Magnesium handling by the distal nephron

项目摘要 镁（Mg 2个以上 Mg（OH）2是许多细胞过程中必不可少的辅因子， 2个以上 稳态可以 会危及生命血清镁 2个以上 通过调节重吸收维持在狭窄的正常范围内， 肾 Mg 远侧 2+通道TRPM 6/7，但重吸收 回旋小管（DCT）仅重吸收一小部分经过滤的Mg， 在这一领域受到严格监管。大多数遗传原因 2个以上 (10%) 低镁血症影响DCT Mg 2+重吸收。在 （NCC） 保持 从 利尿剂 Mg NCC 物种 的 分泌物， 家族性 变化 DCT，通过NaCl协同转运蛋白的Na +重吸收 在跨细胞Mg +重吸收中起关键作用，但连接这两个过程的机制 不清楚低镁血症见于Gitelman综合征和EAST综合征，这两种综合征最终都会出现 NCC活性丧失，以及使用常用噻嗪进行药理学NCC阻断后 慢性地，观察到DCT萎缩，伴随NCC活性的丧失，并且由此导致的对神经细胞的能力丧失。 2+吸收是肾Mg 2+消耗的主要机制。然而，早期的影响 对肾Mg 2+处理抑制作用尚不清楚。我们在小鼠中的初步数据支持了在其他研究中的发现。 噻嗪类药物可暂时降低而非增加尿镁排泄。基于此，我们建议 急性K +负荷后Mg 2+排泄减少，抑制NCC促进下游K + 起到保护血清Mg 2+的作用。与此相反，肾镁处理似乎正常的疾病 高钾血症性高血压（FHHt），其中NCC过度活化。目标1将检验以下假设： 的DCT 2 R i L Na+重吸收在DCT重构之前改变了Mg 2+处理，这是生理上的 相关的我们将使用Gitelman综合征，EAST综合征，噻嗪给药， 和K+负载来测试这个。我们将进行时间过程分析并测量电解质处理的变化， DCT结构具有光学组织清晰和三维成像功能。低镁血症和低钾血症是 临床上常见于一起，例如在顺铂化疗后。低钾血症通常对K+不敏感 补充，除非低镁血症得到解决，但其潜在机制尚未得到证实。 测定目的2将测试两种建议的机制，促进K+分泌沿着连接段 并得到我们初步数据的支持：（i）K+通道ROMK的Mg 2+依赖性去抑制和（ii） 增加DCT的Na+输送。我们将在低镁血症患者中测定NCC、ENaC和ROMK活性， 小鼠和一种新的低镁血症/低钾血症小鼠模型。我们将 还测试了Mg 2+补充是否可以减轻具有诱导型NCC抑制的小鼠模型中的K+损失。 已经提出顶端K+通道Kv1.1产生膜电位，该膜电位提供驱动， Mg 2+沿着DCT进入，因为人Kv1.1突变导致低镁血症。然而，实验 缺乏证据。为了测试这一点，在目标3中，我们将表型一种新的肾小管特异性Kv1.1敲除小鼠， 并确定Kv1.1是否决定早期DCT中的顶膜电位。

Project summary Magnesium (Mg 2+ ) is an essential cofactor in many cellular processes, and disruption of Mg 2+ homeostasis can be life-threatening. Serum Mg 2+ is maintained within a narrow normal range by regulated reabsorption in the kidney. Mg The distal 2+ channel TRPM6/7, but reabsorption convoluted tubule (DCT) reabsorbs only a small fraction of the filtered Mg via the is tightly regulated in this segment. Most genetic causes of 2+ (10%) hypomagnesemia affecting DCT Mg2+ reabsorption. In (NCC) remain from diuretics. Mg NCC species that secretion, Familial changes the DCT, Na + reabsorption via the NaCl cotransporter plays a critical role in transcellular Mg + reabsorption, but the mechanisms linking the two processes unclear. Hypomagnesemia is observed in Gitelman and EAST syndromes, both of which ultimately arise loss of NCC activity, and following pharmacological NCC blockade with t he commonly used thiazide Chronically, DCT atrophy is observed with loss of NCC activity, and the resulting l oss of capacity for 2+ eabsorption s believed to be the major mechanism of renal Mg 2+ wasting. However, the early effects of inhibition on renal Mg 2+ handling are less clear. Our preliminary data in mice support findings in other that thiazides transiently lower, rather than increase, urinary Mg 2+ excretion. Based on this we propose reduced Mg 2+ excretion occurs following an acute K + load, which inhibits NCC to promote downstream K + serving to preserve serum Mg 2+ . In contrast, renal Mg 2+ handling appears norma in the disease Hyperkalemic Hypertension (FHHt), in which NCC is hyperactivated. Aim 1 will test the hypothesis that in DCT 2 r i l Na+ reabsorption modify Mg2+ handling prior to DCT remodeling, and this is physiologically relevant. We will use inducible mouse models of Gitelman syndrome, EAST syndrome, thiazide administration, and K+ loading to test this. We will perform time-course analyses and measure changes in electrolyte handling, and in DCT structure with optical tissue clearing and 3-D imaging. Hypomagnesemia and hypokalemia are commonly seen together clinically e.g. following cisplatin chemotherapy. Hypokalemia is often refractory to K+ supplementation unless hypomagnesemia is resolved, but the underlying mechanisms have not been determined. Aim 2 will test two proposed mechanisms that promote K+ secretion along the connecting segment and are supported by our preliminary data: (i) Mg2+-dependent disinhibition of the K+ channel ROMK and (ii) increased Na+ delivery from DCT. We will determine NCC, ENaC, and ROMK activities in hypomagnesemic mice and a new mouse model of hypomagnesemia/hypokalemia by performing diuretic response tests. We will also test whether Mg2+ supplementation can mitigate K+ losses in mouse models with inducible NCC inhibition. The apical K+ channel Kv1.1 has been proposed to generate the membrane potential that provides the drive for Mg2+ entry along the DCT, since human Kv1.1 mutations cause hypomagnesemia. However, experimental evidence is lacking. To test this, In Aim 3 we will phenotype a novel renal tubule-specific Kv1.1 knockout mouse, and determine whether Kv1.1 determines the apical membrane potential in the early DCT.