIH than do wild-type (WT) mice. AIM 1 tests the hypothesis that CD38 ADPR cyclase participates in the development of AIH such that Ang II produces less pronounced hypertension in CD38-/- (global genetic deficiency) vs. WT mice. Less severe hypertension is also predicted in WT mice with targeted, renal-specific partial knockdown of CD38 induced by siRNA. The severity of AIH is determined by measuring 24-hr arterial pressure (telemetry) in conscious, unrestrained mice before and during chronic Ang II infusion. Also measured are 24 hr urinary excretion of nitrite/nitrate and 8-iso-PGF21 to assess nitric oxide (NO) production and oxidative stress. CD38 mRNA and ADPR cyclase activity will be quantified in preglomerular vessels. AIM 2 assesses the contribution of CD38 ADPR cyclase to renal vasoconstriction and the rightward shift in the pressure-natriuresis relation in AIH. We predict that the kidneys of CD38-deficient mice excrete Na+ more rapidly in response to an acute salt load than WT mice during development of AIH, with excretion rates becoming similar in established AIH. Conscious and anesthetized mice with global knockout and renal-specific knockdown of CD38 will be evaluated in both phases. AIM 3 evaluates the hypothesis that CD38 is the major ADPR cyclase mediating G- protein coupled receptor-elicited Ca2+ signaling involving RyR and Ca2+-induced Ca2+ release in isolated afferent arterioles and renal vasoconstriction in vivo. We predict that Ca2+ signaling and vascular responsiveness to vasoconstrictor agents (Ang II, ET-1, TxA2) are attenuated in CD38-deficient vs. WT mice during control and AIH conditions. Scatchard analysis of radioligand binding will characterize Ang II, ET-1, and TP receptor affinity and/or density in renal microvessels. Our goal is to identify a sequence of events that precede or occur early during the development of hypertension and thus are more likely to be causative than secondary, pressure-dependent consequences. Combining gene-targeted deletion of CD38, global and renal- specific, with pharmacological inhibition of ADPR cyclase and RyR-mediated Ca2+ release will provide important new information that CD38 is the primary cyclase family member mediating Ca2+ signaling in the renal microcirculation and its functional significance in long-term regulation of renal vasoconstriction, Na+ retention and the development of AIH. Successful completion of our novel studies of this underappreciated pathway will significantly advance our understanding of cellular/molecular mechanisms of Ca2+ signaling in the renal microcirculation and regulation of renal vascular reactivity in health and disease, making a major impact on the field. PUBLIC HEALTH RELEVANCE: More than 70 million Americans have hypertension, a major cardiovascular risk factor, highly predictive of other vascular diseases such as atherosclerosis, diabetes mellitus, renal failure, and stroke. Studies are proposed to investigate mechanisms regulating calcium metabolism in smooth muscle cells that contract blood vessels. Abnormal cell signaling can cause arteries to be hyper-responsive to vasoactive hormones and cause the kidneys to retain inappropriate amounts of salt and water, resulting in an expanded blood volume and the development of high blood pressure.