Generation of a tetracycline-inducible NKCC2 expressing MDCKI cell line

In the kidney, the sodium chloride potassium cotransporter NKCC2 of the thick ascending limb (TAL) of the loop of Henle plays an important role in regulation of body salt and water homeostasis. Due to a lack of a suitable polarized epithelial cell line model, the majority of functional and cell biological studies of NKCC2 have been performed in isolated TAL tubules or following expression of NKCC2 in xenopus oocytes. The aim of this study was to generate a MDCKI cell line with stable and tetracycline‐inducible expression of NKCC2. Our previous studies using this approach have demonstrated that this is an excellent approach to study another member of the same protein family, the NaCl cotransporter NCC. FRT‐MDCKI cells transfected with the A‐isoform of human NKCC2 (hNKCC2‐A) had robust NKCC2 mRNA expression after 24 h of tetracycline induction. Enhanced NKCC2 protein expression was achieved by additional treatment of cells with the histone deacetylase inhibitor, valproic acid in conjunction with tetracycline. PNGase F treatment of cell lysates determined that NKCC2 was expressed as highly glycosylated monomers and dimers. NKCC2 was observed both intracellularly and at the apical pole of MDCKI‐hNKCC2‐A cells using immunofluorescence labeling and confocal microscopy. Cycloheximide chase studies determined that the mature form of NKCC2 has a half‐life of around 12 hours, whereas the immature, non‐glycosylated form of NKCC2‐A has a much shorter half‐life. NKCC2 levels in the apical membrane increased with forskolin or hypotonic low chloride stimulation, which were accompanied with significantly increased phosphorylation at Ser126 or Thr96, respectively. We conclude that the MDCKI‐hNKCC2 A cell line is an excellent model for studying NKCC2 function and regulation. Support or Funding Information DFF ‐ Danish Council for Independent Research, Medical Sciences This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .