Stimulation of aquaporin-5 and transepithelial water permeability in human airway epithelium by hyperosmotic stress

Abstract

Osmotic water permeability (P(f )) was measured in spheroid-shaped human nasal airway epithelial explants pre-exposed to increasing levels of hyperosmotic stress. The fluid-filled spheroids, derived from nasal polyps, were lined by a single cell layer with the ciliated apical cell membrane facing the outside. The P(f ) was determined from diameter changes of the spheroids in response to changes in bathing medium osmolarity forth and back between 300 and 225 mOsm x l(-1). Continuous diameter measurements also allowed determination of spontaneous fluid absorption. Hyperosmotic pretreatment (increase from 300 up to 600 mOsm x l(-1)) caused a time- and osmolarity-dependent increase (up to approximately 1.5 times) in epithelial P(f ) which was of similar magnitude in cystic fibrosis (CF) and non-CF spheroids. The effect saturated at approximately 450 mOsm x l(-1) and at approximately 24 h. Expression of aquaporin-5 (AQP5), studied by immunofluorescence and confocal microscopy, showed an increase in parallel with the increase in P(f ) following hyperosmotic stress. The AQP5 was localized both in cytoplasmic vesicles and in apical cell membranes. Spontaneous fluid absorption rates were equal in CF and non-CF spheroids and were not significantly influenced by hyperosmotic stress. The results suggest that hyperosmotic stress is an important activator of AQP-5 in human airway epithelium, leading to significantly increased transepithelial water permeability.

Nasospheroids permit measurements of CFTR-dependent fluid transport

“…The apical membranes face the outside, and basolateral membranes line the lumen of the spherical organoids ( Figure 1). This orientation has been observed by other groups when nondisrupted epithelial sheets are cultured in suspension (40)(41)(42)(43)(44). When CFTR is labeled with a highly specific monoclonal antibody raised against the NBD2 region of CFTR (Ab 528, provided by J.R. Riordan, UNC-Chapel Hill; refs.…”

“…First, we tested non-CF spheroids derived from normal human nasal epithelium (Figure 2). Volume changes in similar spheroids have previously been reported in response to fluid transport changes (40)(41)(42)(43). To test our hypothesis that CFTR activation leads to fluid movement and consequent volume changes of the nasospheroids, we applied forskolin (10 μM) and 3-isobutyl-1-methylxanthine (IBMX,100 μM), a phosphodiesterase inhibitor that maintains intracellular cAMP levels (51), leading to CFTR activation in a cAMP-dependent manner ( Figure 2A).…”

“…Such samples provide a renewable source of differentiated airway epithelial cells from any individual with CF, regardless of their mutation, that does not require tissue expansion and multiple cell divisions prior to performing the assay. To improve the suitability of these samples for both functional and morphological tests, we have developed an organoid culture that is similar to cultures reported previously, where the apical ciliated membrane orients to the media bath (40)(41)(42)(43) but is derived from simple nasal epithelial curettage. This minimally invasive technique does not require polyp excision, which is different from the earlier reports, and utilizes normal nasal epithelial tissue.…”

Kinetics of hyperosmotically stimulated Na-K-2Cl cotransporter in Xenopus laevis oocytes

“…As a result, with the exception of cells located in the outer and inner medulla of the kidney, where the osmolarity changes with the diuretic state of the organism (46,47), few cells experience large fluctuations in their external osmolarity. Most mammalian cells have, however, conserved a variety of response mechanisms to hyperosmolarity, which include the activation of receptors (48) and ion transport mechanisms (2,14,24,34,40,41,54); transcriptional activation of organic solute transporters (7,15,57); and the stimulation of stress response genes (3,9,13,48).…”

Patient-specific three-dimensional explant spheroids derived from human nasal airway epithelium: a simple methodological approach for ex vivo studies of primary ciliary dyskinesia

“…Our method comprises a modified version of the previous well-described model by Pedersen and colleagues, where production of free-floating fluid-filled three-dimensional airway epithelial spheroids derived from protease released epithelium of resected human nasal polyps was performed [9–12]. Their spheroids could be assessed already a few days after polyp resection and have been used for extensive basic research, investigating water permeability and ion transport in healthy and cystic fibrosis airway epithelium [9–12].…”