Regulation of renal Na handling in the collecting duct by local purinergic tone

描述（由申请人提供）：醛固酮敏感远端肾元主要细胞上皮Na+通道（ENaC）的活性是肾盐和水处理的核心，因此是血压调节的核心。事实上，ENaC功能的获得和丧失会导致血压的显著升高和降低。肾素-血管紧张素-醛固酮系统（RAAS）对ENaC的负反馈调节有很好的描述，其中血压的变化最终影响肾脏中ENaC的活性。新出现的证据表明ENaC也受到由局部因素介导的肾脏固有信号的反馈控制。然而，与RAAS的外在调控相比，人们对其内在调控知之甚少。嘌呤能信号因子是主要候选自分泌/旁分泌因子，对远端肾细胞Na+运输很重要。我们的总体观点是，远端肾元盐和水处理受到内在系统的影响，以减少远端对近端事件的补偿，使排泄能够适当地匹配系统条件。这种内在调节的丧失预计会引起或加剧不适当的肾Na+潴留，从而导致高血压。令人惊讶的是，缺乏嘌呤能P2Y2受体或连接蛋白30半通道的小鼠，可能在一定程度上负责远端肾单位ATP的释放，患有与促进肾Na+潴留有关的高血压。考虑到我们强有力的初步结果，我们验证了我们的中心假设，即通过腔内P2Y2受体对远端肾单位局部ATP信号的ENaC活性的生理下调可以调节Na+的重吸收，通过解决四个具体目标：1)确定ATP的生理浓度是否影响哺乳动物远端肾单位的ENaC活性，并确定细胞信号通路和作用机制；2)通过旁分泌/自分泌ATP信号通路受损来确定ENaC调节功能障碍的后果；3)明确连接蛋白30在ENaC ATP调控中的作用；4)通过嘌呤能张力量化全身盐负荷与远端肾元ENaC活性调节之间的关系。我们研究了ENaC的嘌呤能调节，使用了一种综合策略，结合通道活性的直接测量与电生理学和分子遗传学方法，明确地建立了P2Y2受体的作用，并探测了Cx30作为自分泌ATP释放的管道。使用在正常细胞环境中含有天然ENaC的生理相关组织（新鲜分离的小鼠收集管）。此外，本提案的范围是全面探讨嘌呤能调和ATP通过收集管中的Cx30释放对ENaC从动物到分子作用机制的调控。

DESCRIPTION (provided by applicant): The activity of the epithelial Na+ channel (ENaC) in principal cells of the aldosterone-sensitive distal nephron is central to renal salt and water handling, and thus, regulation of blood pressure. Indeed, gain and loss of ENaC function causes marked increases and decreases in blood pressure. Negative-feedback regulation of ENaC by the renin-angiotensin-aldosterone system (RAAS) is well described where changes in blood pressure ultimately affect ENaC activity in the kidney. Emerging evidence suggests that ENaC is also under feedback control by signaling intrinsic to the kidney mediated by local factors. However, compared to extrinsic regulation by RAAS, less is known about intrinsic control. Purinergic signaling factors are leading candidates as autocrine/paracrine factors important to distal nephron Na+ transport. Our overarching idea is that distal nephron salt and water handling is influenced by intrinsic systems to lessen distal compensation of proximal events enabling excretion to appropriately match systemic conditions. Loss of such intrinsic regulation is expected to cause or exacerbate improper renal Na+ retention and thus, hypertension. It is striking that mice engineered to lack purinergic P2Y2 receptors or connexin 30 hemi-channels, which likely are responsible, in part, for ATP release in the distal nephron, have hypertension associated with facilitated renal Na+ retention. In consideration of our strong preliminary results, we test our central hypothesis that physiological down- regulation of ENaC activity in response to local ATP signaling in the distal nephron through luminal P2Y2 receptors tempers Na+ reabsorption by addressing four specific aims: 1) determine whether physiological concentrations of ATP affect ENaC activity in the mammalian distal nephron and determine the cellular signaling pathway and mechanism of action; 2) determine the consequence of dysfunctional regulation of ENaC by compromised paracrine/autocrine ATP signaling; 3) define the role of connexin 30 in ATP regulation of ENaC; and 4) quantify the relation between systemic salt-loading and regulation of ENaC activity in the distal nephron by purinergic tone. We investigate purinergic regulation of ENaC using a comprehensive strategy combining direct measurements of channel activity with electrophysiology with a molecular genetics approach that unequivocally establishes the role of the P2Y2 receptor and probes Cx30 as a conduit for autocrine ATP release. Physiologically relevant tissue (freshly isolated murine collecting duct) containing native ENaC in its normal cellular environment is used. Moreover, the scope of this proposal is comprehensive probing regulation of ENaC by purinergic tone and ATP release via Cx30 in the collecting duct from the animal to molecular mechanism of action. PUBLIC HEALTH RELEVANCE: Hypertension is prevalent in America and continues to rise making it one of the major obstacles for better quality of life and health care in the U.S. A. Appropriate salt handling by the kidneys is critical to proper control of blood pressure. The current proposal investigates the cellular and molecular mechanisms underlying renal salt handling particularly at the distal nephron, the location where systemic salt and water balance is fine-tuned in humans.