Luminal proteolytic activation of collecting duct enac and atp-gated ppx receptors and its potential role in regulation of renal sodium reabsorption

集合管 enac 和 atp 门控 ppx 受体的管腔蛋白水解激活及其在调节肾钠重吸收中的潜在作用

• 批准号: BB/H016449/2
• 金额: $ 7.57万
• 依托单位: University of Kent
• 依托单位国家: 英国
• 项目类别: Training Grant
• 财政年份: 2012
• 资助国家: 英国
• 起止时间: 2012 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FH016449%2F2
• 关键词: Luminal; proteolytic; activation; collecting; duct

The epithelial sodium channel (ENaC) is a transmembrane ion channel that is highly permeable to sodium (Na). ENaC is a key regulator of Na transport across the apical membrane of the distal nephron; it largely determines final urinary Na excretion, and thereby body Na balance and systemic blood pressure. By necessity, its function is tightly regulated through a variety of local and systemic factors. Recently, trypsin, acting from the luminal side (apical cell membrane) of collecting duct principal cells, has been shown to increase ENaC activity in vitro; however, there have been no in vivo studies to test its physiological significance for renal Na reabsorption; urine contains a variety of proteases that could potentially activate ENaC in vivo. The FIRST AIM of this studentship application is to assess directly the effect of luminal trypsin (as a prototypical protease) on ENaC function in vivo using the distal tubule micro-injection technique (with radioactive Na) in rats on a normal Na diet (when baseline ENaC activity is low), in the absence and presence of a trypsin inhibitor, or the ENaC blocker amiloride. Interestingly, a family of ATP-gated ion channels called P2X receptors, which are also expressed apically in the collecting duct principal cells, have a similar structure to ENaC, i.e. they have a large extracellular loop rich in basic amino acid residues that contain the consensus sequence of Arg-X-X-Arg, where X is any residue. As yet, there have been no studies on the effects of proteases on P2 receptor activity. Thus, the SECOND AIM of this studentship application is to assess the effect of extracellular proteases on P2X receptor activity in the presence and absence of ATP using the Xenopus oocyte heterologous expression system, and a twin-electrode voltage-clamp electrophysiological technique. We will examine the effects of proteases on those luminal P2X receptors found in the collecting duct principal cell (i.e. 4 homomeric and 4 heteromeric assemblies). To explore the potential to manipulate protease activation of ENaC (and its interaction with P2X receptors - see below), it is important to have a series of small molecule compounds that can mimic, inhibit, or potentiate, protease activity. The THIRD AIM of this application, to be undertaken in the laboratories of Discovery BioMed Inc (Birmingham, Alabama, USA), is to screen their compound libraries (which come from diverse synthetic and natural-product sources, and are based on the Company's expertise in renal physiology and biochemistry) using modern high-throughput bioassays on mammalian (primarily human)-cell-based platforms to develop suitable pharmacological agents. We have recently shown that renal ENaC activity is inhibited by activation of apical P2X(4 and/or 4/6) receptors when luminal concentrations of Na are high, and that these receptors switch to being potentiators of ENaC activity when concentrations of extracellular Na are lowered. We have proposed that these P2X receptors are Na sensors responsible for the local regulation of ENaC activity. Interestingly, in the original study of trypsin activation of ENaC, amiloride did not block trypsin's effect on ENaC, but in a more recent study it did. This suggests that proteases may act in more than one way to alter ENaC function, and raises the possibility that another local regulator (such as P2X) of ENaC might also be protease-sensitive. The effect of proteolytic factors on ENaC regulation by P2X receptors has not been investigated. The FOURTH AIM of this application is to assess the effect of luminal proteases on P2X-mediated regulation of ENaC activity in vitro using the rat isolated split-open collecting duct technique (with whole-cell patch clamp electrophysiology) in rats on a normal or low Na diet, and to test suitable compounds identified above.

上皮钠通道（ENaC）是对钠（Na）高度渗透的跨膜离子通道。ENaC是Na转运穿过远端肾单位顶膜的关键调节剂;它在很大程度上决定最终尿Na排泄，从而决定身体Na平衡和全身血压。它的功能必然受到各种地方和系统因素的严格管制。最近，胰蛋白酶，从收集管主细胞的腔侧（顶端细胞膜）起作用，已被证明可以增加体外ENaC活性;然而，还没有体内研究来测试其对肾钠重吸收的生理意义;尿液中含有多种蛋白酶，可能在体内激活ENaC。本研究申请的第一个目的是在不存在和存在胰蛋白酶抑制剂或ENaC阻滞剂阿米洛利的情况下，使用远端小管微量注射技术（放射性Na）直接评估管腔胰蛋白酶（作为原型蛋白酶）对正常Na饮食大鼠体内ENaC功能的影响（当基线ENaC活性较低时）。有趣的是，一个称为P2 X受体的ATP门控离子通道家族，也在集合管主细胞的顶部表达，具有与ENaC相似的结构，即它们具有富含碱性氨基酸残基的大的细胞外环，所述碱性氨基酸残基含有Arg-X-X-Arg的共有序列，其中X是任何残基。到目前为止，还没有研究蛋白酶对P2受体活性的影响。因此，本研究申请的第二个目的是评估细胞外蛋白酶对P2 X受体活性的影响，在存在和不存在ATP的情况下，使用非洲爪蟾卵母细胞异源表达系统，和双电极电压钳电生理技术。我们将研究蛋白酶对集合管主细胞中发现的管腔P2 X受体（即4个同源和4个异源组装体）的影响。为了探索操纵ENaC的蛋白酶活化（及其与P2 X受体的相互作用-参见下文）的潜力，重要的是具有一系列可以模拟、抑制或增强蛋白酶活性的小分子化合物。本申请的第三个目的是在Discovery BioMed Inc（Birmingham，亚拉巴马，美国）的实验室中进行，在基于哺乳动物（主要是人类）细胞的平台上使用现代高通量生物测定筛选其化合物文库（来自不同的合成和天然产物来源，并基于本公司在肾脏生理学和生物化学方面的专业知识），以开发合适的药理学试剂。我们最近已经表明，肾ENaC活性抑制激活顶端P2 X（4和/或4/6）受体时，管腔浓度的Na高，这些受体切换到ENaC活性的增强剂时，细胞外Na的浓度降低。我们已经提出，这些P2 X受体是负责ENaC活性的局部调节的Na传感器。有趣的是，在胰蛋白酶激活ENaC的最初研究中，阿米洛利并没有阻断胰蛋白酶对ENaC的作用，但在最近的一项研究中，它做到了。这表明蛋白酶可能以多种方式改变ENaC功能，并提出了ENaC的另一种局部调节因子（如P2 X）也可能是蛋白酶敏感的可能性。蛋白水解因子对P2 X受体调控ENaC的作用尚未研究。本申请的第四个目的是在正常或低Na饮食的大鼠中，使用大鼠分离的分裂开放集合管技术（具有全细胞膜片钳电生理学）评估管腔蛋白酶对P2 X介导的ENaC活性的体外调节的作用，并测试上述鉴定的合适化合物。