Site-specific regulation of the epithelial sodium channel (ENaC) and the renal outer medullary K+ channel (ROMK) in the aldosterone-sensitive distal nephron (ASDN)

醛固酮敏感远端肾单位 (ASDN) 中上皮钠通道 (ENaC) 和肾外髓 K 通道 (ROMK) 的位点特异性调节

• 批准号: 391676485
• 负责人: Professor Dr. Christoph Korbmacher
• 金额: --
• 依托单位: Institut für Zelluläre und Molekulare Physiologie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2017
• 资助国家: 德国
• 起止时间: 2016-12-31 至 2022-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/391676485?language=en
• 关键词: Site; specific; regulation; epithelial; sodium

Pathophysiological disturbances of Na+ or K+ homeostasis result in potentially life threatening disorders. This justifies the need to investigate cellular and molecular mechanisms involved in renal Na+ and K+ handling. The aldosterone-sensitive distal nephron (ASDN) comprises the late distal convoluted tubule (DCT2), connecting tubule (CNT), cortical collecting duct (CCD) and its distal parts. In the ASDN the epithelial sodium channel (ENaC) and the renal outer medullary K channel (ROMK) are critically important for the final adjustment of tubular Na+ absorption and K+ secretion, respectively. Aldosterone stimulates ENaC through the mineralocorticoid receptor (MR). This promotes tubular Na+ absorption and enhances the electrical driving force for ROMK mediated renal K+ secretion. Recent evidence supports the idea that ENaC and ROMK are expressed and regulated in a site-specific manner. This has important implications for Na+ homeostasis and blood pressure control as well as for K+ homeostasis. This renewal proposal is a logical continuation of our current project with a focus on patch-clamp studies in microdissected murine renal tubules. In addition, we will use a differentiated mouse CCD cell line (mCCDcl1) and native mouse distal colon in a comparative approach to reveal common and tissue-specific regulatory mechanisms of ENaC mediated Na+ transport. Based on published findings and preliminary experiments, we have two key objectives: (1) Identify common and distinct mechanisms of ENaC regulation in DCT2/CNT versus CNT/CCD; (2) Explore site-specific mechanisms of ROMK regulation in the distal nephron. Whole-cell and outside-out patch-clamp recordings will be performed in the transitional zones between DCT2 and early CNT (DCT2/CNT) and between late CNT and early cortical collecting duct (CNT/CCD). In combination with transgenic mouse models this provides a powerful approach to address unresolved physiological questions regarding the site-specific regulation of ENaC and ROMK in native tubules. In particular, we will use inducible nephron-specific MR deficient (MR KO) mice, a nephron specific glucocorticoid receptor (GR) deficient mouse model and angiotensin II type 1A (AT1A) receptor deficient (Agtr1A-/-) mice to explore the differential effects of MR, GR and AT1A deficiency on ENaC and ROMK activity in DCT2/CNT versus CNT/CCD. Recent evidence suggest that mTORC2 plays a role in ENaC regulation in response to changes in dietary K+ intake and local [K+]. To explore the role of mTORC2 in site-specific ENaC and ROMK regulation, we will use a novel mouse model (TRKO mice) with doxycycline-inducible tubular specific mTORC2 deficiency. With the experiments detailed in our project proposal, we expect to obtain new findings regarding the mechanisms involved in the site-specific regulation and function of ENaC and ROMK. This will enhance our understanding of the physiological interplay of these channels in Na+ and K+ homeostasis.

Na+或K+稳态的病理生理紊乱导致潜在的危及生命的疾病。这证明需要研究肾脏Na+和K+处理的细胞和分子机制。 醛固酮敏感性远端肾单位（aldosterone-sensitive distal nephron，ASDN）由晚远曲小管（late distal coronary tubule，DCT 2）、连接小管（connecting tubule，CNT）、皮质集合管（coronary collecting duct，CCD）及其远端组成。在ASDN中，上皮钠通道（ENaC）和肾外髓钾通道（ROMK）分别对肾小管Na+吸收和K+分泌的最终调节至关重要。醛固酮通过盐皮质激素受体（MR）刺激ENaC。这促进了肾小管Na+吸收并增强了ROMK介导的肾K+分泌的电驱动力。最近的证据支持ENaC和ROMK以位点特异性方式表达和调节的观点。这对Na+稳态和血压控制以及K+稳态具有重要意义。这个更新的建议是一个合乎逻辑的延续，我们目前的项目，重点是膜片钳研究在显微镜下的小鼠肾小管。此外，我们将使用一个分化的小鼠CCD细胞系（mCCDcl 1）和本地小鼠远端结肠的比较方法，以揭示共同的和组织特异性的ENaC介导的Na+转运的调节机制。基于已发表的研究结果和初步实验，我们有两个关键目标：（1）确定在DCT 2/CNT与CNT/CCD中ENaC调节的共同和不同机制;（2）探索远端肾单位中ROMK调节的位点特异性机制。将在DCT 2和早期CNT（DCT 2/CNT）之间以及晚期CNT和早期皮质集合管（CNT/CCD）之间的过渡区进行全细胞和外向膜片钳记录。与转基因小鼠模型相结合，这提供了一种强有力的方法来解决未解决的生理学问题，关于天然小管中ENaC和ROMK的位点特异性调节。特别是，我们将使用诱导型肾单位特异性MR缺陷（MR KO）小鼠，肾单位特异性糖皮质激素受体（GR）缺陷小鼠模型和血管紧张素II 1A型（AT 1A）受体缺陷（Agtr 1A-/-）小鼠，以探讨MR，GR和AT 1A缺陷对ENaC和ROMK活性的差异影响，在DCT 2/CNT与CNT/CCD。最近的证据表明，mTORC 2在ENaC调节中起作用，以响应膳食K+摄入量和局部[K+]的变化。为了探索mTORC 2在位点特异性ENaC和ROMK调节中的作用，我们将使用一种新的小鼠模型（TRKO小鼠），其具有多西环素诱导的肾小管特异性mTORC 2缺陷。通过我们项目建议书中详细描述的实验，我们期望获得有关ENaC和ROMK位点特异性调节和功能机制的新发现。这将增强我们对这些通道在Na+和K+稳态中的生理相互作用的理解。