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钠通道(ENaC)的重要作用 盐敏感性和血管紧张素病理中集合管(CD)主细胞介导的重吸收 II(Ang II)依赖型高血压。相反，醛固酮介导的ENaC活性在 高血钾是刺激钾分泌所必需的，没有容量滞留的迹象。在这里，我们建议 CD独特的马赛克结构，由电非耦合的主细胞和嵌入的细胞组成， 有助于单独控制Na+、K+和Cl-的通量，以根据体积确定其传输轮廓 对Na+/K+交换的滞留(Na+和Cl-重吸收)(耦合Na+重吸收和K+分泌)。我们进一步 提供了强有力的初步证据支持ClC-K2/b通道介导的氯离子通量在 在这一过程中嵌入细胞。使用新鲜分离的小鼠CD，我们发现ClC-K2/b活性和 表达与膳食中的氯离子摄入量呈负相关，而与K+的摄入量无关。此外，Ang II还表现出多组分 Cd嵌入细胞对ClC-K2/b活性、转运和通道丰度的刺激作用 提示其在低氯饮食和容量耗竭时激活ClC-K2/b中起重要作用。一如既往地，老鼠 缺乏血管紧张素1型受体(AT1R)的患者血容量降低，肾脏ClC-K2/b表达降低。 总体而言，我们假设CLC-K2/b在嵌入细胞中的功能主要受饮食中氯离子摄入量的调节 可能是以依赖血管紧张素II的方式。这种随意的阴离子ClC-K2/b介导的氯-内流降低了ENaC- 产生了K+分泌的驱动力，使Cd能够从Na+/K+交换中调节Cd的转运 低血容量时高钾血症(仅醛固酮升高)到氯化钠重吸收(醛固酮和血管紧张素转换酶) II是增加的)。伴随着ENaC和ClC-K2/b的过度刺激，Cd的操作转移到了氯化钠 重吸收模式，从而参与血管紧张素转换酶II诱导的高血压的病理过程。为了解决这个中心问题 假设，我们制定了3个具体目标： SA1：检测饮食提示对Cd中ClC-K2/b活性和表达的调节。建立 血管紧张素转换酶II和醛固酮在此过程中的贡献和优势。 SA2：明确Ang II调控ClC-2的作用机制和细胞信号通路 光盘中的k2/b活动。 SA3：在病理学中建立CD中ClC-K2/b活性增强的病理生理分支 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。 综上所述，这一建议试图极大地扩展我们对电解液主要是如何运输的理解 而嵌入的细胞整合在一起，使CD对饮食和内分泌的输入做出适当的反应。此外，它还 提供生理上相关的方法来靶向Cd中依赖ClC-K2/b的Cl-重吸收，作为一种工具 在临床环境中对抗盐敏感和血管紧张素Ⅱ依赖型高血压。

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.