PF-06869206 is a selective inhibitor of renal P_itransport: evidence from in vitro and in vivo studies

Abstract

Plasma phosphate (Pi) levels are tightly controlled, and elevated plasma Pi levels are associated with an increased risk of cardiovascular complications and death. Two renal transport proteins mediate the majority of Pi reabsorption, the Na+-phosphate cotransporters Npt2a and Npt2c, with Npt2a accounting for 70-80% of Pi reabsorption. The aim of this study was to determine the in vitro effects of a novel Npt2a inhibitor (Npt2a-I, PF-06869206) in opossum kidney (OK) cells as well as determine its selectivity in vivo in Npt2a knockout (Npt2a-/-) mice. In OK cells, Npt2a-I caused dose-dependent reductions of Na+-dependent Pi uptake (IC50: ~1.4 μmol/L), while the unselective Npt2 inhibitor phosphonoformic acid (PFA) resulted in a ~20% stronger inhibition of Pi uptake. The dose-dependent inhibitory effects were present after 24-hour incubation with both low and high Pi media. Michaelis-Menten kinetics in OK cells identified a ~2.4-fold higher Km for Pi in response to Npt2a inhibition with no significant change in apparent Vmax. Higher PTH concentrations decreased Pi uptake equivalent to the maximal inhibitory effect of Npt2a-I. In vivo, the Npt2a-I induced a dose-dependent increase in urinary Pi excretion in wild-type mice (ED50: ~23 mg/kg), which was completely absent in Npt2a-/- mice, alongside a lack of decrease in plasma Pi. Of note, the Npt2a-I-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-I on open probability of the epithelial Na+ channel, ENaC, in the cortical collecting duct.

Npt2a as a target for treating hyperphosphatemia

“…Comparable results were found in acutely isolated rat proximal tubule cells: PF-06869206 inhibited ∼55% of 32 P uptake at the maximum tested concentration of 30 µmol L −1 and PFA inhibited 70% of Na + -dependent P i uptake [ 44 ]. Michaelis–Menten kinetics in OK cells identified no significant difference in apparent maximum velocity of reaction; however, showed a ∼2.4-fold higher substrate concentration for P i in response to PF-06869206, which is consistent with a competitive mode of inhibition [ 8 ].…”

“…In OK cells, PF-06869206 caused a dose-dependent inhibition of Na + -dependent 32 P i uptake (IC 50 ∼1 µmol L −1 ), reaching a maximum inhibitory effect of ∼70% at 100 µmol L −1 . The nonselective Npt2 inhibitor PFA inhibited ∼90% of Na + -dependent P i uptake, indicating that, in addition to Npt2a, Npt2c is inhibited [ 8 ]. Comparable results were found in acutely isolated rat proximal tubule cells: PF-06869206 inhibited ∼55% of 32 P uptake at the maximum tested concentration of 30 µmol L −1 and PFA inhibited 70% of Na + -dependent P i uptake [ 44 ].…”

“…BAY-767 caused a ∼1.7-fold increase in fractional urinary P i excretion over a 16 h period at the highest dose tested (10 mg kg −1 ) [ 40 ]. Studies by 2 different groups confirmed the specificity of PF-06869206 by using Npt2a −/− mice which completely lack a change in urinary P i excretion [ 8 , 44 ]. In terms of the increase in urinary P i excretion, Npt2c −/− mice were indistinguishable from wild-type mice.…”

“…The overall contribution of some of these transporters in P i reabsorption has been elucidated by using genetically modified mice. The renal contribution of Npt2a has been estimated to be ∼70%, based on studies in brush border membrane vesicles of Npt2a −/− mice [ 7 ], and is associated with increased urinary P i excretion and reduced plasma P i levels [ 8 ]. The contribution of Npt2c to P i reabsorption is less clear.…”

Kidney physiology: our future is now

“…His current research focuses on the characterization of channels, transporters, receptors, and signaling molecules in the physiology and pathophysiology of the kidney including arterial hypertension. His work has been instrumental in determining the physiology of sodiumglucose cotransporters and the pharmacology of SGLT2 and Npt2a inhibitors using clearance and micropuncture studies in anesthetized mice as well as the measurement of glomerular filtration rate in awake mice using single-bolus and two-compartment kinetic models (8). Dr. Rieg is actively involved in APS; he has served on the Animal Care and Experimentation Committee and the Joint Program Committee, and he is currently the Chair of the Epithelial Transport Group and serves on the APS Awards Committee.…”