Abstract
Plasma phosphate (P(i)) levels are tightly controlled, and elevated plasma P(i) levels are associated with an increased risk of cardiovascular complications and death. Two renal transport proteins mediate the majority of P(i) reabsorption: Na(+)-phosphate cotransporters Npt2a and Npt2c, with Npt2a accounting for 70-80% of P(i) reabsorption. The aim of the present study was to determine the in vitro effects of a novel Npt2a inhibitor (PF-06869206) in opossum kidney (OK) cells as well as determine its selectivity in vivo in Npt2a knockout (Npt2a(-/-)) mice. In OK cells, Npt2a inhibitor caused dose-dependent reductions of Na(+)-dependent P(i) uptake (IC(50): ~1.4 μmol/L), whereas the unselective Npt2 inhibitor phosphonoformic acid (PFA) resulted in an ~20% stronger inhibition of P(i) uptake. The dose-dependent inhibitory effects were present after 24 h of incubation with both low- and high-P(i) media. Michaelis-Menten kinetics in OK cells identified an ~2.4-fold higher K(m) for P(i) in response to Npt2a inhibition with no significant change in apparent V(max). Higher parathyroid hormone concentrations decreased P(i) uptake equivalent to the maximal inhibitory effect of Npt2a inhibitor. In vivo, the Npt2a inhibitor induced a dose-dependent increase in urinary P(i) excretion in wild-type mice (ED(50): ~23 mg/kg), which was completely absent in Npt2a(-/-) mice, alongside a lack of decrease in plasma P(i). Of note, the Npt2a inhibitor-induced dose-dependent increase in urinary Na(+) excretion was still present in Npt2a(-/-) mice, a response possibly mediated by an off-target acute inhibitory effect of the Npt2a inhibitor on open probability of the epithelial Na(+) channel in the cortical collecting duct.