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 pro...