Probing intracellular Cl- in a WNK signaling-dependent transporting epithelium

 描述（由申请方提供）：肾上皮细胞离子转运障碍导致高血压和高钾血症，从而增加心血管并发症和死亡的风险。来自人类和小鼠模型的数据表明，抑制无赖氨酸（WNK）激酶在转运肾上皮细胞中具有改善高血压和高钾血症的潜力。最近的工作表明，在体外，Cl-结合到WNK活性位点并抑制其活化。然而，尚不清楚Cl-是否调节WNK依赖的跨上皮离子转运。为了理解这一点，需要测量和操纵WNK调节上皮中的Cl-的能力。然而，这在哺乳动物肾小管中难以实现。申请人的长期目标是通过使用果蝇肾小管更好地理解疾病相关的上皮离子转运机制。这种创新的方法利用了在生理上可接近的运输上皮中进行遗传操作的便利性。本提案的总体目标是开发方法来测量和操纵细胞内的氯离子浓度在苍蝇肾小管。其基本原理是使该项目的未来阶段，研究细胞内Cl-是否以及如何通过调节WNK信号转导来调节跨上皮离子转运。申请人先前已经证明，低渗刺激苍蝇肾小管中的WNK依赖性跨上皮离子转运。在其他肾上皮细胞中，低渗诱导肿胀后细胞内Cl-有两个阶段的减少。首先，细胞内Cl-浓度稀释性降低。随后随着细胞经历KCl外排介导的代偿性调节体积减少，Cl-进一步减少。外排通过钾-氯协同转运蛋白（KCCs）和/或通过通道的平行K+和Cl-外排发生。在果蝇培养的细胞中，雌激素样肽-1是膨胀激活的Cl-通道。这里的假设是，在低渗诱导的肿胀的条件下抑制雌激素-1将钝化细胞内Cl-的降低，而组成型活性KCC的表达将在等渗条件下降低细胞内Cl-，在没有细胞肿胀的情况下。该假设将在三个具体目标中进行测试：在目标1中，申请人将确定用于测量果蝇肾小管中细胞内Cl-的最佳方法，测试使用转基因比率荧光Cl-传感器的可行性。目的2将确定是否bestrophin-1介导肿胀诱导的Cl-流出小管上皮细胞通过测量细胞内Cl-在等渗和低渗条件下，有或没有bestrophin-1抑制敲低或显性负转基因表达。在目标3中，将突变引入预测的磷酸调节丝氨酸和苏氨酸中以产生组成型活性KCC。将在等渗（非溶胀）条件下测量细胞内Cl-。所提出的研究是重要的，因为它将促进靶向WNK依赖性上皮离子转运过程的药物的开发，对高血压和高钾血症具有有益作用，由于细胞内Cl-调节激酶的独特机制，降低了脱靶效应的风险。

 DESCRIPTION (provided by applicant): Disordered renal epithelial ion transport results in hypertension and hyperkalemia, which confer increased risk of cardiovascular complications and death. Data from humans and mouse models indicate that inhibition of with-no-lysine (WNK) kinases in transporting renal epithelia has the potential to ameliorate hypertension and hyperkalemia. Recent work has shown that in vitro, Cl- binds to the WNK active site and inhibits its activation. What is unknown, however, is whether Cl- regulates WNK-dependent transepithelial ion transport. To understand this, the ability to measure and manipulate Cl- in a WNK-regulated epithelium is required. However, this is difficult to achieve in mammalian renal tubules. The applicant's long-term goal is to better understand disease-relevant epithelial ion transport mechanisms by using the Drosophila melanogaster renal tubule. This innovative approach harnesses the ease of genetic manipulation in a physiologically accessible transporting epithelium. The overall objective of this proposal is to develop methods to measure and manipulate intracellular Cl- concentrations within the fly renal tubule. The rationale is to enable a future phase of the project, studying whether and how intracellular Cl- regulates transepithelial ion transport through regulation of WNK signaling. The applicant has previously demonstrated that hypotonicity stimulates WNK-dependent transepithelial ion transport in the fly renal tubule. In other renal epithelia, there is a two-phase decrease in intracellular Cl- after hypotonicity-induced swelling. First, there is a dilutional decrease in intracellular Cl-. This is followed by a further decrease in Cl- as cells undergo compensatory regulatory volume decrease mediated by efflux of KCl. Efflux occurs through potassium-chloride cotransporters (KCCs), and/or parallel K+ and Cl- efflux through channels. In Drosophila cultured cells, bestrophin-1 is the swell-activated Cl- channel. The hypothesis here is that inhibition of bestrophin-1 under conditions of hypotonicity-induced swelling will blunt the lowering of intracellular Cl-, while expression of constitutively active KCC will lower intracellular Cl- in isotonic conditions, in the absence of cellular swelling. This hypothesis will be tested in three specific aims: In aim 1, the applicant will determine the optimal means for measuring intracellular Cl- in the Drosophila renal tubule, testing the feasibility of using a transgenic ratiometric fluorescent Cl- sensor. Aim 2 will determine whether bestrophin-1 mediates swell-induced Cl- efflux from tubule epithelial cells by measuring intracellular Cl- in isotonic and hypotonic conditions, with or without bestrophin-1 inhibition by knockdown or dominant-negative transgene expression. In aim 3, mutations will be introduced into predicted phospho-regulatory serines and threonines to generate a constitutively active KCC. Intracellular Cl- will be measured in isotonic (non-swell) conditions. The proposed research is significant, because it will facilitate development of drugs targeting WNK-dependent epithelial ion transport processes, with beneficial effects on hypertension and hyperkalemia, that have decreased risk of off- target effects due to the unique mechanism of kinase regulation by intracellular Cl-.