Role of Renal and Intestinal AC6 and NHE3 for Phosphate Homeostasis

肾和肠 AC6 和 NHE3 对磷酸盐稳态的作用

• 批准号: 9160960
• 负责人: Timo Rieg
• 金额: $ 5.68万
• 依托单位: VETERANS MEDICAL RESEARCH FDN/SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-08-25 至 2017-02-01
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9160960
• 关键词: Address; Adenylate Cyclase; Adult; Affect; Animal Model; Apical; Biochemistry; Biological Process; Cardiovascular system; Cell membrane; Chronic Kidney Failure; Confocal Microscopy; Coupled; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Data; Development; Electron Microscopy; Energy Metabolism; Ensure; Environment; Equilibrium; Excretory function; Exhibits; Failure; Feedback; Functional disorder; GTP-Binding Protein alpha Subunits, Gs; Genetic study; Goals; Health; Homeostasis; Hormones; Inositol; Intestinal Mucosa; Intestines; Kidney; Kidney Calculi; Knock-out; Knockout Mice; Laboratories; Lead; Link; Lysosomes; Mediating; Micropuncture; Modeling; Morphology; Mus; Mutant Strains Mice; Names; Nutrient; PTH gene; Pathway interactions; Patients; Phospholipase C; Plasma; Play; Positioning Attribute; Prevention strategy; Protein Biosynthesis; Protein Isoforms; Protein Kinase C; Proteins; Publishing; Regulation; Role; Signal Pathway; Signal Transduction; Skeletal Development; Skeletal bone; Small Intestines; Testing; Vitamin D; adenylyl cyclase 6; apical membrane; base; bone; cardiovascular risk factor; clinically relevant; fibroblast growth factor 23; inorganic phosphate; mortality; mouse model; neutralizing antibody; novel; novel therapeutics; research study; sodium-phosphate cotransporter proteins; trafficking; tripolyphosphate; uptake; wasting

The precise regulation of the body's phosphate level is a critical task. Nearly all patients with chronic kidney disease (CKD) exhibit hyperphosphatemia which is associated with increased cardiovascular mortality. Renal reabsorption of Pi in the proximal tubule is hormonally regulated and requires fibroblast growth factor 23 (FGF23) and parathyroid hormone (PTH). The latter has been described to signal via cyclic adenosine monophosphate (cAMP), generated by adenylyl cyclases (AC), and retrieve Na+-Pi cotransporters 2a and 2c (Npt2a and Npt2c) and Na+/H+ exchanger 3 (NHE3) from the apical cell membrane. The overarching goal of this proposal is to determine the roles of adenylyl cyclase 6 (AC6) and NHE3 in Pi homeostasis by analyzing the intestine-kidney axis. We identified that AC6 is the most important isoform for PTH-mediated cAMP formation and Pi homeostasis. In contrast to the expected pathophysiology resulting in impaired Pi excretion, lack of AC6 causes renal Pi wasting with 80% of Npt2a residing in lysosomes. To avoid further Pi loss, PTH and FGF23 levels would be expected to be suppressed; however, lack of AC6 is associated with significantly elevated levels of both hormones indicating that this Pi loss cannot be countered hormonally. While regulation of the milieu intérieur would require intestinal Pi uptake to be enhanced or unchanged, we found that lack of AC6 causes an almost complete absence of intestinal Npt2b. This paradox highlights that AC6 plays a role in a so far unidentified negative feedback loop that suppresses Pi regulating hormones. Since PTH also targets NHE3, we generated a novel kidney-specific NHE3 knockout mouse to determine the contribution of NHE3 for Pi homeostasis. While this model has normal Npt2a abundance, Npt2c abundance is diminished, providing a novel link between NHE3 and Npt2c that has never been shown before. In Aim 1, we will determine the role of renal AC6 in Pi homeostasis under normal conditions and CKD. In Aim 2, we will delineate the contribution of 3 specific signaling pathways for Pi homeostasis: i) Gαs protein coupled AC6/cAMP/protein kinase A; ii) Gαq/11 protein coupled phospholipase C(PLC)/inositol triphosphate/Ca2+/protein kinase C; and iii) FGF23. A novel mouse model with defective PLC (named DSEL mouse) and AC6 signaling will allow us to study the contribution of each of these pathways in regulating expression of Npt2a/c in the proximal tubule. To determine the contribution of FGF23 signaling we will pharmacologically antagonize FGF23 via a novel neutralizing antibody. Aim 3 will determine if there is a linkage between NHE3 and Npt2c for Pi homeostasis. Based on the hypothesis that NHE3 is regulated by PTH, we will use our kidney-specific NHE3 knockout mouse to study Npt2a/c trafficking and colocalization and determine to which extent NHE3 is required for renal Pi homeostasis. Aim 4 will determine if AC6 and/or NHE3 play a role in intestinal Pi uptake and Pi homeostasis by utilizing novel intestinal mucosa-specific AC6 and intestinal mucosa-specific NHE3 knockout mice. Modulating PTH, FGF23 and active vitamin D levels will test for the regulation of Npt2b.

精确调节体内磷酸盐水平是一项关键任务。几乎所有慢性肾病患者 慢性肾病（CKD）表现出高磷血症，与心血管死亡率增加相关。肾 近曲小管中 Pi 的重吸收受激素调节，需要成纤维细胞生长因子 23 (FGF23) 和甲状旁腺激素 (PTH)。后者被描述为通过环腺苷发出信号 单磷酸盐 (cAMP)，由腺苷酸环化酶 (AC) 产生，并恢复 Na+-Pi 协同转运蛋白 2a 和 2c （Npt2a 和 Npt2c）和来自顶端细胞膜的 Na+/H+ 交换器 3 (NHE3)。总体目标是 该提案旨在通过分析确定腺苷酸环化酶 6 (AC6) 和 NHE3 在 Pi 稳态中的作用 肠-肾轴。我们发现 AC6 是 PTH 介导的 cAMP 最重要的亚型 形成和 Pi 稳态。与预期的导致 Pi 排泄受损的病理生理学相反， AC6 缺乏会导致肾 Pi 消耗，80% 的 Npt2a 驻留在溶酶体中。为了避免进一步的 Pi 损失，PTH FGF23 水平预计会受到抑制；然而，缺乏 AC6 与显着相关 两种激素水平升高表明这种 Pi 损失无法通过激素来抵消。监管的同时 内部环境的变化需要肠道 Pi 的吸收增强或不变，我们发现缺乏 AC6 导致肠道 Npt2b 几乎完全缺失。这个悖论凸显了 AC6 在 迄今为止，尚未发现抑制 Pi 调节激素的负反馈回路。由于 PTH 还针对 NHE3，我们培育了一种新型肾脏特异性 NHE3 敲除小鼠，以确定 NHE3 对肾脏的贡献 Pi 稳态。虽然该模型具有正常的 Npt2a 丰度，但 Npt2c 丰度减少，提供了 NHE3 和 Npt2c 之间的新颖联系以前从未被证明过。在目标 1 中，我们将确定以下角色： 正常情况下和 CKD 情况下肾脏 AC6 在 Pi 稳态中的作用。在目标 2 中，我们将描述 3 的贡献 Pi 稳态的特定信号通路：i) Gαs 蛋白偶联 AC6/cAMP/蛋白激酶 A； ii) Gαq/11 蛋白偶联磷脂酶C(PLC)/三磷酸肌醇/Ca2+/蛋白激酶C；和iii) FGF23。一本小说 具有缺陷 PLC 的小鼠模型（称为 DSEL 小鼠）和 AC6 信号传导将使我们能够研究 这些途径中的每一个在调节近曲小管中 Npt2a/c 的表达中的贡献。确定 为了研究 FGF23 信号传导的贡献，我们将通过一种新的中和方法在药理学上拮抗 FGF23 抗体。目标 3 将确定 NHE3 和 Npt2c 之间是否存在与 Pi 稳态相关的联系。基于 假设NHE3受PTH调节，我们将使用我们的肾脏特异性NHE3敲除小鼠来研究 Npt2a/c 运输和共定位，并确定肾 Pi 稳态需要 NHE3 的程度。 目标 4 将利用新型技术确定 AC6 和/或 NHE3 是否在肠道 Pi 摄取和 Pi 稳态中发挥作用。 肠粘膜特异性 AC6 和肠粘膜特异性 NHE3 敲除小鼠。调节 PTH、FGF23 活性维生素 D 水平将测试 Npt2b 的调节。