Non‐Polarized mpkCCD Cell Model for Aquaporin‐2 Phosphorylation and Membrane Trafficking Study

Abstract

Aquaporin‐2 (AQP2) is a water channel protein expressed in the kidney collecting duct principal cells responsible for water reabsorption. AQP2's ability to regulate water reabsorption depends on its phosphorylation states and trafficking to and from the apical plasma membrane regulated by the peptide hormone vasopressin. The mpkCCD cells have been an instrumental collecting duct cell model for studying AQP2 phosphorylation and trafficking. However, the cells require a time‐consuming polarization process before they can respond to vasopressin. We sought to circumvent this issue by examining AQP2 phosphorylation and trafficking in non‐polarized AQP2‐overexpressing mpkCCD cells treated with forskolin, which elevates intracellular cyclic AMP (cAMP), a second messenger of vasopressin. We found that forskolin induced AQP2 phosphorylation at serine 269 (S269) by 1.6 folds as observed in polarized mpkCCD and isolated rat inner medullary collecting duct cells treated with vasopressin. Before forskolin stimulation, most AQP2 was intracellular and was not phosphorylated at S269 by confocal immunofluorescence microscopy. In response to forskolin, AQP2 was found in the plasma membrane and was phosphorylated at S269, similar to those observed in the polarized mpkCCD cells and native collecting duct cells stimulated with vasopressin. As a proof of principle, we found that small hairpin RNA‐mediated protein kinase A knockdown reduced AQP2 phosphorylation at S269 and membrane localization in the non‐polarized mpkCCD cells treated with forskolin. Thus, non‐polarized AQP2‐overexpressing mpkCCD cells can be used in lieu of polarized mpkCCD cells when time is a limiting factor including transient small hairpin RNA‐mediated gene knockdown experiments.Support or Funding InformationThis work was supported by the Ministry of Science and Technology, TAIWAN (MOST 107‐2320‐B‐002‐057‐MY3).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.