Regulation of K+ balance by distal nephron TRPV4 channel

摘要 钾平衡的严密调节是正常生理的基础。钾动态平衡紊乱与 有一系列与心血管和肾脏相关的病理。远端肾单位(DN)，包括 连接小管(CNT)和集合管(CD)，是受控钾转运的主要部位。 肾脏，这对于匹配尿钾排泄和不同的饮食钾摄入量是必不可少的。在这个提案中，我们假设 在糖尿病肾病中大量表达的机械敏感的钙通透性TRPV4通道是一个关键 肾钾处理能否通过Maxi-K(BK)刺激血流依赖性钾分泌的决定因素 通道，抑制H-K-ATPase依赖的K重吸收。我们提供了大量的证据支持我们 糖尿病肾病中的TRPV4活性受饮食中钾摄入量的调节，其功能障碍导致显著的 全身性钾平衡的扭曲。TRPV4-/-小鼠在糖尿病肾病和糖尿病肾病模型中BK通道活性显著降低。 当饮食中钾的摄入量较高时，会出现高钾血症。相反，TRPV4缺失会增加H-K 当饮食中钾摄入量低时，ATPase活性和防止低钾血症。总体而言，我们假设 在饮食补钾方案的适应过程中，TRPV4起着生理上相关的利钾因子的作用 通过刺激BK介导的K分泌和抑制H-K ATPase依赖的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 饮食钾摄入量对糖尿病肾病患者TRPV4调节的信号决定因素。SA2：建立 TRPV4在糖尿病肾病中BK介导的钾分泌中的作用及其病理生理意义 它对全身钾平衡的破坏的后果。SA3：探索以下方面的机制和相关性 TRPV4对糖尿病肾病钾重吸收的调节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武器库，其中包括监控TRPV4 膜片钳电生理、细胞内钙和H-K-ATPase介导的pH对BK通道活性的影响 啮齿类动物和人类天然糖尿病肾病细胞的测量，共聚焦免疫荧光显微镜，平衡 传统动物和基因操纵动物膳食钾摄入量调控的研究。总体而言，我们 以期直接证明机械敏感的TRPV4通道在糖尿病肾病中的生理学相关性。 系统水平，并将确定TRPV4功能障碍对系统性K的病理生理影响 动态平衡。此外，这将促使基于靶向TRPV4的新策略的开发来管理 临床环境中高钾和低钾血症危及生命的状态。

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.