Regulation of Flow-Induced K+ Wasting

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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.