Role of Aquaporin 2 in cell volume regulation

PAULA FORD; VALERIA RIVAROLA; OSVALDO CHARA; LUCIANO GALICIA; MARCEL BLOT-CHABAUD; FRANCOISE CLUZEAUD; NICOLETTE FARMAN; MARIO PARISI; CLAUDIA CAPURRO

Bruselas - Bélgica

Conferencia; IV International Conference on Aquaporins; 2005

Objectives: In the mammalian kidney, cortical collecting duct cells are exposed to changes in environment osmolality, consequently cell volume regulation may be especially important for maintenance of intracellular homeostasis. The aim of the present work was to study cell volume regulation after hypo or hypertonic challenges, in two cortical collecting duct cell lines: one not expressing aquaporins and other stably transfected with aquaporin 2. Material and methods: We used a cellular line established from rat cortical collecting duct (RCCD1), which maintains the cellular types and the transport characteristics of the native duct. Cell volume was evaluated using a fluorescent probe technique, adapted to confluent epithelial monolayers grown on coverslips. Fluorescence data were acquired every 10 s by use of a charge coupled device camera connected to a computer. The acquisition of single-cell kinetic signal can be simultaneously recorded with the intracellular pH. Results: Experiments with hypertonic mannitol (impermeable solute) media demonstrated that, independently of Aquaporin-2 expression, cortical collecting duct cells shrink but fail to show regulatory volume increase. When the bathing media was made hyperosmotic by the addition of urea (permeable solute) both RCCD1 cell lines responded with a transient osmotic shrinkage. However, cell volume recovery was completely abolished in the presence of phloretin, a specific inhibitor of urea transporters. This result is due to solute diffusion and not volume regulation. In contrast, under hypotonic shocks, regulatory volume decrease occurs and the activation of these mechanisms is more rapid in Aquaporin-2 transfected cells. This regulatory response takes place in parallel with intracellular acidification, which is faster in cells expressing Aquaporin-2. The acidification and the initial regulatory volume decrease response were inhibited by glibenclamide and barium chloride only in Aquaporin-2 cells. Conclusions: These results suggest that increases in the osmotic water permeability due to the expression of Aquaporin-2, are critical for a rapid activation of regulatory volume decrease mechanisms, which would be linked to cystic fibrosis transmembrane conductance regulator and to barium-sensitive potassium channels