Production of an AQP2‐Enriched Clonal mpkCCD Cell Line

The collecting duct cell line mpkCCD (developed by A. Vandewalle) expresses vasopressin‐regulated water channel AQP2 endogenously, but at low levels relative to native inner medullary collecting ducts (IMCDs). To obtain an optimized model for study of AQP2 regulation, we sub‐cloned a line that expresses AQP2 at high levels (mpkCCD‐BY11) from the original mpkCCD cell line. AQP2 expression in these cells was found to be induced to levels similar to the native IMCD cells when grown on membrane filter and exposed to 0.1 nM synthetic vasopressin analog dDAVP at the basolateral medium. Semiquantitative immunoblotting revealed 5.0‐fold enrichment of AQP2 protein relative to the original cells, while AQP2 mRNA was increased 1.7‐fold. Immunoblotting of the mpkCCD‐BY11 cells showed dose‐ and time‐dependent increases in AQP2 abundance in response to dDAVP. Immunoblotting with phospho‐specific antibodies showed increased phosphorylation at Ser‐256 and Ser‐269 of AQP2 in response to dDAVP, similar to the native IMCD cells. When exposed to dDAVP, AQP2 was found at the apical plasma membrane by confocal fluorescence microscopy. Upon withdrawal of dDAVP, AQP2 internalized into intracellular vesicles in 4 hours. Re‐applying dDAVP caused AQP2 to redistribute to the apical plasma membrane in 0.5 hour. Thus, the mpkCCD‐BY11 cells recapitulated vasopressin‐induced AQP2 trafficking as seen in the native IMCD principal cells.