Combination treatment with tenapanor and sevelamer synergistically reduces urinary phosphorus excretion in rats

The majority of patients with chronic kidney disease (CKD) receiving dialysis do not reach target serum phosphorus concentrations, despite treatment with phosphate binders. Tenapanor is a non-binder, sodium/hydrogen exchanger isoform 3 (NHE3) inhibitor that reduces paracellular intestinal phosphate absorption. This pre-clinical study evaluated the effect of tenapanor and varying doses of sevelamer carbonate on urinary phosphorus excretion, a direct reflection of intestinal phosphate absorption. We measured 24-hour urinary phosphorus excretion in male rats assigned to groups dosed orally with vehicle or tenapanor (0.3 mg/kg/day) and provided a diet containing varying amounts of sevelamer (0-3% w/w). We also evaluated the effect of the addition of tenapanor or vehicle on 24-hour urinary phosphorus excretion to rats on a stable dose of sevelamer (1.5% w/w). When administered together, tenapanor and sevelamer decreased urinary phosphorus excretion significantly more than either tenapanor or sevelamer alone across all sevelamer dose levels. The Bliss statistical model of independence indicated that the combination was synergistic. A stable sevelamer dose (1.5% w/w) reduced mean (±standard error of the mean) urinary phosphorus excretion by 42±3% compared with vehicle; together, tenapanor and sevelamer reduced residual urinary phosphorus excretion by an additional 37±6% (P < 0.05). While both tenapanor and sevelamer reduce intestinal phosphate absorption individually, administration of tenapanor and sevelamer together results in more pronounced reductions in intestinal phosphate absorption than if either agent is administered alone. Further evaluation of combination tenapanor plus phosphate binder treatment in patients receiving dialysis with hyperphosphatemia is warranted.

A common NHE3 single-nucleotide polymorphism has normal function and sensitivity to regulatory ligands

Na+/H+ exchanger NHE3 mediates the majority of intestinal and renal electroneutral sodium absorption. Dysfunction of NHE3 is associated with a variety of diarrheal diseases. We previously reported that the NHE3 gene ( SLC9A3) has more than 400 single-nucleotide polymorphisms (SNPs) but few nonsynonymous polymorphisms. Among the latter, one polymorphism (rs2247114-G>A), which causes a substitution from arginine to cysteine at amino acid position 799 (p.R799C), is common in Asian populations. To improve our understanding of the population distribution and potential clinical significance of the NHE3-799C variant, we investigated the frequency of this polymorphism in different ethnic groups using bioinformatics analyses and in a cohort of Japanese patients with cardiovascular or renal disease. We also characterized the function of human NHE3-799C and its sensitivity to regulatory ligands in an in vitro model. NHE3-799C had an allele frequency of 29.5–57.6% in Asian populations, 11.1–23.6% in European populations, and 10.2–22.7% in African populations. PS120/FLAG-NHERF2 fibroblasts stably expressing NHE3-799C had lower total protein expression but a higher percentage of surface expression than those expressing NHE3-799R. NHE3-799C had similar basal activity to NHE3-799R and was similarly stimulated or inhibited, by serum or forskolin, respectively. Tenapanor, a small-molecule NHE3 inhibitor, dose-dependently inhibited NHE3-799R and NHE3-799C activities. The IC50 values of tenapanor for NHE3-799C and NHE3-799R were significantly different, but both were in the nanomolar range. These results suggest that NHE3-799C is a common variant enriched in Asian populations, is not associated with compromised function or abnormal regulation, and is unlikely to contribute to clinical disease. NEW & NOTEWORTHY This study reports results on the functional significance of human NHE3-799C under basal conditions and in response to regulatory ligands, including a novel NHE3 inhibitor called tenapanor. We demonstrate that NHE3-799C is a common variant of NHE3 that is enriched in Asian populations; however, in contrast to our previous studies using rabbit NHE3, its presence seems to have limited clinical significance in humans and is not associated with compromised function or abnormal transport regulation.

Abstract 011: An Orally Absorbable Potent NHE3 Inhibitor Attenuates Angiotensin II-induced Hypertension Primarily by Inhibiting NHE3 in the Proximal Tubule of the Kidney

We have recently shown that angiotensin (ANG II)-induced hypertension was attenuated in mice with global ( Nhe3 -/- ) and Nhe3 -/- mice with transgenic rescue of the NHE3 gene selectively in small intestines (tg Nhe3 -/- ), suggesting an important role of NHE3 in the development of ANG II-dependent hypertension. In this study, we specifically tested whether the pharmacological inhibition of NHE3 mainly in the proximal tubules of the kidney attenuates ANG II-dependent hypertension induced by a low and slow pressor dose of ANG II supplemented with a high salt diet. Overall, 9 groups (n=5-12) of adult male C57BL/6J mice were infused with or without ANG II (500 μg/kg/day, i.p. via minipump) and supplemented with or without a 2% NaCl diet to slowly and moderately increase systolic blood pressure (SBP) in 2 weeks. ANG II alone increased SBP from 116 ± 2 mmHg to 140 ± 2 mmHg ( p <0.01), and supplement of ANG II with a 2% NaCl diet further increased SBP to 147 ± 4 mmHg ( p <0.05). Concurrent treatment with an orally active, absorbable NHE3 inhibitor AVE0657 (Sanofi-Aventis; 20 mg/kg/day, p.o.) significantly decreased SBP to 125 ± 4 mmHg in ANG II-infused mice ( p <0.01), and to 134 ± 6 mmHg in ANG II-infused mice supplemented with 2% NaCl ( p <0.01), respectively. Further treatment with AVE0657 and losartan, an AT 1 receptor blocker (20 mg/kg/day, p.o.), completely normalize SBP in mice treated with ANG II and 2% NaCl to control (115 ± 5 mmHg, p <0.01). In the kidney, AVE0657 significantly increased 24h urinary Na + excretion from 157.1 ± 6.7 to 207.7 ± 8.1 μmol/24h ( p <0.01) without altering 24h urine excretion or SBP. Furthermore, AVE0657 did not significantly alter 24 h fecal Na + excretion in non ANG II-infused (4.99 ± 0.37 μmol/24h, n.s.) or ANG II-infused mice (4.19 ± 0.67 μmol/24h, n.s.), compared with control (4.02 ± 0.20 μmol/24h, n.s. ) or global Nhe3 -/- mice (50.8 ± 0.8 μmol/24h, p <0.01). Since small intestines in the gut and the proximal tubules of the kidney express the vast majority of NHE3 in the body, these results provide preclinical evidence and perspectives that orally absorbable NHE3 inhibitors may be pharmacologically beneficial to prevent and treat hypertension induced by ANG II and a high salt, mainly by inhibiting NHE3 in the proximal tubule of the kidney.

OSR1-sensitive small intestinal Na+ transport

The oxidative stress responsive kinase 1 (OSR1) contributes to WNK (with no K)-dependent regulation of renal tubular salt transport, renal salt excretion, and blood pressure. Little is known, however, about a role of OSR1 in the regulation of intestinal salt transport. The present study thus explored whether OSR1 is expressed in intestinal tissue and whether small intestinal Na+/H+ exchanger (NHE), small intestinal Na+-glucose cotransport (SGLT1), and/or colonic epithelium Na+ channel (ENaC) differ between knockin mice carrying one allele of WNK-resistant OSR1 ( osr1 +/KI) and wild-type mice ( osr1 +/+). OSR1 protein abundance was determined by Western blotting, cytosolic pH from BCECF fluorescence, NHE activity from Na+-dependent realkalinization following an ammonium pulse, SGLT1 activity from glucose-induced current, and colonic ENaC activity from amiloride-sensitive transepithelial current in Ussing chamber experiments. As a result, OSR1 protein was expressed in small intestine of both osr1 +/KI mice and osr1 +/+ mice. Daily fecal Na+, K+, and H2O excretion and jejunal SGLT1 activity were lower, whereas small intestinal NHE activity and colonic ENaC activity were higher in osr1 +/KI mice than in osr1 +/+ mice. NHE3 inhibitor S-3226 significantly reduced NHE activity in both genotypes but did not abrogate the difference between the genotypes. Plasma osmolarity, serum antidiuretic hormone, plasma aldosterone, and plasma corticosterone concentrations were similar in both genotypes. Small intestinal NHE3 and colonic α-ENaC protein abundance were not significantly different between genotypes, but colonic phospho-β-ENaC (ser633) was significantly higher in osr1 +/KI mice. In conclusion, OSR1 is expressed in intestine and partial WNK insensitivity of OSR1 increases intestinal NHE activity and colonic ENaC activity.

Abstract 625: Role Of Proximal Na+ Reabsorption In The Fructose-induced Hypertension

Methods/Results: Wistar rats were fed for 2 or 8 weeks with control diet (CTRL) or isocaloric 60% fructose diet (HF). Systolic blood pressure (SBP) was measured weekly by tail cuff plethysmography. There was no difference in the SBP (mmHg) after 2 weeks of HF intake (110 ± 1.7 CTRL x 121 ± 5.8 HF); however, there was an increase in SBP after 8 weeks (111.9 ± 1.3 CTRL x 143.3 ± 1.5 HF, P < 0.0001). Urine of 24h collected in metabolic cages showed a reduction in urine flow (mL/min) (2 weeks - 0.018 ± 0.002 CTRL x 0.009 ± 0.001 HF, P = 0.0007 and 8 weeks - 0.017 ± 0.002 CTRL x 0.007 ± 0.0004 HF, P = 0.001). Glomerular filtration rate (GFR) was not altered after 2 weeks (1.31 ± 0.3 CTRL x 1.22 ± 0.5 HF), but was decreased after 8 weeks (1.82 ± 0.4 CTRL x 0.37 ± 0.1 HF, P = 0.001). Na+ fractional excretion (Na-FE), in % of creatinine clearance, was decreased after 2 weeks (0.37 ± 0.05 CRTL x 0.13 ±0.03 HF), but was increased after 8 weeks (0.18 ± 0.06 CTRL x 0.54 ± 0.14 HF, P = 0.038). After 2 or 8 weeks, rats were subjected to in situ microperfusion experiments and had their proximal tubules (TP) perfused with an alkaline solution to investigate Na+ reabsorption by means of bicarbonate flux (JHCO3-, in nmol/cm2 x s). JHCO3- was increased after 2 weeks (1.78 ± 0.09 CTRL x 2.7 ± 0.14 HF), but was reduced after 8 weeks (2.00 ± 0.09 CRTL x 1.19 ± 0.04 HF). The perfusion of PT with S3226, a specific NHE3 inhibitor, showed that fructose acts stimulating NHE3 activity after 2 weeks (0.82 ± 0.09 CRTL + S3226 x 1.83 ± 0.14 HF + S3226, P < 0.0001) and inhibiting NHE3 activity after 8 weeks (0.85 ± 0.09 CRTL x 0.22 ± 0.04 HF, P < 0.0001). Conclusions: Reduced Na+-FE and urine flow, associated with increased NHE3 activity after 2 weeks of HF suggest that, initially, fructose leads to a state of Na+ overload, which may contribute to the development of hypertension observed after 8 weeks of HF intake. Kidney damage was increased after 8 weeks, since GFR was decreased, explaining the persistence of reduced urine flow, despite reduced NHE3 activity. These data, together with the increase in Na-FE and installation of hypertension, suggest that the mechanism of pressure-natriuresis was activated after 8 weeks, in order to compensate for volume expansion. Our data suggest a role of increased Na+ reabsorption in the development of fructose-induced hypertension.

Evidence for NHE3-mediated Na transport in sheep and bovine forestomach

Na absorption across the cornified, multilayered, and squamous rumen epithelium is mediated by electrogenic amiloride-insensitive transport and by electroneutral Na transport. High concentrations of amiloride (>100 μM) inhibit Na transport, indicating Na+/H+ exchange (NHE) activity. The underlying NHE isoform for transepithelial Na absorption was characterized by mucosal application of the specific inhibitor HOE642 for NHE1 and S3226 for NHE3 in Ussing chamber studies with isolated epithelia from bovine and sheep forestomach. S3226 (1 μM; NHE3 inhibitor) abolished electroneutral Na transport under control conditions and also the short-chain fatty acid-induced increase of Na transport via NHE. However, HOE642 (30 μM; NHE1 inhibitor) did not change Na transport rates. NHE3 was immunohistochemically localized in membranes of the upper layers toward the lumen. Expression of NHE1 and NHE3 has been previously demonstrated by RT-PCR, and earlier experiments with isolated rumen epithelial cells have shown the activity of both NHE1 and NHE3. Obviously, both isoforms are involved in the regulation of intracellular pH, pHi. However, transepithelial Na transport is only mediated by apical uptake via NHE3 in connection with extrusion of Na by the basolaterally located Na-K-ATPase. The missing involvement of NHE1 in transepithelial Na transport suggests that the proposed “job sharing” in epithelia between these two isoforms probably also applies to forestomach epithelia: NHE3 for transepithelial transport and NHE1 for, among others, pHi and volume regulation.