Mo2029 Differential Activity of NHE3 and NHE2 in the Small Intestine of Irradiated Mice

Abstract

Tight junctions are formed by mature enterocytes and create an efficient barrier that has the necessary machinery for electrolyte and nutrient absorption. Radiotherapy causes epithelial barrier dysfunction leading to increased macromolecular translocation into the systemic compartment causing endotoxemia and inflammatory response. Recently, it was shown that glucose and some amino acids (AAs) activated active anion secretion and/ or increased paracellular permeability, whereas our mitigating amino acids mixture (MAAM) comprised of lysine, glycine, tryptophan, tyrosine, aspartic acid, isoleucine, threonine, valine, and serine increased electrolyte absorption and decreased paracellular permeability. The mechanism by which these amino acids tightened the mucosal barrier was not known. Therefore, studies were undertaken to determine the mechanism by which MAAM tightened the mucosal barrier. NIH Swiss mice were irradiated using a 137Cs source. Radiation dose-dependent and time-dependent (0, 6, 12, 24, 48, 72, and 144 hr after irradiation) changes in transepithelial electrical resistance (TEER) and dilution potential (DP) and the relative permeability of Cland Na+ (PCl/PNa) were measured. Transmission electron microscopy (TEM) images, Western blot analysis, and immunohistochemistry (IHC) for the cell junction protein complex (Claudin 1, 2, 5, Nectin, and E-cadherin) were performed at similar time points. Radiation resulted in a dose-dependent increase in conductance (43.3 ± 1.2, 29.5 ± 1.2, 48.2 ± 2.3, 48.9 ± 1.7, 38.7 ± 1.2 mS at 0, 1, 3, 5, 7 Gy, respectively). Similarly, time-dependent changes in conductance showed a maximal increase occurring in the first 6 hr (46.3 ± 1.5); there was no significant difference with increasing time after irradiation. Treatment with MAAM resulted in a significant decrease in conductance. DP studies showed that MAAM restored ion selectivity. TEM showed disruption of the cell junction complex and formation of clear space between the cells as early as 6 hr after irradiation. The changes persisted for the duration of the experiment (6 days). MAAM prevented cell junction disruption in mice; this was identifiable at 6 hr after irradiation. Western analysis and IHC showed a radiation dose-dependent increase in Claudin 1, 2, 5, Nectin, and E-cadherin except at 7 Gy. MAAM further increased Claudin 1, 2, 5, Nectin, and E-cadherin across all radiation doses. MAAM treatment decreased paracellular permeability. TEM showed disruption of the cell junction complex that peaked at 6 hours and was corrected using MAAM. Western analysis and IHC showed that radiation-induced alterations in the cell junction protein complex were further corrected in the MAAM group. We conclude that the disruption in the barrier results from changes in the cell junction complex and that MAAM restores the barrier function by correcting these changes.