Net K Secretion in Thick Ascending Limb of Mice on Low Na High K Diet

 描述(申请人提供)：高钾血症是慢性肾脏疾病(CKD)患者的常见情况，原因是肾脏排钾能力下降以及肾素-血管紧张素-醛固酮系统抑制剂的使用。旧石器时代和地中海饮食的钠含量低，钾含量高，众所周知，它们对心血管疾病和慢性肾脏病有保护作用。然而，在钠摄入量低的情况下，肾脏如何处理如此高的钾负荷仍不清楚。这对于选择有效的利尿剂和预防服用这些饮食的患者的低钾血症和高钾血症的严重后果至关重要。例如，我们在这项研究中的初步发现表明，在低钠高钾饮食中，消耗K的环状利尿剂可能变得节钾并减少K的排泄。在粗大的升支(TAL)，Na、K和Cl-通过Na-K-2Cl-共转运体(NKCC2)被重新吸收，大部分K被心尖髓外K通道(ROMK)循环回到管腔，以促进NKCC2对盐的重吸收。长期以来，人们一直认为，高K饮食引起的高K离子浓度会抑制以基底侧为主的K通道，导致膜去极化，抑制Cl-的退出，从而抑制NKCC2在TAL中的重吸收。然而，我们的实验室最近发现，在低钠高钾饮食(LNaHK)的小鼠中，TAL中有相当大的速尿敏感的Na重吸收和净K分泌。与传统观点相反，我们还发现在LNaHK饮食中NKCC2的表达增加。我们推测，在LNaHK饮食中，心尖ROMK上调，并成为主要的K通道，不受高K浓度的影响。NKCC2被上调，使Na供应给基侧Na-K ATPase，并带来更多的K被分泌。与以前的研究不同，我们现在有更先进的工具来进一步研究TAL中的离子运输，例如基因敲除小鼠。这一假设认为，LNaHK饮食诱导的高水平的醛固酮(ALDO)和血管紧张素II(Ang II)增加了TAL顶膜上的NKCC2和ROMK活性，从而导致K的净分泌。有两个特定的目的将研究TAL净K分泌的调节机制：特定目的1.确定NKCC2在TAL净K分泌中的作用和调节。具体目标2：确定ROMK在TAL净K分泌中的作用和调节。我们将用免疫印迹和实时定量聚合酶链式反应检测LNaHK组小鼠TAL中NKCC2和ROMK的表达，并与对照饲料进行比较。我们将使用显微穿刺法、原子吸收光谱法和膜片钳技术来检测NKCC2和ROMK在TAL中的功能活性。我们还将进行肾上腺切除术(ADX)，并使用醛固酮(ALDO)替代物和肾素-血管紧张素-醛固酮系统抑制剂来阐明ALDO和Ang II对NKCC2的调节。通过实施这一建议，我们将更好地了解低钠高钾饮食对心脏保护性人群肾脏K的处理。

 DESCRIPTION (provided by applicant): Hyperkalemia is a common condition in chronic kidney disease (CKD) patients because of decreased ability of kidney to excrete K as well as the use of inhibitors of renin-angiotensin-aldosterone system. Paleolithic and Mediterranean diets have low Na, high K contents and are well known to be protective against cardiovascular diseases and CKD. However, it remains unclear how the kidneys handle such a high K load in the context of low Na intake. This is crucial to choosing effective diuretics and preventing the drastic consequences of hypokalemia and hyperkalemia for patients on these diets. For instance, our preliminary findings in this proposal suggest that the K-wasting loop diuretics may become K-sparing and decrease K excretion on low Na high K diet. In the thick ascending limb (TAL), Na+, K+, and Cl- are reabsorbed via Na+-K+-2Cl- cotransporter (NKCC2), and most of the K+ is recycled back into lumen by apical Renal Outer Medullary K channel (ROMK) to facilitate NaCl reabsorption by NKCC2. It has been long understood that the high interstitial K concentration induced by high K diet would inhibit the predominant basolateral K channels, leading to membrane depolarization and inhibition of Cl- exit, and thus inhibit NaCl reabsorption via NKCC2 in the TAL. However, our lab has recently discovered that in mice on a low Na, high K diet (LNaHK), there is a considerable furosemide-sensitive Na+ reabsorption as well as a net K+ secretion in the TAL. Contrary to the traditional view, we have also found that the NKCC2 expression is increased on LNaHK diet. We hypothesize that on LNaHK diet, apical ROMK is upregulated and becomes the predominant K channel which is not affected by high interstitial K+ concentration. NKCC2 is upregulated to supply Na+ to the basolateral Na+-K+ ATPase and bring in more K+ to be secreted. Unlike previous studies, we now have more advanced tools to further investigate the ion transport in TAL such as knockout mice. The hypothesis of this proposal is that the high levels of aldosterone (aldo) and angiotensin II (Ang II) induced by LNaHK diet increase NKCC2 and ROMK activities in the apical membrane of the TAL, thereby causing a net K+ secretion. Two specific aims will investigate the mechanisms that regulate net K secretion in TAL: Specific Aim 1. Determine the role and regulation of NKCC2 in the net K+ secretion in TAL. Specific Aim 2: Determine the role and regulation of ROMK in the net K+ secretion in TAL. We will determine the expression of NKCC2 and ROMK in TAL using western blot and real- time PCR in mice on LNaHK compared to control diet. We will measure the functional activity of NKCC2 and ROMK in TAL using micropuncture, atomic absorptive spectrometry, and patch clamp techniques. We will also perform adrenalectomy (ADX) and use aldosterone (aldo) replacement and inhibitors of renin-angiotensin- aldosterone system to elucidate the regulation of NKCC2 by aldo and Ang II. By fulfilling this proposal, we will have a better understanding of the renal K handling in people on the cardioprotective low Na high K diets.