Regulation of Flow-Induced K+ Wasting

描述(由申请人提供)：该项目的总体目标是阐明局部嘌呤能信号在调节晚期远端小管的皮质收集管(CCD)的血流依赖性、不适当的K+分泌方面的作用。众所周知，向远端小管晚期液体输送增强的状态会导致K+分泌增加，从而导致K+排泄/K+浪费。这种依赖流动的K+损耗广泛发生在容量扩张、使用环状利尿剂的情况下，以及巴特综合征和吉特曼综合征等缺盐肾小管疾病中。它会很快导致低钾血症、容量耗竭和低血压。虽然这种依赖于流的K+流失的机制被认为涉及到流诱导的钙内流，进而激活远端小管上的钙依赖的“BK”K+通道，但其机制仍然存在争议且知之甚少，特别是关于BK对钙的敏感性以及该通道是否能完全解释K+的丢失。我们最近发现，钙离子可通透的TRPV4通道是钙离子内流敏感的关键途径，现在发现它的流量依赖性在很大程度上受上游的、流敏感的、嘌呤能信号(局部ATP释放)的调节，该信号与PLC/DAG/PKC通路相结合来激活TRPV4。重要的是，我们发现了一种新的钙依赖的K+通道SK3，它比BK具有更高的钙亲和力，BK在CCD细胞的管腔边缘高度表达，并被Flow激活。SK3的激活导致膜超极化，导致钙离子内流增加，我们推测这反过来将支持低亲和力BK通道的激活。 我们的假设是，高肾小管流量激活TRPV4(通过嘌呤能信号)，TRPV4介导的钙内流首先激活SK3，促进钙内流，然后激活BK，从而通过两个K+通道流诱导K+分泌。本研究的目的有两个：1)阐明SK3和BK K+通道在CDC中的功能和相互依赖关系，并阐明通过TRPV4流诱导的钙信号调节这些通道从而产生流敏感K+分泌的机制；2)通过对K+排泄转基因动物模型中流动敏感信号成分的关键评估，验证嘌呤能信号和增强的小管流激活流依赖K+排泄的分子模型。该项目在使用细胞培养模型和天然的分裂开放的CCDs来定义调控途径的关键方面(使用钙成像、电生理学、免疫荧光、生化/分子策略)方面具有创新性，并在K+排泄失调的转基因动物模型中验证了发现。这些研究的结果将为我们理解肾脏流动敏感K+排泄的分子基础提供新的见解，并将确定潜在的新的治疗靶点，以开发K+耗竭病理的治疗策略。

DESCRIPTION (provided by applicant): The overall goal of the project is to elucidate the role of local purinergic signaling in regulating flow-dependent, inappropriate, K+ secretion by the cortical collecting ducts (CCD) of the late distal tubule. It is well known that states of enhance fluid delivery to the late distal tubule induces enhanced K+ secretion which results in excess K+ excretion/K+ wasting. Such flow-dependent K+ wasting is wide-spread occurring in conditions of volume expansion, loop-diuretic use and in salt-losing tubulopathies, such as Bartter and Gitelman syndromes. It can quickly lead to hypokalemia, volume depletion, and low blood pressure. While the mechanism of this flow- dependent K+ wasting is thought to involve flow-induced Ca2+ influx which, in turn, activates the Ca2+- dependent "BK" K+ channel in the late distal tubule, the mechanism remains controversial and poorly understood, especially with regard to the Ca2+ sensitivity of BK and whether this channel can fully account for the K+ lose. We recently identified the Ca2+-permeable TRPV4 channel as the key flow-sensitive Ca2+ influx pathway and now show that its flow-dependence is largely regulated by upstream, flow-sensitive, purinergic signaling (local ATP release) coupled to the PLC/DAG/PKC pathway to activate TRPV4. Importantly, we have identify a new Ca2+-dependent K+ channel, SK3, with a much higher Ca2+ affinity than BK, which is highly expressed at the luminal border of CCD cells and is activated by flow. Activation of SK3 hyperpolarizes the membrane leading to enhanced Ca2+ influx, which we postulate would, in turn, support activation of the low- affinity BK channel. Our hypothesis is that high tubular flow activates TRPV4 (via purinergic signaling) and that the TRPV4-mediated Ca2+ influx first activates SK3, enhancing Ca2+ influx, and subsequently activating BK leading to flow-induced K+ secretion by both K+ channels. The study has two aims: 1) To elucidate the function and interdependency of SK3 and BK K+ channels in CCD, and to elucidate the mechanism by which flow- induced Ca2+ signaling through TRPV4 regulates these channels to give rise to flow-sensitive K+ secretion, and 2) To verify the molecular model by which purinergic signaling and enhanced tubular flow activate flow- dependent K+ excretion by critical assessment of flow-sensitive signaling components in genetically modified animal models of K+ excretion. The project is innovative in the use of cell culture models and native, split- opened CCDs to define key aspects of the regulatory pathways (using Ca2+ imaging, electrophysiology, immunofluorescence, biochemical/molecular strategies), with verification of the findings in genetically modified animal models of dysregulate K+ excretion. The outcome of these studies will provide new insights into our understanding of the molecular basis of flow-sensitive K+ excretion in the kidney and will identify potential new therapeutic targets for development of treatment strategies in K+ wasting pathologies.