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

基本信息

批准号：
9519555
负责人：
Timo Rieg
金额：
$ 33.52万
依托单位：
UNIVERSITY OF SOUTH FLORIDA
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-02-02 至 2021-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9519555
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 Goals Health Homeostasis Hormonal Hormones Impairment 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 Pharmacology 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 experimental study fibroblast growth factor 23 inorganic phosphate mortality mouse model neutralizing antibody novel novel therapeutics sodium-phosphate cotransporter proteins trafficking tripolyphosphate uptake wasting

项目摘要

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.

人体磷酸盐水平的确切调节是一项关键任务。疾病（CKD）表现出与心血管死亡率增加的高磷酸化血症。 PI在近端小管中的重吸收是荷尔蒙经常定期正常的纤维细胞生长因子23 （FGF23）和甲状旁腺激素（PTH）。单磷酸盐（CAMP），由腺苷酸（AC）产生，并返回Na+-Pi共转运蛋白2A和2C （NPT2A和NPT2C）和Na+/H+ Exchangeer 3（NHE3）来自顶端细胞膜。该提议是通过分析来确定腺苷酸环化酶6（AC6）和NHE3在稳态中的作用肠道轴轴。形成和PI稳态与预期的病理生理形成对比缺乏AC6会导致80％居住在溶酶体中的NPT2A的肾脏PI。预计FGF23水平将被抑制）两种激素的升高表明该PI损失不能在调节时用荷尔蒙进行抵消 MIRIEU INTERIEUR需要增强或不变，我们发现缺乏 AC6几乎没有肠NPT2B的压缩。到目前为止，抑制PI调节激素的未识别的需求循环。 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。 NHE3和NPT2C之间从未在AIM 1中进行的新链接。在正常条件下的PI稳态中的肾脏AC6，在AIM 2中。 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 蛋白质的磷脂酶C（PLC）/肌醇三磷酸/Ca2+/蛋白激酶C和III 具有缺陷PLC（命名为DSEL鼠标）和AC6信号传导的小鼠模型将使我们能够研究您这些途径在调节近端小管中NPT2A/C的表达方面的贡献 FGF23信号传导的贡献我们将通过新颖的中和药物学对FGF23进行拮抗抗体3将确定基于PI的NHE3和NPT2C之间的联系假设NHE3由PTH定期 NPT2A/C运输和共定位，并确定肾脏PI稳态需要在多大程度上NHE3。 AIM 4将通过利用新颖肠粘膜特异性AC6和肠粘膜特异性NHE3敲除小鼠活性维生素D水平将用于调节NPT2B。