Background: Osmoregulation, which maintains the osmolarity of fluid surrounding cells, is a key feature of ionocytes across species and has been studied extensively in fish adaptation to environmental changes in salinity.In the mammalian kidney, osmoregulation is coordinated by principal cells and intercalated cells of the collecting ducts, which control water movement; acid-base regulation; and Na + , Cl -, K + , and Ca 2+ homeostasis.Given the overlap in expressed channels between pulmonary ionocytes and kidney principal cells and intercalated cells, we hypothesized that pulmonary ionocytes may also participate in osmoregulation by airway epithelia.Methods: To mimic fish gill ionocytes in freshwater and seawater environments, we created hypertonic and hypotonic air-liquid interface (ALI) culture media.FOXI1-Cre ERT2 lineage-traced proximal airway stem cells were differentiated under hypertonic or hypotonic conditions in ALI culture.At full differentiation (21 days), we quantified ionocyte numbers and ionocyte subtype gene expression patterns.Results: We exposed actively differentiating ferret basal cells to slightly hypertonic media (+77 mOsm/L NaCl) and observed a marked increase (7.32-fold; p < 0.0015) in the number of lineage-traced pulmonary ionocytes at full differentiation (21 days).Under hyperosmotic stress, greater numbers of ionocytes was correlated with high mRNA expression of key ionocyte markers FOXI1 and ASCL3.We have previously shown that three ionocyte subtypes exist (Type-A, Type-B, Type-C) in ferret airway ALI culture.We found that hyperosmotic stress increased expression of Type-A (BSND) and Type-C (CXCL17) ionocyte marker genes but decreased expression of the Type-B ionocyte marker ID3.The observed downregulation in CFTR expression under hyperosmotic stress is consistent with the expansion of Type-C ionocytes, which express significantly ( p = 2 × 10 -11 ) less CFTR than other ionocyte subtypes.Hyperosmotic stress imposed on fully differentiated ALI cultures did not alter the frequency of lineage-traced pulmonary ionocytes, suggesting that a hyperosmotic environment affects progenitor cell specification of Type-A and Type-C ionocytes.Exposure of differentiating ferret basal cells to hypotonic media (-77 mOsm/L) also led to expansion of ATP6V1G3 + ionocytes when fully differentiated at the ALI.Ongoing gene expression profiling is defining the dominant ionocyte subtype under hypotonic conditions.Conclusions: These findings indicate that osmolarity affects basal cell specification of pulmonary ionocytes and suggests that there are specialized ionocyte functions that adapt to environmental changes in airway osmolarity.Hyperosmotic fluid at the basolateral surface of the airway would be expected to be extracted water from cells and the airway surface liquid (ASL), leading to cell shrinkage and ASL dehydration.Our data suggest that apical membrane Cl -permeability is constrained by CFTR-expressing ionocytes and that the observed hyperosmotic downregulation of CFTR may be a compensatory mechanism to limit ASL dehydration.This finding may be analogous to the inactivation of ionocyte CFTR expression in fish gills during seawater to freshwater transition.
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