Intestinal cell line studies indicated luminal Na homeostasis is essential for proton-coupled peptide absorption, because the driving force of PepT1 activity is supported by the apical Na/H exchanger NHE3. However, there is no direct evidence demonstrating the importance of in vivo luminal Na for peptide absorption in animal experiments. To investigate the relationship between luminal Na homeostasis and peptide absorption, we took advantage of claudin 15 deficient (cldn15) mice, whereby Na homeostasis is disrupted. We quantitatively assessed the intestinal segment responsible for peptide absorption using radiolabeled non-hydrolyzable dipeptide (glycylsarcosine, Gly-Sar) and non-absorbable fluid phase marker polyethylene glycol (PEG) 4000 in vivo. In wild-type (WT) mice, the concentration ratio of Gly-Sar to PEG 4000 decreased in the upper jejunum, suggesting the upper jejunum is responsible for peptide absorption. Gly-Sar absorption was decreased in the jejunum of cldn15 mice. To elucidate the mechanism underlining these impairments, a Gly-Sar-induced short-circuit ( I) current was measured. In WT mice, increments of Gly-Sar-induced I were inhibited by the luminal application of a NHE3 specific inhibitor S3226 in a dose-dependent fashion. In contrast to in vivo experiments, robust Gly-Sar-induced I increments were observed in the jejunal mucosa of cldn15 mice. Gly-Sar-induced I was inhibited by S3226 or a reduction of luminal Na concentration, which mimics low luminal Na concentrations in vivo. Our study demonstrates that luminal Na homeostasis is important for peptide absorption in native epithelia and that there is a cooperative functional relationship between PepT1 and NHE3.

Many nutrients are absorbed via Na+ cotransport systems, and therefore it is predicted that nutrient absorption mechanisms require a large amount of luminal Na+. It is thought that Na+ diffuses back into the lumen via paracellular pathways to support Na+ cotransport absorption. However, direct experimental evidence in support of this mechanism has not been shown. To elucidate this, we took advantage of claudin-15 deficient (cldn15−/−) mice, which have been shown to have decreased paracellular Na+ permeability. We measured glucose-induced currents (ΔIsc) under open- and short-circuit conditions and simultaneously measured changes in unidirectional 22Na+ fluxes (ΔJNa) in Ussing chambers. Under short-circuit conditions, application of glucose resulted in an increase in ΔIsc and unidirectional mucosal to serosal 22Na+ (∆JNaMS) flux in both wild-type and cldn15−/− mice. However, under open-circuit conditions, ΔIsc was observed but ∆JNaMS was strongly inhibited in wild-type but not in cldn15−/− mice. In addition, in the duodenum of mice treated with cholera toxin, paracellular Na+ conductance was decreased and glucose-induced ∆JNaMS increment was observed under open-circuit conditions. We concluded that the Na+ which is absorbed by Na+-dependent glucose cotransport is recycled back into the lumen via paracellular Na+ conductance through claudin-15, which is driven by Na+ cotransport induced luminal negativity.