Proceedings of The Physiological Society

Europhysiology 2018 (London, UK) (2018) Proc Physiol Soc 41, PCB095

Poster Communications

Measuring alveolar epithelial fluid reabsorption with confocal microscopy

F. Freimuth1,2, C. Bauerdick1,2, A. Halavatyi3,2, H. Mairbäurl1,2

1. Medical Clinic VII, Sports Medicine, University Hospital Heidelberg, Heidelberg, Germany. 2. Translational Lung Research Center (TLRC), German Center of Lung Research (DZL), Heidelberg, Germany. 3. EMBL, Heidelberg, Germany.

Alveolar reabsorption controls the thickness of the layer of alveolar lining fluid (ALF), which is of importance for alveolar gas exchange. In cultured cells, reabsorption is not measured directly but estimated from electrophysiological and biochemical measurements of activity and expression of ion transporters. The aim of this study was to establish a method to determine the rate of alveolar fluid clearance and the steady-state thickness of ALF by confocal microscopy. Rat primary alveolar epithelial cells were grown to confluence on clear transwell filters in a tissue culture incubator in normoxia and hypoxia (1.5% O2) with or without dexamethasone (1µM). For measurement of ALF two wells of a 6-well plate with glass bottom were filled with water and pre-warmed. The plate was closed with a special lid having an opening cut out that was tightly covered with a PCR-foil. After removal of apical culture medium filters were placed on spacers into the glass bottom plate, 150µl of culture medium were added to the basolateral compartment, and filters were equilibrated for 20 min. Culture medium (20µl) with and without amiloride (10µM) containing 2 mg/ml of tetramethylrhodamine-labeled dextran (10kD) were applied to the apical surface by puncturing the PCR-foil with a micro-syringe, and the plate was immediately transferred to a confocal microscope (Leica SP8) equipped with a climatized chamber. X/Z-scans were immediately started (t0; time after adding dextran ~ 40 sec) and were repeated after 1, 2, 3.5, 5, 8, 10, and 13 min; images of 5 positions per filter were stored for automated off-line measurement of ALF-height at each time-point using MATLAB software. The rate of reabsorption was calculated from the decrease in ALF using the first three time-points. Steady-state height of ALF was determined from the 10 and 13 min time-points. Results show that there was no difference in ALF(t0) between control and dexamethasone treated cells in normoxia. Amiloride did not affect ALF(t0) in normoxic control cells. However, amiloride increased ALF(t0) significantly in hypoxic cells and in normoxic and hypoxic cells after treatment with dexamethasone. In contrast, there was no statistically significant difference in the calculated rate of reabsorption between the conditions. There was also no difference between conditions in the steady state height of ALF estimated from 10 and 13 min time-points. Results indicate that measurements of the calculated neither rate of reabsorption nor steady-state ALF-height reflect the differences in Na-transport rates measured electro-physiologically, which show inhibition by hypoxia and amiloride and stimulation of transport with dexamethasone in normoxia and hypoxia (Güney et al; PM:17873005). At best, values of ALF(t0) reflect differences in Na-transport, indicating that reabsorption rates are too fast to be detected by sequential confocal X/Y-scans.

Where applicable, experiments conform with Society ethical requirements