Regulation of Flow-Induced K+ Wasting

流量引起的钾浪费的调节

• 批准号: 8914606
• 负责人: ROGER Gordon O'NEIL
• 金额: $ 22.8万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCI CTR HOUSTON
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-08-20 至 2017-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8914606
• 关键词: 1,2-diacylglycerol; Accounting; Affinity; Animal Model; Bartter Disease; Biochemical; Cell Culture Techniques; Cell physiology; Cells; Coupled; Couples; Dependence; Development; Diglycerides; Disease; Distal; Diuretics; Duct (organ) structure; Electrolyte Balance; Electrophysiology (science); Equilibrium; Excretory function; Fluid Balance; Genetically Modified Animals; Goals; Health; Hypertension; Hypokalemia; Hypotension; Image; Immunofluorescence Immunologic; Intercalated Cell; KCNJ1 gene; Kidney; Lead; Link; Liquid substance; Mediating; Membrane; Modeling; Molecular; Molecular Models; Morbidity - disease rate; Mus; Nephrons; Outcome Study; Pathology; Pathway interactions; Phospholipase C; Play; Potassium; Potassium Channel; Process; Protein Kinase C; Proteins; Publishing; Regulation; Regulatory Pathway; Role; Signal Pathway; Signal Transduction; Sodium Chloride; Stimulus; Syndrome; System; Testing; Therapeutic Agents; Transport Process; Tubular formation; base; blood pressure reduction; epithelial Na+ channel; extracellular; innovation; insight; large-conductance calcium-activated potassium channels; molecular modeling; mortality; new therapeutic target; novel therapeutics; receptor; response; therapeutic target; treatment strategy; wasting

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.

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远端小管的皮层收集管（CCD）调节流动依赖，不适当的K+分泌方面的作用。众所周知，增强流体向远端小管的递送状态会诱导增强的K+分泌，从而导致多余的K+排泄/K+浪费。这种依赖流动的K+浪费是在体积膨胀，环节使用的条件下以及在盐失去的肾纤维病（例如Bartter和Gitelman综合征）中发生的广泛扩展。它可以迅速导致低钾血症，体积耗竭和低血压。虽然认为这种依赖性的K+浪费的机制涉及流动诱导的Ca2+涌入，这反过来又激活了晚期远端小管中依赖的Ca2+ - 依赖性的“ BK” K+通道，但该机制仍然有争议，尤其是在BK的CA2+敏感性方面尤其是对BK的敏感性，并且是否可以充分考虑K+ Loss。我们最近将Ca2+可渗透的TRPV4通道确定为关键流动敏感的Ca2+涌入途径，现在表明其流动依赖性在很大程度上受到上游，流动敏感的，嘌呤能信号（局部ATP释放）耦合到PLC/DAG/DAG/DAG/PKC途径，以激活TRPV4。重要的是，我们已经确定了一个新的Ca2+依赖性K+通道SK3，其Ca2+亲和力比BK高得多，BK高度表达在CCD细胞的腔界面，并被流动激活。 SK3的激活超极使膜导致CA2+涌入增强，我们假设这将支持低亲和力BK通道的激活。 我们的假设是，高管状流动激活TRPV4（通过嘌呤能信号传导），并且TRPV4介导的Ca2+流入首先激活SK3，增强Ca2+流入，并随后激活BK，从而导致两个K+通道引起流动诱导的K+分泌。 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评估K+排泄的转基因动物模型中流动敏感的信号传导成分。该项目在使用细胞培养模型和本地分裂开放的CCD方面具有创新性，以定义调节途径的关键方面（使用CA2+成像，电生理学，免疫荧光，生化/分子策略），并在基因修改的动物模型中对发现的发现的发现验证了失去失调的K+ Excretirals K+ Excretion Incretient。这些研究的结果将为我们对肾脏流动敏感K+排泄的分子基础的理解提供新的见解，并将确定潜在的新的治疗靶标，以开发K+浪费病理学的治疗策略。