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

Am J Physiol Renal Physiol. 2020 Sep 1;319(3):F541-F551. doi: 10.1152/ajprenal.00146.2020. Epub 2020 Aug 3.

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: Na+-phosphate cotransporters Npt2a and Npt2c, with Npt2a accounting for 70-80% of Pi 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 Pi uptake (IC50: ~1.4 μmol/L), whereas the unselective Npt2 inhibitor phosphonoformic acid (PFA) resulted in an ~20% stronger inhibition of Pi uptake. The dose-dependent inhibitory effects were present after 24 h of incubation with both low- and high-Pi media. Michaelis-Menten kinetics in OK cells identified an ~2.4-fold higher Km for Pi in response to Npt2a inhibition with no significant change in apparent Vmax. Higher parathyroid hormone concentrations decreased Pi uptake equivalent to the maximal inhibitory effect of Npt2a inhibitor. In vivo, the Npt2a inhibitor 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 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.

Keywords: Npt2a knockout; chronic kidney disease; inhibitor; opossum kidney cells; phosphate.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Biological Transport / drug effects
  • Cell Line
  • Gene Expression Regulation / drug effects
  • Mice
  • Mice, Inbred C57BL
  • Mice, Knockout
  • Opossums
  • Parathyroid Hormone / pharmacology
  • Patch-Clamp Techniques
  • Phosphates / metabolism*
  • Random Allocation
  • Sodium-Phosphate Cotransporter Proteins, Type IIa / genetics
  • Sodium-Phosphate Cotransporter Proteins, Type IIa / metabolism*

Substances

  • Parathyroid Hormone
  • Phosphates
  • Slc34a1 protein, mouse
  • Sodium-Phosphate Cotransporter Proteins, Type IIa