Effect of an NHE3 inhibitor in combination with an NPT2b inhibitor on gastrointestinal phosphate absorption in Rodent models

Abstract

Many of the pathological consequences of chronic kidney disease can be attributed to an elevation in serum phosphate levels. Current therapies focused on decreasing intestinal phosphate absorption to treat hyperphosphatemia are inadequate. The most effective therapeutic strategy may be to target multiple absorptive pathways. In this study, the ability of a novel inhibitor of the intestinal sodium hydrogen exchanger 3 (NHE3), LY3304000, which inhibits paracellular, diffusional uptake of phosphate, to work in combination with an inhibitor of the active transporter, sodium dependent phosphate cotransporter 2b (NPT2b), LY3358966, was explored. LY3304000 modestly inhibited the acute uptake of phosphate into plasma of rats, while surprisingly, it doubled the rate of phosphate uptake in mice, an animal model dominated by NPT2b mediated acute phosphate uptake. In rats, LY3004000 and LY3358966 work in concert to inhibit acute phosphate uptake. On top of LY3358966, LY3304000 further decreased the acute uptake of phosphate into plasma. Studies measuring the recovery of radiolabeled phosphate in the intestine demonstrated LY3304000 and LY3358966 synergistically inhibited the absorption of phosphate in rats. We hypothesize the synergism is because the NHE3 inhibitor, LY3304000, has two opposing effects on intestinal phosphate absorption in rats, first it decreases diffusion mediated paracellular phosphate absorption, while second, it simultaneously increases phosphate absorption through the NPT2b pathway. NHE3 inhibition decreases proton export from enterocytes and raises the cell surface pH. In vitro, NPT2b mediated phosphate transport is increased at higher pHs. The increased NPT2b mediated transport induced by NHE3 inhibition is masked in rats which have relatively low levels of NPT2b mediated phosphate transport, by the more robust inhibition of diffusion mediated phosphate absorption. Thus, the inhibition of NPT2b mediated phosphate transport in rats in the presence of NHE3 inhibition has an effect that exceeds its effect in the absence of NHE3 inhibition, leading to the observed synergism on phosphate absorption between NPT2b and NHE3 inhibition.

Fig 1. Chemical structure of LY3304000.

Fig 2. In vitro human NHE3 activity of LY3304000.

Fig 3. Effect of LY3304000 on acute phosphate uptake in mice.

Fig 4. Effect of LY3304000 on acute phosphate uptake in rats.

Fig 5. Effect of LY3304000 on sodium and phosphate (measured as phosphorus) excreted in urine in rats.

Fig 6. Acute phosphate uptake in rats treated with a fixed, 1.2 mg/kg dose of LY3358966 and increasing doses of LY3304000.

Fig 7

LY3304000, LY3358966, and an LY3304000/LY3358966 Combination Inhibited Radioactive Phosphate Absorption in Plasma (A) and Increased Its Recovery in the Gastrointestinal Tract (B) in Rats.

Fig 8

Effect of pH on Human (A), Mouse (B) or Rat (C) NPT2b Activity in Vitro.