Physiology of ClC-K2/b Cl- channel in the collecting duct

大量的基础和临床证据证实了增强型上皮Na+通道（ENaC）的重要作用 介导的重吸收在集合管（CD）的主要细胞的病理盐敏感性和血管紧张素 II（Ang II）依赖性高血压。与此相反，醛固酮介导的ENaC活性增加， 高钾血症是刺激钾分泌所必需，而没有容量保留的迹象。在这里，我们建议 CD的独特镶嵌结构，由电性未偶联的主细胞和插入细胞组成， 用于分别控制Na+、K+和Cl-通量以确定其从体积的传输轮廓 Na +/K+交换（Na+重吸收与K+分泌耦合）。我们进一步 产生了强有力的初步证据，支持ClC-K2/B通道介导的Cl-通量在 在这一过程中的细胞。使用新鲜分离的鼠CD，我们发现ClC-K2/B活性和 表达与膳食Cl-而不是K+摄入量呈负相关。此外，Ang II表现出多组分 对CD插入细胞中ClC-K2/B活性、运输和通道丰度的刺激作用 暗示其在低Cl-饮食和容量消耗期间ClC-K 2/B活化中的重要作用。一致性，小鼠 缺乏血管紧张素1型受体（AT 1 R）的人是低血容量的，并且具有降低的肾C1 C-K2/B表达。 总的来说，我们假设在闰细胞中ClC-K2/B的功能主要受饮食中Cl-摄入的调节 可能是以血管紧张素II依赖的方式。这种任意阴离子ClC-K2/b介导的Cl-内流降低了ENaC- 产生K+分泌的驱动力，使得能够在过程中从Na+/K+交换调节CD转运谱 高钾血症（仅醛固酮升高）对低血容量期间NaCl重吸收（醛固酮和Ang 二是增加）。同时过度刺激ENaC和ClC-K2/B b使CD操作转移到NaCl 重吸收模式，从而有助于血管紧张素II诱导的高血压的病理。为了解决这个中心问题， 假设，我们制定了3个具体目标： SA 1：检查饮食提示对CD中ClC-K 2/B活性和表达的调节。建立 血管紧张素II和醛固酮在这一过程中的贡献和优势。 SA 2：定义作用机制并描述Ang II调节ClC的细胞信号通路- CD中的K2/B活性。 SA 3：确定病理学中CD中ClC-K2/B活性增强的病理生理学后果 使用靶向通道缺失的转基因小鼠研究血管紧张素II依赖性高血压。 总之，该建议旨在大大扩展我们对电解质转运原理的理解， 和嵌入细胞整合，以适当的CD响应饮食和内分泌输入。而且还 提供了生理学相关的手段，靶向CD中的ClC-K2/b依赖性Cl-重吸收，作为一种工具， 在临床环境中对抗盐敏感性和Ang II依赖性高血压。

Abundant basic and clinical evidence identify a critical role of the augmented epithelial Na+ channel (ENaC) mediated reabsorption in the collecting duct (CD) principal cells in the pathology of salt-sensitive and Angiotensin II (Ang II)-dependent hypertension. On the contrary, aldosterone-mediated increases in ENaC activity during hyperkalemia are necessary to stimulate potassium secretion with no sign of volume retention. Here, we propose that the unique mosaic structure of the CD, consisting of electrically uncoupled principal and intercalated cells, is instrumental for separate control of Na+, K+ and Cl- fluxes to determine its transport profile from volume retention (Na+ and Cl- reabsorption) to Na+/K+ exchange (coupling Na+ reabsorption to K+ secretion). We further generated strong preliminary evidence supporting essential role of ClC-K2/b channel mediated Cl- flux in intercalated cells in this process. Using freshly isolated murine CDs, we found that ClC-K2/b activity and expression is inversely related to dietary Cl- but not K+ intake. Moreover, Ang II exhibits multicomponent stimulatory effects on ClC-K2/b activity, trafficking and channel abundance in the CD intercalated cells implicating its important role in ClC-K2/b activation during low Cl- diet and volume depletion. Consistently, mice lacking Angiotensin type 1 receptors (AT1R) are hypovolemic and have reduced renal ClC-K2/b expression. Overall, we hypothesize that ClC-K2/b function in intercalated cells is primarily regulated by dietary Cl- intake likely in an Ang II-dependent manner. This discretional anionic ClC-K2/b-mediated Cl- influx reduces the ENaC- generated driving force for K+ secretion enabling to tune CD transport profile from Na+/K+ exchange during hyperkalemia (only aldosterone is elevated) to NaCl reabsorption during hypovolemia (both aldosterone and Ang II are increased). Concomitant over-stimulation of ENaC and ClC-K2/b shifts the CD operation to NaCl reabsorptive mode, thereby contributing to the pathology of Ang II-induced hypertension. To address this central hypothesis, we developed 3 specific aims: SA1: Examine regulation of ClC-K2/b activity and expression in the CD by dietary cues. Establish the contribution and supremacy of Ang II and aldosterone in this process. SA2: Define the mechanism of action and delineate the cellular signaling pathway of Ang II regulation of ClC- K2/b activity in the CD. SA3: Establish pathophysiological ramifications of augmented ClC-K2/b activity in the CD in the pathology of Ang II-dependent hypertension using transgenic mice with targeted channel deletion. In summary, this proposal seeks to greatly expand our understanding how electrolyte transport in principal and intercalated cells integrate for proper CD response to dietary and endocrine inputs. Moreover, it also provides physiologically relevant means to target ClC-K2/b-dependent Cl- reabsorption in the CD as a tool to fight salt-sensitive and Ang II-dependent hypertension in clinical setting.