Fluorescence Quenching to Measure dDAVP-induced Cell Volume Changes in Kidney Collecting Duct Cell / 대한신장학회지

ABSTRACT

PURPOSE: Cell volume regulation is critical in kidney collecting duct cells which are subjected to large transepithelial osmotic gradients and stimulation of vasopressin. The present study aimed at validating the usefulness of the fluorescence quenching method to measure rapid changes in the cell volume of the kidney collecting duct cells in response to changes of extracellular osmolality and/or dDAVP (V2 receptor agonist) stimulation. METHODS: M-1 cell (a mouse cortical collecting duct cell line) was used and the data presented traces of cellular fluorescence in M-1 cells loaded with calcein collected over time as extracellular osmolality was repeatedly changed or dDAVP was treated. And the "initial relative rate of cell volume changes" was calculated. RESULTS: M-1 cells loaded with calcein revealed that fluorescence was increased when exposed to low extracellular osmolality (250 mOsm/KgH2O), whereas it was decreased by high extracellular osmolality (350 mOsm/KgH2O). This could reflect volume-dependent changes in fluorescence intensity in the range of quenching concentrations. The calculated "initial relative rate of cell volume changes" in M-1 cells during 1 sec was increased-7-fold by dDAVP treatment (10(-8)M, 2 min), compared with vehicle treatment when extracellular osmolality was changed from 350 to 250 mOsm/KgH2O. CONCLUSION: This study suggests that a fluorescence quenching method could be exploited for investigating an effect of dDAVP or other drugs/chemicals on the relative rate of cell volume changes in the kidney collecting duct cells.