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.

人体磷酸盐水平的精确调节是一项关键任务。几乎所有患有慢性肾脏的患者 疾病（CKD）表现出与心血管死亡率升高有关的高磷酸血症。肾脏 PI在近端小管中的重吸收受荷尔蒙调节，需要成纤维细胞生长因子23 （FGF23）和甲状旁腺激素（PTH）。后者已被描述为通过循环腺苷发出信号 单磷酸（CAMP），由腺苷循环（AC）产生，并检索Na+-Pi共转运蛋白2A和2C （NPT2A和NPT2C）以及来自顶端细胞膜的Na+/H+交换器3（NHE3）。总体目标 该建议是通过分析来确定腺苷酸环化酶6（AC6）和NHE3在PI稳态中的作用 肠kidney轴。我们确定AC6是PTH介导的CAMP最重要的同工型 形成和PI稳态。与预期的病理生理相反，导致PI排泄受损， 缺乏AC6会导致肾脏PI浪费，其中80％的NPT2A居住在溶酶体中。为避免进一步的PI损失，PTH 预计FGF23水平将被抑制；但是，缺乏AC6与 两种骑马的水平升高，表明这种PI损失不能用荷尔蒙对抗。同时调节 在环境中，intérieur需要增强或不变，我们发现缺乏 AC6几乎完全没有肠NPT2B。这种悖论强调了AC6在A中起作用 到目前为止，抑制PI调节激素的未识别的负反馈回路。因为PTH也针对 NHE3，我们生成了一种新颖的肾脏特异性NHE3敲除鼠标，以确定NHE3对 PI稳态。虽然该模型的NPT2A丰度正常，但NPT2C的丰度减少，提供了 NHE3和NPT2C之间从未显示过的新颖链接。在AIM 1中，我们将确定 PI稳态中的肾脏AC6在正常条件下和CKD下。在AIM 2中，我们将描述3的贡献 PI稳态的特定信号通路：i）GαS蛋白偶联的AC6/CAMP/蛋白激酶A； ii）GαQ/11 蛋白偶联的磷脂酶C（PLC）/肌醇三磷酸/Ca2+/蛋白激酶C；和iii）FGF23。小说 具有缺陷PLC（命名为DSEL鼠标）和AC6信号传导的鼠标模型将使我们能够研究 这些途径在近端小管中NPT2A/C的调节表达中的贡献。确定 FGF23信号的贡献我们将通过新颖的中和物理对FGF23进行物理拮抗 抗体。 AIM 3将确定PI稳态之间的NHE3和NPT2C之间是否存在联系。基于 假设NHE3受PTH调节，我们将使用肾脏特异性的NHE3基因敲除小鼠研究 NPT2A/C的贩运和共定位，并确定肾脏PI稳态所需的范围NHE3。 AIM 4将确定AC6和/或NHE3是否使用新颖 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。