Regulation of K+ balance by distal nephron TRPV4 channel

摘要 钾平衡的严格调节是正常生理的基础。 K 稳态紊乱与 具有广泛的心血管和肾脏相关病理。远端肾单位 (DN)，包括 连接小管（CNT）和集合管（CD）是细胞内控制钾转运的主要场所。 肾脏，这对于使尿钾排泄与不同的膳食钾摄入量相匹配至关重要。在这个提案中，我们假设 DN 中大量表达的机械敏感 Ca2 渗透性 TRPV4 通道是一个关键 肾钾处理的决定因素，能够通过 maxi-K (BK) 刺激流量依赖性 K 分泌 通道并抑制 H -K ATP 酶依赖性 K 重吸收。我们产生了丰富的支持证据 DN 中的 TRPV4 活性受膳食钾摄入量的调节，其功能障碍会导致显着的 系统钾平衡的扭曲。 TRPV4 -/- 小鼠 DN 中的 BK 通道活性显着降低，并且 当膳食钾摄入量高时，会出现高钾血症。相反，TRPV4 缺失增强了 H -K 当膳食钾摄入量较低时，ATP 酶活性可防止低钾血症。总的来说，我们假设 TRPV4 在适应饮食钾方案期间充当生理相关的利尿因子 通过刺激 BK 介导的 K 分泌并抑制 H -K ATP 酶依赖性 K 重吸收。 为了解决这一中心假设，我们制定了三个具体目标： SA1：检查分子和 通过膳食钾摄入量对 DN 中 TRPV4 进行调节的信号决定因素。 SA2：建立 TRPV4 功能对于 DN 中 BK 介导的 K 分泌的重要性并定义病理生理学 它对系统钾平衡的破坏的影响。 SA3：探索机制和相关性 TRPV4 调节 DN 中 K 重吸收。为了使这个项目取得成果，我们招募了强大的 协作专业知识并实施全面的实验库，其中包括监测 TRPV4 和 BK 通过膜片钳电生理学、细胞内 Ca2 和 H -K ATP 酶介导的 pH 通道活动 测量、啮齿动物和人类天然 DN 细胞的共焦免疫荧光显微镜、平衡 对传统动物和基因改造动物的膳食钾摄入量进行控制的研究。总的来说，我们 期望直接证明 DN 中机械敏感 TRPV4 通道的生理相关性 系统水平并将定义 TRPV4 功能障碍对系统 K 的病理生理学影响 体内平衡。此外，这将促使开发基于针对 TRPV4 的新策略来管理 临床环境中危及生命的高钾血症和低钾血症。

SUMMARY Tight regulation of K+ balance is fundamental for normal physiology. Disturbed K+ homeostasis is associated with a wide spectrum of cardiovascular- and kidney-related pathologies. The distal nephron (DN), including the connecting tubule (CNT) and the collecting duct (CD), is the major site of controlled potassium transport in the kidney, which is essential to match urinary K+ excretion to varying dietary K+ intake. In this proposal, we posit that the mechanosensitive Ca2+-permeable TRPV4 channel abundantly expressed in the DN is a critical determinant of renal potassium handling capable of stimulating flow-dependent K+ secretion via maxi-K (BK) channel and inhibiting H+-K+ ATPase-dependent K+ reabsorption. We generated abundant supportive evidence that the TRPV4 activity in the DN is regulated by dietary potassium intake with its dysfunction causing significant distortions of systemic K+ balance. TRPV4 -/- mice have a markedly reduced BK channel activity in the DN and develop hyperkalemia when dietary potassium intake is high. On the contrary, TRPV4 deletion augments H+-K+ ATPase activity and protects against hypokalemia when dietary potassium intake is low. Overall, we hypothesize that TRPV4 serves as a physiologically relevant kaliuretic factor during adaptations to dietary potassium regimen by stimulating BK-mediated K+ secretion and inhibiting H+-K+ ATPase-dependent K+ reabsorption. To address this central hypothesis, we developed three specific aims: SA1: Examine molecular and signaling determinants of TRPV4 regulation in the DN by dietary K+ intake. SA2: Establish the importance of TRPV4 function for BK-mediated K+ secretion in the DN and define pathophysiological ramifications of its disruption on systemic K+ balance. SA3: Explore the mechanism and relevance of regulation of K+ reabsorption in the DN by TRPV4. To bring this project to fruition, we recruit strong collaborative expertise and implement a comprehensive experimental arsenal which involves monitoring TRPV4 and BK channels activity with patch clamp electrophysiology, intracellular Ca2+ and H+-K+ ATPase-mediated pH measurements, confocal immunofluorescent microscopy in native DN cells of rodents and humans, balance studies upon manipulation of dietary K+ intake in conventional and genetically manipulated animals. Overall, we expect to directly demonstrate the physiological relevance of mechanosensitive TRPV4 channel in the DN at the systemic level and will define pathophysiological ramifications of TRPV4 dysfunction on systemic K+ homeostasis. Furthermore, this will urge development of novel strategies based on targeting TRPV4 to manage life-threatening states of hyper- and hypokalemia in clinical setting.