Membrane Trafficking of Renal Ion Transport Proteins in Potassium Homeostasis

描述（由申请人提供）：肾脏调节远端肾单位的K+分泌，以响应饮食摄入量的变化，维持K+稳态。远端K+分泌涉及K+通过顶端K+通道（如ROMK）从细胞流出到管腔中。通过上皮Na+通道ENaC的Na+重吸收为K+分泌提供电驱动力。Na-Cl协同转运蛋白NCC活性的增加可能会减少Na+向ENaC的递送，从而减少K+分泌。在支持的作用，NCC以及ROMK在维持K+稳态的饮食变化的K+摄入量，高K+负荷的啮齿类动物增加ROMK的密度，同时降低NCC的密度在远端肾单位。ROMK和NCC的上游调控仍然不完全清楚。WNK 1是一种蛋白激酶，其基因突变导致表达增加，导致假性醛固酮减少症II型（PHA 2），一种常染色体显性疾病，特征为高血压和高钾血症。WNK 1具有几种选择性剪接变体，包括普遍存在的全长长WNK 1（L-WNK 1）和较短的肾特异性WNK 1（KS-WNK 1）。基于细胞的表达研究表明，L-WNK 1激活NCC并抑制ROMK。KS-WNK 1本身不调节NCC和ROMK，但分别逆转L-WNK 1介导的NCC和ROMK的激活和抑制。我们研究的长期目标是了解正常生理和疾病状态下的K+稳态。在这里，我们将研究三个具体目标。目的1：探讨L-WNK 1在肾脏Na+、K+转运中的生理作用及其机制。目的二是验证KS-WNK 1拮抗L-WNK 1对肾脏Na+和K+转运的调节，以及饲料K+的变化影响L-/KS-WNK 1调节NCC和ROMK的比例。目的3：探讨醛固酮与L-和KS-WNK 1通路在肾脏Na+、K+转运及对膳食K+变化反应中的相互作用。我们已经产生了L-和/或KS-WNK 1基因表达改变的小鼠模型。将通过测量血液和尿液电解质以及Na+和K+转运蛋白对饮食Na+或K+扰动和利尿剂的反应表达，研究这些小鼠的肾脏Na+和K+转运。还将通过离体CCD小管的体外微灌注研究K+分泌。将进行手术肾上腺切除术，以检查醛固酮和WNK 1通路之间的相互作用。这些研究将为WNK 1在正常生理和疾病状态下肾脏Na+和K+转运中的体内作用提供重要见解。

DESCRIPTION (provided by applicant): The kidney adjusts K+ secretion in the distal nephron in response to variations in dietary intake to maintain K+ homeostasis. The distal K+ secretion involves K+ efflux from the cell into the lumen through apical K+ channels such as ROMK. Na+ reabsorption via the epithelial Na+ channel ENaC provides the electrical driving force for K+ secretion. An increase in the activity of the Na-Cl cotransporter NCC may decrease Na+ delivery to ENaC thus diminishing K+ secretion. In support for the role of NCC as well as ROMK in maintaining K+ homeostasis during dietary variations of K+ intake, high K+ loading in rodents increases the density of ROMK while decreasing the density of NCC in the distal nephron. The upstream regulation of ROMK and NCC remains incompletely understood. WNK1 is a protein kinase of which gene mutations resulting in increased expression cause pseudohypoaldosteronism type II (PHA2), an autosomal-dominant disease characterized by hypertension and hyperkalemia. WNK1 has several alternatively spliced variants including a ubiquitous full- length long WNK1 (L-WNK1) and a shorter kidney-specific WNK1 (KS-WNK1). Cell-based expression studies have shown that L-WNK1 activates NCC and inhibits ROMK. KS-WNK1, by itself, does not regulate NCC and ROMK but reverses L-WNK1-mediated activation and inhibition of NCC and ROMK, respectively. The long- term goal of our research is to understand K+ homeostasis in the normal physiology and in diseased states. Here, we will examine three specific aims. Aim 1 is to examine the physiological role and mechanism of L- WNK1 in the regulation of renal Na+ and K+ transport. Aim 2 is to examine the hypothesis that KS-WNK1 antagonizes L-WNK1 regulation of renal Na+ and K+ transport and changes in dietary K+ affect the ratio of L- over KS-WNK1 to regulate NCC and ROMK. Aim 3 is to examine the interplay between aldosterone and L- and KS-WNK1 pathway in the regulation of renal Na+ and K+ transport and the response to variations in dietary K+. We have generated mouse models with genetically altered expression of L-and/or KS-WNK1. Renal Na+ and K+ transport in these mice will be studied by measuring blood and urinary electrolytes and expression of Na+ and K+ transporters in response to dietary Na+ or K+ perturbations and to diuretics. K+ secretion will also be studied by in vitro microperfusion of isolated CCD tubules. Surgical adrenalectomy will be performed to examine interactions between aldosterone and WNK1 pathway. These studies will provide important insights to the in vivo role of WNK1 in renal Na+ and K+ transport in the normal physiology and in diseased states.