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

 描述（由申请方提供）：高钾血症是慢性肾脏疾病（CKD）患者的常见疾病，因为肾脏排泄钾的能力降低以及使用肾素-血管紧张素-醛固酮系统抑制剂。旧石器时代和地中海饮食具有低Na，高K含量，并且众所周知可以预防心血管疾病和CKD。然而，目前尚不清楚肾脏如何在低钠摄入的情况下处理如此高的钾负荷。这对于选择有效的利尿剂和预防低钾血症和高钾血症对这些饮食患者的严重后果至关重要。例如，我们的初步研究结果表明，在低钠高钾饮食中，耗钾袢利尿剂可能会成为保钾剂，减少钾的排泄。在粗升支（TAL）中，Na+、K+和Cl-通过Na+-K+-2Cl-共转运蛋白（NKCC 2）重吸收，大部分K+通过顶端肾外髓钾通道（ROMK）再循环回到管腔，以促进NKCC 2对NaCl的重吸收。长期以来，高钾饮食诱导的高间质钾浓度会抑制主要的基底外侧钾通道，导致膜去极化和Cl-出口抑制，从而抑制TAL中通过NKCC 2的NaCl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中存在相当大的呋塞米敏感性Na+重吸收以及净K+分泌。与传统观点相反，我们还发现NKCC 2表达在LNaHK饮食中增加。我们推测，在LNaHK饮食，顶端ROMK上调，并成为主要的K通道，这是不受高间质K+浓度。NKCC 2被上调以向基底外侧Na+-K+ ATP酶供应Na+并分泌更多的K+。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中的离子转运，例如敲除小鼠。该建议的假设是，由LNaHK饮食诱导的高水平的醛固酮（aldosterone，aldo）和血管紧张素II（angiotensin II，Ang II）增加了TAL顶端膜中的NKCC 2和ROMK活性，从而引起净K+分泌。两个具体的目标将调查的机制，调节净钾分泌TAL：具体目标1。确定NKCC 2在TAL中净K+分泌中的作用和调节。具体目标2：确定ROMK在TAL的净K+分泌中的作用和调节。我们将使用蛋白质印迹和真实的-时间PCR在LNaHK小鼠中与对照饮食相比确定TAL中NKCC 2和ROMK的表达。我们将使用显微穿刺、原子吸收光谱法和膜片钳技术测量TAL中NKCC 2和ROMK的功能活性。我们还将进行肾上腺切除术（adrenalectomy，ADX），并使用醛固酮（aldosterone，aldo）替代和肾素-血管紧张素-醛固酮系统抑制剂来阐明aldo和Ang II对NKCC 2的调节。通过实现这一建议，我们将有一个更好的了解肾脏钾处理的人对心脏保护的低钠高钾饮食。

 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.