Background: Inorganic phosphate (PO₄^3-, P_i) plays a critical role in skeletal development and bone integrity, energy metabolism, cell signaling, and regulation of protein synthesis. P_i levels in the body are balanced by dietary uptake in the small intestine and urinary excretion by the kidneys. In healthy individuals, an increase in serum P_i induces parathyroid hormone (PTH) and fibroblast growth factor 23 (FGF23) secretion. In kidney proximal tubule cells, PTH activates the G_s protein-coupled PTH 1 receptor (PTH₁R) to stimulate a G_sα/AC/cAMP/PKA pathway; which subsequently reduces the expression and luminal membrane abundance of the Na-phosphate cotransporters Npt2a and Npt2c in the proximal tubules of the kidney leading to reduced renal P_i absorption and increased urinary P_i excretion (Fig. 1).

In addition a PTH/Gα_q/11/PLC/IP₃/Ca²⁺/PKC pathway has been speculated to contribute to the PTH-dependent down-regulation of Npt2a expression. Importantly, the PTH-PKC pathway is unable to compensate for the impairment of PTH-PKA signaling in patients with pseudohypoparathyroidism type Ia (PHP-Ia) or type Ib (PHP-Ib). These patients develop PTH-resistant hyperphosphatemia because of inherited mutations that abolish the activity of G_sα in the renal proximal tubule. These clinical findings indicate a dominant role for the PTH-PKA pathway in the PTH-dependent regulation of Npt2a expression and urinary P_i excretion. PTH also increases 1α-hydroxylase secretion by the kidney, which increases 1,25-dihydroxyvitamin D₃ production and enhances intestinal P_i absorption through Npt2b. A balance between these mechanisms allows plasma P_i levels to remain within a narrow range to maintain physiological processes (Fig. 2).

Problem statement and objectives: Critical to maintaining body P_i balance are the effects of PTH, which are mediated via a G_sα/AC/cAMP/PKA pathway. However, which AC isoform(s) mediate PTH-induced effects, either directly or indirectly, remains unknown. This proposal will determine the role of AC6 in body P_i homeostasis using various AC6 gene knockout mice. Furthermore, we hypothesize that altered sensitivity to PTH in the kidneys of AC6 gene modified mice will lead to changes in FGF23 and 1,25-dihydroxyvitamin D₃ levels and thus indirect alterations in intestinal P_i transport and bone mineralization. The research is broken down into 3 research objectives:   

1)      Objective I will assess the role of AC6 in renal P_i transport and molecular pathways of P_i  uptake

2)      Objective II will assess P_i uptake in the intestine and if changes occur secondary to renal effects

3)      Objective III will study the effects of altered P_i homeostasis on bone morphology/mineralization

Perspectives for the field: The experiments outlined will employ a novel, mostly in vivo, approach to determine the role of AC6 in P_i homeostasis. Our studies will provide mechanistic insight into the role of AC6 in P_i handling by the kidneys (including a transport profile along the proximal tubule), intestinal P_i transport, and bone mineralization – which combined makes our studies of high importance for various fields. We will generate two novel mutant mouse models to decipher the contribution of AC6 for renal versus intestinal P_i homeostasis that will provide unprecedented insights into the regulation of P_i homeostasis. Currently, no specific protein involved in P_i homeostasis has become a pharmacological target for activation and/or blockade making it necessary to study basic physiological questions to provide new targets for clinical exploitation.