Luminal Na⁺ homeostasis has an important role in intestinal peptide absorption in vivo

Abstract

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.

Na+-Coupled Nutrient Cotransport Induced Luminal Negative Potential and Claudin-15 Play an Important Role in Paracellular Na+ Recycling in Mouse Small Intestine

“…The unidirectional mucosal to serosal 22 Na + flux (J Na MS : 51.4 ± 2.3 µmol/cm 2 /h) was larger than the serosal to mucosal flux (J Na SM : 24.6 ± 1.7 µmol/cm 2 /h). This result suggests that Na + absorption occurred in the baseline conditions.…”

“…As shown in Figure 1A, the addition of glucose to the mucosal side increased I sc (∆I sc 13.7 ± 0.8 µmol/cm 2 /h) in wild-type mice. J Na MS was also significantly increased ( Figure 1B, ∆J Na MS , 12.5 ± 0.8 µmol/cm 2 /h, n = 5) after luminal application of glucose, while the unidirectional serosal to mucosal 22 Na + flux (J Na SM ) was not significantly changed after the addition of luminal glucose (Figure 1B, 25.5 ± 1.1 vs. 23.5 ± 1.1 µmol/cm 2 /h, p = 0.21, before and after the addition of glucose, respectively). Activation of SGLT1 increases unidirectional mucosal to serosal 22 Na + fluxes under short-circuit conditions in wild-type mice: Glucose-induced short-circuit current changes (I sc ) (A) and 22 Na + unidirectional flux changes (J Na ) (B) were measured simultaneously in Ussing chambers, as described in the Materials and Methods.…”

“…J Na MS was also significantly increased ( Figure 1B, ∆J Na MS , 12.5 ± 0.8 µmol/cm 2 /h, n = 5) after luminal application of glucose, while the unidirectional serosal to mucosal 22 Na + flux (J Na SM ) was not significantly changed after the addition of luminal glucose (Figure 1B, 25.5 ± 1.1 vs. 23.5 ± 1.1 µmol/cm 2 /h, p = 0.21, before and after the addition of glucose, respectively). Activation of SGLT1 increases unidirectional mucosal to serosal 22 Na + fluxes under short-circuit conditions in wild-type mice: Glucose-induced short-circuit current changes (I sc ) (A) and 22 Na + unidirectional flux changes (J Na ) (B) were measured simultaneously in Ussing chambers, as described in the Materials and Methods. After measure basal I sc and J Na during the initial 30 min (squares represent mean of 9 and 8 measurements mucosal to serosal (M to S) and serosal to mucosal (S to M), respectively), glucose was added to the mucosal side, indicated by arrows (n = 9 and 8, M to S and S to M, respectively).…”

“…Non-metabolizable sugar alpha methyl-d-glucose (αMDG) increase of I sc (E) and J Na (F) (n = 6 and 6, M to S and S to M, respectively). Closed squares indicate mucosal to serosal unidirectional Na + fluxes (J Na MS ) and open squares indicate mucosal to serosal 22 Na + fluxes (J Na SM ). Each point represents the mean ± SE.…”

“…In this study, to investigate whether paracellular Na + conductance through claudin-15 is involved in this Na + recycling system and to elucidate the role of Na + -nutrient cotransport induced luminal negativity for Na + recycling, we measured glucose-induced short-circuit currents (∆I sc ) under open-and short-circuit conditions in Ussing chambers. In addition, we simultaneously measured changes in unidirectional mucosal to serosal 22 Na + (∆J Na MS ) flux in wild-type mice and compared them with those of 15 deficient (cldn15 −/− ) mice. Furthermore, to understand the mechanism of oral rehydration therapy, which is based on the notion that the Na + that is absorbed by Na + -glucose cotransport enters into systemic circulation [16], we measured glucose-induced unidirectional ∆J Na MS in cholera toxin-diarrhea model mice.…”