Membrane Trafficking of Renal Ion Transport Proteins in Potassium Homeostasis

描述（由申请人提供）：肾脏调节远端肾元的K+分泌，以响应饮食摄入的变化，维持K+稳态。远端K+分泌涉及K+从细胞通过顶端K+通道（如ROMK）外排到管腔。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共转运体NCC活性的增加可能会减少Na+向ENaC的传递，从而减少K+的分泌。为了支持NCC和ROMK在K+摄入量变化期间维持K+稳态中的作用，啮齿动物的高K+负荷增加了ROMK的密度，同时降低了远端肾元中NCC的密度。ROMK和NCC的上游调控尚不完全清楚。WNK1是一种蛋白激酶，其基因突变导致表达增加导致假醛固酮减少症II型（PHA2），这是一种常染色体显性疾病，以高血压和高钾血症为特征。WNK1有几种选择性剪接变体，包括普遍存在的全长长WNK1 （L-WNK1）和较短的肾特异性WNK1 （KS-WNK1）。基于细胞的表达研究表明，L-WNK1激活NCC并抑制ROMK。KS-WNK1本身不调节NCC和ROMK，但分别逆转l - wnk1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K+的体内平衡。在这里，我们将研究三个具体目标。目的1：探讨L- WNK1在肾脏Na+和K+转运调控中的生理作用和机制。目的2是验证KS-WNK1拮抗L- wnk1调控肾脏Na+和K+转运以及饮食K+变化影响L- / KS-WNK1调节NCC和ROMK的假设。目的3是研究醛固酮与L-和KS-WNK1通路在调节肾脏Na+和K+转运以及对饮食K+变化的反应中的相互作用。我们已经建立了l -和/或KS-WNK1基因表达改变的小鼠模型。通过测量血液和尿液电解质以及饮食中Na+或K+扰动和利尿剂对Na+和K+转运蛋白表达的反应，研究这些小鼠肾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。外科肾上腺切除术将进行检查醛固酮和WNK1途径之间的相互作用。这些研究将为WNK1在正常生理和病变状态下肾脏Na+和K+转运中的体内作用提供重要的见解。

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.