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.
O'Neil Flow-sensitive potassium secretion in the late distal nephron and collecting system are associated with altered fluid and electrolyte balance that can rapidly lead to hypokalemia, volume depletion and low blood pressure, which contribute to morbidity and mortality around the world. The current project will begin to elucidate the mechanisms by which mechanosensitive kidney segments contribute to and/or cause altered extracellular volume levels and hypertension in health and disease. The long- term goal is to identify new proteins/processes that can be used as targets for development of new therapeutic agents to ameliorate these dysfunctional states.
|Mamenko, Mykola V; Boukelmoune, Nabila; Tomilin, Viktor N et al. (2017) The renal TRPV4 channel is essential for adaptation to increased dietary potassium. Kidney Int 91:1398-1409|