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

描述(由申请人提供)：在对醛固酮敏感的远端肾单位的主细胞中，上皮钠通道(ENaC)的活动是肾脏盐和水处理的中心，从而调节血压。事实上，ENaC功能的增强和丧失会导致血压的显著上升和下降。肾素-血管紧张素-醛固酮系统(RAAS)对ENaC的负反馈调节被很好地描述为血压的变化最终影响肾脏ENaC的活动。新的证据表明，ENaC也处于反馈控制之下，通过局部因素介导的肾脏固有信号。然而，与RAAS的外在调控相比，人们对内在控制的了解较少。嘌呤能信号因子是重要的自分泌/旁分泌因子，对远端肾单位钠转运具有重要作用。我们的总体想法是，远端肾单位盐和水的处理受到内在系统的影响，以减少近端事件的远端补偿，使排泄能够适当地匹配全身条件。这种内在调节的丧失预计会导致或加剧不当的肾脏钠滞留，从而导致高血压。令人惊讶的是，被设计为缺乏嘌呤能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的ATP释放，并与促进肾脏钠滞留相关的高血压。考虑到我们强有力的初步结果，我们检验了我们的中心假设，即通过腔内P2Y2受体对局部ATP信号做出反应而导致远端肾单位ENaC活性的生理性下调通过解决四个特定目标来抑制钠的重吸收：1)确定生理浓度的ATP是否影响哺乳动物远端肾单位的ENaC活性，并确定细胞信号途径和作用机制；2)确定旁分泌/自分泌ATP信号对ENaC的功能失调调节的后果；3)确定连接蛋白30在ENaC的ATP调节中的作用；以及4)通过嘌呤能张力定量系统盐负荷与调节远端肾单位ENaC活动之间的关系。我们使用一种综合的策略来研究ENaC的嘌呤能调节，该策略将直接测量通道活动与电生理学和分子遗传学方法相结合，明确地确定了P2Y2受体的作用，并探测Cx30作为自分泌ATP释放的管道。使用含有正常细胞环境中的天然ENaC的生理相关组织(新鲜分离的小鼠集合管)。此外，这项建议的范围是从动物到分子作用机制的全面探索，通过集合管道中的嘌呤能张力和通过Cx30释放的ATP来调节ENaC。 与公共健康相关：高血压在美国很普遍，而且还在继续上升，这使它成为美国改善生活质量和医疗保健的主要障碍之一。肾脏的适当盐分处理对于正确控制血压至关重要。目前的建议研究了肾脏盐处理的细胞和分子机制，特别是在远端肾单位，这是人类全身盐和水平衡微调的位置。

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.