Kidney Glycolysis as the Mammalian Phosphate Sensor

摘要 磷酸盐(PI)是生命所必需的，在骨骼矿化、细胞信号和能量方面发挥着重要作用 新陈代谢。然而，PI水平是如何被检测到的是未知的，这表明在 人类生物学。骨源性激素FGF23通过减少肾脏PI重吸收对PI升高作出反应 和1，25(OH)2D的产生，但PI不直接刺激骨FGF23和中间体的产生 PI过量和FGF23合成之间的步骤仍然不清楚。最近，我们发现了一个肾脏到- 骨信号轴，糖酵解的副产物肾脏衍生的甘油-3-磷酸(G-3-P)在其中循环 并触发FGF23的产生。在初步数据中，我们发现PI给药(在喂食的小鼠中)触发 肾脏糖酵解和G-3-P生成急剧增加，其他器官未见变化。 在这里，我们提出了一个中心假设，即肾脏近端小管细胞糖酵解是哺乳动物 磷酸盐传感器，G-3-P和FGF23的上游。目标1将确定糖酵解和 PI刺激G-3-P产生过程中的糖异生作用我们将检验PI刺激肾脏G-3-P的假设 生产在联邦状态下发生，但在糖异生条件下减弱；我们将检查两个 生理上相关的糖异生刺激、禁食和代谢性酸中毒。此外，我们还将展示 使用同位素标记和糖酵解抑制剂，PI刺激的G-3-P需要糖酵解， 糖异生和甘油三酯的合成。目标2将确定甘油-3-磷酸的作用 脱氢酶1(GPD1)，催化G-3-P合成的酶，在系统PI的动态平衡中。使用 本实验室建立了GPD1基因敲除动物，我们将检验GPD1介导的G-3-P和 FGF23的产生是预防高磷血症、血管钙化和慢性骨质疏松症所必需的 膳食PI负荷；我们将比较0.6%、1.2%和2%的PI饮食，并评估GPD1在添加或不添加GPD1的作用 诱发甲状旁腺功能减退症。此外，我们将评估外源性G-3-P是否可以拯救有害的人 GPD1缺乏对PI动态平衡的影响目标3将证明钠依赖的共转运蛋白 Npt2a使肾脏对糖酵解PI的感觉具有特异性。我们将检验PI刺激的假设 肾脏的糖酵解需要Npt2a，通过18F-FDG PET/MRI和代谢组图谱进行评估；我们将 在静脉注射的比较中也要考虑肠道的PI摄取。而不是口服PI。在体外，我们将测试 将Npt2a引入无基础Npt2a/c表达的细胞是否会导致PI反应性糖酵解 和G-3-P的产生，正如在原代人和小鼠肾近端小管细胞中观察到的那样。如果成功， 这些研究将确定一种新的哺乳动物传感器，对人类生物学和疾病具有广泛的影响， 并将支持治疗磷酸盐稳态紊乱的新药理靶点。最后，这一点 计划书将由一个具有协作记录的团队执行，该团队在肾脏领域拥有专业知识 代谢、PI和FGF23、骨生物学和代谢成像。

ABSTRACT Phosphate (Pi) is essential for life, playing fundamental roles in bone mineralization, cell signaling, and energy metabolism. However, how Pi levels are detected is unknown, representing a significant gap in knowledge in human biology. The bone-derived hormone FGF23 responds to elevated Pi by reducing kidney Pi reabsorption and 1,25(OH)2D production, but Pi does not directly stimulate bone FGF23 production and the intermediate steps between Pi excess and FGF23 synthesis have remained obscure. Recently, we identified a kidney-to- bone signaling axis whereby kidney-derived glycerol-3-phosphate (G-3-P), a byproduct of glycolysis, circulates to bone and triggers FGF23 production. In preliminary data, we find that Pi administration (in fed mice) triggers an acute increase in glycolysis and G-3-P production in the kidney, with no change observed in other organs. Here, we advance the central hypothesis that kidney proximal tubular cell glycolysis is the mammalian phosphate sensor, upstream of G-3-P and FGF23. Aim 1 will determine the role of glycolysis and gluconeogenesis in Pi-stimulated G-3-P production. We will test the hypothesis that Pi-stimulated kidney G-3-P production occurs in the fed state, but is attenuated under gluconeogenic conditions; we will examine two physiologically relevant gluconeogenic stimuli, fasting and metabolic acidosis. In addition, we will show that glycolysis is required for Pi-stimulated G-3-P using isotope labeling and inhibitors of glycolysis, gluconeogenesis, and triglyceride synthesis. Aim 2 will establish the role of glycerol-3-phosphate dehydrogenase 1 (Gpd1), the enzyme that catalyzes G-3-P synthesis, in systemic Pi homeostasis. Using a Gpd1 knockout animal generated in our laboratory, we will test the hypothesis that Gpd1 mediated G-3-P and FGF23 production is required to prevent hyperphosphatemia, vascular calcification, and bone loss with chronic dietary Pi loading; we will compare 0.6%, 1.2%, and 2% Pi diets and assess the role of Gpd1 with or without induced hypoparathyroidism. Further, we will assess whether exogenous G-3-P can rescue the deleterious effects of Gpd1 deficiency on Pi homeostasis. Aim 3 will demonstrate that the sodium-dependent cotransporter Npt2a confers kidney specificity to glycolytic Pi sensing. We will test the hypothesis that Pi-stimulated glycolysis in the kidney requires Npt2a, as assessed by 18F-FDG PET/MRI and metabolomic profiling; we will also consider intestinal Pi uptake in a comparison of i.v. versus oral Pi administration. In vitro, we will test whether the introduction of Npt2a to cells without basal Npt2a/c expression confers Pi-responsive glycolysis and G-3-P production, as observed in primary human and mouse kidney proximal tubule cells. If successful, these studies will identify a new mammalian sensor, with broad implications for human biology and disease, and will endorse new pharmacologic targets for treating disorders of phosphate homeostasis. Finally, this proposal will be executed by a team with a track record of collaboration, spanning expertise in kidney metabolism, Pi and FGF23, bone biology, and metabolic imaging.