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

 描述(申请人提供)：肾上皮细胞离子转运障碍会导致高血压和高血钾，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，抑制肾上皮细胞运输中的非赖氨酸(WNK)激酶具有改善高血压和高钾血症的潜力。最近的研究表明，在体外，氯-与WNK活性部位结合并抑制其激活。然而，尚不清楚的是，氯离子是否调节依赖于WNK的跨上皮细胞离子转运。为了理解这一点，需要能够测量和操纵WNK调节的上皮细胞中的氯离子。然而，这在哺乳动物的肾小管中很难实现。申请者的长期目标是通过使用果蝇的黑腹大鼠肾小管更好地了解与疾病相关的上皮离子转运机制。这一创新的方法利用了在生理上可访问的运输上皮中进行基因操作的简便性。这项建议的总体目标是开发测量和操纵苍蝇肾小管内细胞内氯离子浓度的方法。其基本原理是使该项目的未来阶段成为可能，研究细胞内氯离子是否以及如何通过调节WNK信号来调节跨上皮细胞离子的运输。申请人先前已经证明，低渗刺激依赖于WNK的跨上皮细胞离子在飞翔的肾小管中的转运。在其他肾上皮细胞中，在低渗诱导的肿胀后，细胞内氯离子呈两相减少。首先，细胞内氯离子呈稀释性减少。随之而来的是氯离子的进一步减少，因为细胞经历了由KCl外流介导的代偿性调节性体积减少。外流通过钾-氯共转运体(KCC)发生，和/或平行的钾和氯-通过通道外流。在果蝇培养细胞中，Bestrophin-1是膨胀激活的氯离子通道。这里的假设是，在低张诱导的肿胀条件下抑制Bestrophin-1将钝化细胞内Cl-的降低，而在没有细胞肿胀的情况下，成分活性KCC的表达将在等渗条件下降低细胞内的Cl-。这一假设将在三个具体目标中得到验证：在目标1中，申请人将确定测量果蝇肾小管细胞内氯离子的最佳方法，测试使用转基因比率荧光氯离子传感器的可行性。目的研究Bestrophin-1是否参与膨胀诱导的肾小管上皮细胞氯外流，方法是在等渗和低渗条件下检测细胞内氯离子，并通过基因敲除或显性负性转基因表达抑制Bestrophin-1的表达。在目标3中，突变将被引入到预测的磷酸调节丝氨酸和苏氨酸中，以产生构成活性的KCC。细胞内的氯离子将在等渗(非膨胀)条件下进行测量。这项拟议的研究具有重要意义，因为它将促进针对WNK依赖的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的效果，这些药物由于细胞内Cl-调节激酶的独特机制而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-.