Proceedings of The Physiological Society

Europhysiology 2018 (London, UK) (2018) Proc Physiol Soc 41, PCB098

Poster Communications

Contribution of tricellulin to paracellular water permeability in epithelia of different tightness

C. M. Ayala Torres1, R. Rosenthal1, S. Krug1, J. Schulzke1, M. Fromm1

1. Institut für Klinische Physiologie, Charité - Universitätsmedizin Berlin, Campus Benjamin Franklin, Hindenburgdamm 30, 12203 Berlin, Germany, Berlin, Germany.

Introduction: The existence of a paracellular pathway for water through the tight junctions (TJ) was discussed controversially for many years until a molecular component of the paracellular water transport, claudin-2, was identified. The contribution of the tricellular TJ (the site where three cell meet) and its major component tricellulin was not elucidated so far. Methods: Knockdown (KD) of tricellulin was performed by transfection with shRNA targeting this protein in HT-29/B6 (human colon) or in MDCK C7 (dog kidney) cells. The expression of tricellulin, claudins, and aquaporins was investigated in KD and control cells by Western blot. The transepithelial water flux was measured using a modified Ussing chamber setup. Water flux was induced by an osmotic gradient of 100 mM mannitol or 100 mM 4 kDa-dextran. Results: Tricellulin KD did not alter the expression of claudins or AQPs. In HT-29/B6 cells, tricellulin KD caused a decrease of transepithelial resistance (TER) from 1858±88 Ω×cm2 to 969±27 Ω×cm2. However, water flux did not significantly change in tricellulin KD at osmotic gradients induced either by mannitol or by 4 kDa-dextran. Since in HT-29/B6 cells claudin-2 is genuinely expressed, a large component of water transport may have travelled through the bicellular TJ and a possible contribution of the tricellular TJ may not have reached significant levels. In MDCK C7 cells the tricellulin KD resulted in a decrease of TER from 12.7±0.3 kΩ×cm2 to 9.2±0.3 kΩ×cm2, but in contrast to HT-29/B6 cells, here we observed an increased water flux compared to controls under the mannitol-induced osmotic gradient (controls: 5.17±0.51 µl/h/cm2, KD: 7.10±0.41 µl/h/cm2, p<0.05). The same effect was observed under a gradient induced by 4 kDa-dextran. That in these cells an effect of tricellulin KD on water transport became apparent can be explained by the fact that MDCK C7 cells lack any claudin-2 expression and therefore the large background otherwise mediated by the bicellular TJ was absent, uncovering a significant contribution of the tricellular TJ. Current size assumptions of the tricellular TJ comprise a diameter of ∼100 Å that would be suitable for movement of water molecules (∼3 Å), however through a tube of ~1 µm length. Conclusion: In HT-29/B6 cells a tricellulin KD does not significantly alter transepithelial water transport due to the presence of the large contribution of other water-conducting proteins as claudin-2. In contrast, tricellulin KD in MDCK C7 cells which lack claudin-2 results in an increase in transepithelial water transport. Thus, the contribution of tricellulin and the tricellular TJ to transepithelial water transport depends on the tightness of the epithelia. In the absence of claudin-2, the tricellular TJ, controlled by tricellulin expression, contributes to paracellular water transport.

Where applicable, experiments conform with Society ethical requirements