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

低钠高钾饮食的小鼠粗升肢的净钾分泌

• 批准号: 9049067
• 负责人: Bangchen Wang
• 金额: $ 3.17万
• 依托单位: UNIVERSITY OF NEBRASKA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-17 至 2020-09-16
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9049067
• 关键词: Adrenalectomy; Affect; Aldosterone; Angiotensin II; Angiotensin II Receptor; Antihypertensive Agents; Apical; Back; Bumetanide; Cardiovascular Diseases; Cells; Chronic Kidney Failure; Diet; Distal; Diuresis; Diuretics; Drug usage; Effectiveness; Enzyme Inhibitor Drugs; Enzyme Inhibitors; Excretory function; Furosemide; Hypertension; Hypokalemia; Intake; Ion Transport; Kidney; Knockout Mice; Limb structure; Measures; Mediating; Mediator of activation protein; Mediterranean Diet; Membrane; Micropuncture; Mus; Na(+)-K(+)-Exchanging ATPase; Nephrons; Patch-Clamp Techniques; Patients; Potassium; Potassium Channel; Recycling; Regulation; Renin-Angiotensin-Aldosterone System; Role; Spectrometry; Structure of ascending limb of Henle' s loop; Thick; Time; Vegetarian diet; Western Blotting; apical membrane; basolateral membrane; dietary salt; driving force; hyperkalemia; inhibitor/antagonist; interstitial; prevent; public health relevance; receptor; salt intake; sodium-potassium chloride cotransporter 2 protein; tool; uptake; wasting

 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.

 描述（申请人提供）：高钾血症是慢性肾病（CKD）患者的常见病症，因为肾脏排钾能力下降以及旧石器时代和地中海饮食中肾素-血管紧张素-醛固酮系统抑制剂的使用。众所周知，钠、高钾含量可以预防心血管疾病和慢性肾病，但目前尚不清楚肾脏如何在钠摄入量低的情况下处理如此高的钾负荷。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，我们在本提案中的初步研究结果表明，消耗钾的袢利尿剂可能会成为钾保留剂，并减少低钠高钾的排泄。 K 饮食在厚升肢 (TAL) 中，Na+、K+ 和 Cl- 通过 Na+-K+-2Cl- 协同转运蛋白 (NKCC2) 被重吸收，并且大部分。 K+ 通过心尖肾外髓 K 通道 (ROMK) 循环回到管腔，以促进 NKCC2 对 NaCl 的重吸收。长期以来，人们一直认为高 K 饮食诱导的高间质 K 浓度会抑制主要的基底外侧 K ，从而导致 K 的增加。膜去极化和抑制 Cl- 退出，从而通过 TAL 中的 NKCC2 抑制 NaCl 重吸收。 Na、高钾饮食（LNaHK）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 LNaHK 饮食中 NKCC2 表达增加。坚持认为，在 LNaHK 饮食中，顶端 ROMK 上调并成为不受高间质 K+ 浓度影响的主要 K 通道，NKCC2 上调以向体内供应 Na+。与之前的研究不同，我们现在拥有更先进的工具来进一步研究 TAL（例如基因敲除小鼠）中的离子转运。该提议的假设是醛固酮水平较高。 LNaHK 饮食诱导的 (aldo) 和血管紧张素 II (Ang II) 增加 TAL 顶膜中的 NKCC2 和 ROMK 活性，从而引起净 K+ 分泌。目标将研究调节 TAL 中净 K+ 分泌的机制： 具体目标 1. 确定 NKCC2 在 TAL 中净 K+ 分泌中的作用和调节 具体目标 2： 确定 ROMK 在 TAL 中净 K+ 分泌中的作用和调节。我们将使用 LNaHK 小鼠中的蛋白质印迹和实时 PCR 来确定 TAL 中 NKCC2 和 ROMK 的表达，并与对照饮食进行比较。使用微穿刺、原子吸收光谱法和膜片钳技术在 TAL 中检测 NKCC2 和 ROMK 我们还将进行肾上腺切除术 (ADX) 并使用醛固酮 (aldo) 替代品和肾素-血管紧张素-醛固酮系统抑制剂来阐明 NKCC2 的调节。 Ang II。通过完成这项提案，我们将更好地了解具有心脏保护作用的低钠高钾患者的肾脏钾处理情况。饮食。