Transepithelial water transport is essential for any living organism; however, currently available approaches for its direct measurement are insufficient. We established a novel method using D₂O dilution to quantify extracellular aqueous volumes, and demonstrate its suitability to measure transepithelial water transport. The water content of H₂O/D₂O mixtures was quantified with a volume resolution of 24 nl using attenuated total reflection mid-range infrared spectroscopy. To establish this approach for investigating epithelial transport we cultivated NCI-H441 cells as air liquid interface at different Dexamethasone concentrations ([Dex]). Increased ENaC activity was identified in epithelia cultivated at [Dex] above 3 nM. The apical surface liquid (ASL) volume remained unaffected by Dex at 1.2 µl per 0.33 cm² surface area when cells were cultivated at [Dex] of 3 nM and above. To measure water transport, 25 µl of isotonic NaCl solution was added to the apical compartment for 16 h. The remaining volume was measured afterwards. Resorption was observed, when cells were cultivated at [Dex] above 3 nM. It increased with increasing [Dex] and became saturated at 2.8 ±0.06 µl/cm²*h (mean ±SEM) in epithelia cultivated at [Dex] of 100 nM and above. Amiloride reduced water resorption by approximately 70%. Amiloride sensitivity of water transport versus sensitivity of I_SC was reduced (IC₅₀ 3.77 ±0.9 mM and 0.66 ±0.06 mM for water transport and I_SC respectively).

The extraordinary high volume resolution along with its inherent robustness and accuracy makes the deuterium oxide dilution method extremely suitable to study water transport and ASL homeostasis.