Inhibition of sodium/hydrogen exchanger 3 in the gastrointestinal tract by tenapanor reduces paracellular phosphate permeability

Abstract

Hyperphosphatemia is common in patients with chronic kidney disease and is increasingly associated with poor clinical outcomes. Current management of hyperphosphatemia with dietary restriction and oral phosphate binders often proves inadequate. Tenapanor, a minimally absorbed, small-molecule inhibitor of the sodium/hydrogen exchanger isoform 3 (NHE3), acts locally in the gastrointestinal tract to inhibit sodium absorption. Because tenapanor also reduces intestinal phosphate absorption, it may have potential as a therapy for hyperphosphatemia. We investigated the mechanism by which tenapanor reduces gastrointestinal phosphate uptake, using in vivo studies in rodents and translational experiments on human small intestinal stem cell-derived enteroid monolayers to model ion transport physiology. We found that tenapanor produces its effect by modulating tight junctions, which increases transepithelial electrical resistance (TEER) and reduces permeability to phosphate, reducing paracellular phosphate absorption. NHE3-deficient monolayers mimicked the phosphate phenotype of tenapanor treatment, and tenapanor did not affect TEER or phosphate flux in the absence of NHE3. Tenapanor also prevents active transcellular phosphate absorption compensation by decreasing the expression of NaPi2b, the major active intestinal phosphate transporter. In healthy human volunteers, tenapanor (15 mg, given twice daily for 4 days) increased stool phosphorus and decreased urinary phosphorus excretion. We determined that tenapanor reduces intestinal phosphate absorption predominantly through reduction of passive paracellular phosphate flux, an effect mediated exclusively via on-target NHE3 inhibition.

Fig. 1.. Effects of tenapanor on phosphate absorption in vivo in rats.

(A) Effect of tenapanor (10 μM) and sodium-free buffer on radioactive phosphate absorption in the rat jejunum in vivo loop model compared with vehicle [dimethyl sulfoxide (DMSO)] (n = 5 to 7 per group). (B) Urinary phosphate excretion 4 hours after an oral (p.o.) bolus of varying phosphate concentrations (0.15 to 1.5 M) in rats pretreated with tenapanor (0.5 mg/kg) or vehicle (acidified water, 0.01% Tween 80) (n = 6 per group). (C) Urinary phosphate excretion in rats at different dietary phosphate intakes at baseline and after 4 days of treatment with tenapanor (0.5 mg/kg) or vehicle (acidified water, 0.01% Tween 80) (n = 7 per group). (D) Urinary phosphate and urinary sodium excretion 4 hours after a fixed quantity (5 g) of high-phosphorus (1.2%) meal in rats treated with tenapanor (0.15 mg/kg) or vehicle (acidified water, 0.01% Tween 80) (n = 8 per group). Cecal (E) sodium delivery, (F) phosphate delivery, (G) water volume, (H) sodium concentration, (I) phosphate concentration, (J) potassium concentration, (K) chloride concentration, (L) calcium concentration, and (M) magnesium concentration in rats fed a fixed quantity of high-phosphorus (1.2%) meal and treated with vehicle (acidified water, 0.01% Tween 80) or tenapanor (0.15 mg/kg) (n = 6 per time point per group). Means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; two-way analysis of variance (ANOVA), with post hoc testing at each time point with Bonferroni’s correction (A to C and E to M); pairwise comparisons by Student’s t test (D).

Fig. 2.. Effects of phosphate concentration gradient and tenapanor on phosphate absorption in human duodenum monolayer cultures.

(A) Correlation between phosphate flux and initial apical phosphate concentration in human duodenum monolayer cultures from two separate donors (1, 2) after overnight incubation. The reproducibility of phosphate absorption was assessed in experiments from two separate passages for one donor (n = 4 per group). (B) Basolateral phosphate concentration, phosphate flux, and (D) transepithelial electrical resistance (TEER) at different apical phosphate concentrations (1 to 30 mM) in human duodenum monolayer cultures after 4 hours of treatment with tenapanor (1 μM) or vehicle (DMSO) with an initial starting basolateral phosphate concentration of 0 mM (n = 4 to 16 per group). (E) Apical phosphate retention, (F) apical phosphate concentration, (G) basolateral phosphate concentration, and (H) apical fluid volume at different initial apical phosphate concentrations (1 to 30 mM) in human duodenum monolayer cultures after overnight treatment with tenapanor (1 μM) or vehicle (DMSO) with an initial starting basolateral phosphate concentration of 1 mM; data from the donor used to test reproducibility in (A). (I) Effect of tenapanor (1 μM) or vehicle (DMSO) on bidirectional phosphate flux at varying phosphate concentration gradients (1 to 10 mM) in human duodenum monolayer cultures (n = 4 per group). Means ± SEM. *P < 0.05, ***P < 0.001, ****P < 0.0001 versus vehicle; two-way ANOVA, with post hoc testing at each concentration with Bonferroni’s correction (B to I).

Fig. 3.. TEER and pHi in human intestinal cell monolayer cultures.

Effects of tenapanor (1 μM) versus vehicle (DMSO) on TEER after the change from acidic (pH 6.0) apical media at baseline to fresh neutral pH apical media to restore the gradient for NHE3-mediated proton efflux in (A) human duodenum and (B) human ileum cell monolayer cultures (n = 3 per group). (C) TEER after the change from acidic (pH 6.0) apical media at baseline to fresh apical media at different pH in human duodenum monolayer cultures (n = 3 per group). (c) Effect of tenapanor (1 μM) on TEER at varying apical media pH in human duodenum monolayer cultures normalized to equivalent pH level treated with vehicle (DMSO) (n = 3 per group). Recovery of pHi, initiated by the addition of a sodium-containing media and measured using pH-sensitive BCECF-AM dye after acid loading in sodium-free media in (E) human duodenum and (F) human ileum monolayer cells at different apical pH in the presence of tenapanor (1 μM) or vehicle (DMSO). RLU, relative luminescence units. (G) Concentration-response effect of tenapanor on rate of the recovery from intracellular acidification at neutral and acidic apical pH in human ileum monolayer cultures. (H) Change in pHi, measured using BCECF-AM dye, over time after the change to acidic, neutral, or alkaline pH apical media in human ileum monolayer cultures. Effect of tenapanor (1 μM) versus vehicle (DMSO) on change in (I) pHi and (J) TEER over time in human ileum monolayer cultures (n = 3 to 6 per group). Means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 versus vehicle (A, B, and I), versus pH 7.3 (C and D), or versus pH 6.0 (H); two-way ANOVA, with post hoc testing at each concentration with Bonferroni’s correction (A to D, H, and I); non-linear regression analysis, log (inhibitor) versus response (three parameters) (G).

Fig. 4.. Effect of apical pH on phosphate flux and effect of tenapanor on phosphate flux at different apical pH in ileum monolayers and jejunum tissue ex vivo.

Effect of varying apical pH on basolateral phosphate flux in human ileum epithelial cell monolayer cultures after an overnight incubation at an initial apical phosphate concentration of 10 or 30 mM and an initial basolateral phosphate concentration of 0 mM, with (A) vehicle (DMSO) or (B) tenapanor (1 μM; the effect of tenapanor is represented as change from vehicle). (C) Phosphate flux measured with radioactive tracer and (D) paracellular phosphate permeability (pPO₄³⁻) measured by biionic dilution potential in mouse jejunum strips at pH 6.0 and 8.0 (n = 4 to 6 per group). Means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; one-way ANOVA, with post hoc testing at each pH with Bonferroni’s correction (A and B); pairwise comparisons by Student’s t test (C and D).

Fig. 5.. Effect of tenapanor on paracellular permeability measured by direct biophysical methods and the chemical driving forces for paracellular absorption in vivo in rats.

Effect of tenapanor at pH 8.0 on (A) TEER, (B) sodium permeability (pNa⁺), (C) chloride permeability (pCl⁻), (D) sodium-to-chloride permeability (pNa⁺/pCl⁻), and (E) phosphate permeability (pPO ³⁻) in human duodenum monolayer cultures mounted in Ussing chambers. Effect of tenapanor on (F) TEER, (G) pNa⁺, (H) pCl⁻, (I) pNa⁺/pCl⁻, and (J) pPO₄³⁻ across mouse jejunum in Ussing chambers. Luminal concentrations of (K) sodium, (L) chloride, and (M) phosphate in the proximal and distal small intestine in untreated rats trained to eat a standardized meal (n = 6 per group). Means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; pairwise comparisons by Student’s t test (A to J); two-way ANOVA, with post hoc testing at each time point with Bonferroni’s correction (K to M).

Fig. 6.. Paracellular phosphate absorption in NHE3 KO human ileum monolayer cultures.

(A) DNA sequencing showing CRISPR/Cas9-mediated gene editing of NHE3 resulting in nucleotide deletions and insertions. The single-guide RNA (sgRNA) targeting human NHE3 genomic DNA exon 2 (sgNHE3–13) is shown in red, and the protospacer adjacent motif (PAM) region is shown in blue. The NHE3 KO line (sgNHE3–13 clone 45) contains two different types of mutations (Mut 1 and Mut 2), shown in gray boxes. (A) Western blot showing NHE3 and β-actin (ACTB) protein expression in control and NHE3-edited cells and (C) apical media showing acidification (yellow) or absence of acidification (pink) in control and NHE3-edited cells. Recovery from intracellular acidification after acid loading in the presence of tenapanor or vehicle (DMSO) in (D) control (nontargeting sgControl clone 4) and (E) NHE3 KO (sgNHE3–13 clone 45) human ileum epithelial cell clones. (F) Apical volume, (G) apical sodium, (H) apical phosphate, and (I) apical pH in control and NHE3 KO human ileum monolayers after overnight incubation (n = 6 per group). (J) TEER after the change from acidic (pH 6.0) apical media at baseline to fresh neutral pH apical media to restore the gradient for NHE3-mediated proton efflux in control and NHE3 KO monolayers (n = 4 per group). (K) Change in apical-to-basolateral phosphate absorption and (L) change in apical phosphate concentration with tenapanor (1 μM), normalized to the effect of vehicle (DMSO) in NHE3 KO and control monolayers (n = 4 per group). (M) Change in TEER with tenapanor (1 μM) relative to vehicle (DMSO) after the change to neutral pH apical media in control and NHE3 KO monolayers (n = 4 per group). Means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; pairwise comparisons by Student’s t test (F to I, K, and L); two-way ANOVA, with post hoc testing at each time point with Bonferroni’s correction (J and M).

Fig. 7.. Effects of tenapanor on NaPi2b expression in rats and NaPi2b activity in mouse ileum monolayer cultures and in vivo in mice.

(A) NaPi2b mRNA expression in different intestinal segments after 14 days of tenapanor (0.5 mg/kg) or vehicle (acidified water, 0.01% Tween 80) treatment in rats (n = 8 per group). (B) Immunohistochemistry showing NaPi2b expression in rat jejunum after in vivo treatment with tenapanor or vehicle. (C) Apical and basolateral phosphate concentrations after overnight incubation at different initial apical phosphate concentrations (1 to 5 mM) in mouse ileum monolayer cultures with initial basolateral phosphate concentration of 1 mM (n = 6 per group). (D) Apical phosphate concentrations after a 4-hour, 2-day, or 3-day incubation with tenapanor (1 μM), NTX-9066 (NaPi2b inhibitor; 1 μM), or vehicle (DMSO) in mouse ileum monolayer cultures (n = 6 per group). BQL, below quantification limit. (E) Phosphate absorption with tenapanor (10 μM) versus vehicle in NaPi2b KO and control [wild-type (WT)] mouse ileum in an in vivo ileum loop model (n = 4 to 5 per group). Means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; two-way ANOVA, with post hoc testing at each segment with Bonferroni’s correction (A); one-way ANOVA, with post hoc testing at each concentration (C) or time point (D) with Bonferroni’s correction.

Fig. 8.. Effects of tenapanor on paracellular macromolecule absorption in rats and on phosphate, sodium, and potassium absorption in healthy human volunteers.

(A) Radioactive phosphate and (B) radioactive mannitol absorption in vivo in rats after treatment with vehicle (acidified water, 0.01% Tween 80) or different doses of tenapanor. dpm, disintegrations per minute; AUC, area under the curve. (C) Small intestinal glucose content after initiation of a standardized meal in rats pretreated with vehicle or tenapanor (0.15 mg/kg) or vehicle (acidified water, 0.01% Tween 80) (n = 4 to 7 per group). (D) Stool and (E) urinary phosphorus, (F) urinary sodium, and (G) urinary potassium excretion at baseline (day −1) and after 4 days of tenapanor treatment [15 mg twice daily (b.i.d.)] in healthy volunteers. Means ± SEM (A to C), means ± SD (D to G). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; two-way ANOVA, with post hoc testing at time point with Bonferroni’s correction (A to C); pairwise comparisons by Student’s t test (D to G).