Apical Expression and Activity of Na‐Cl Cotransporters (NCC) in MDCK I TetOff Cells Does Not Require NCC Phosphorylation

Abstract

NCC is regulated in the renal DCT by trafficking between apical and subapical membranes. In NCC‐transfected oocytes and non‐epithelial cells, trafficking is regulated by the WNK4‐SPAK/OSR1 cascade leading to phosphorylation of NCC at Thr58 (NCCpT58). To investigate the connection between phosphorylation, trafficking and activity, we aimed to generate a stable renal epithelial cell line expressing apical NCC. MDCK I TetOff cells were transfected with rat NCC, then cultured to full confluency (7d) +/− Doxycycline (Dox). Dox+ had no NCC expression, while Dox− had robust expression by immunoblot with anti‐rat NCC. By immunofluorescence, NCC was expressed throughout the cell including apical membrane; additionally, a fraction of NCC was biotinylatable from the apical surface. There was no detectable NCCpT58. Thiazide‐sensitive 22Na+ uptake was detected in Dox− (not Dox+) and was not increased by acute low Cl− medium or Angiotensin II, suggesting that these responses may require NCCpT58. By RT‐PCR with canine kidney as + control: SPAK, OSR1, WNK1, WNK4, but not WNK3, were detected in transfected MDCK cells. In summary, a cell line has been generated that expresses active NCC in the apical membranes of MDCK cells, suggesting that apical localization and activity do not require NCCpT58. Activation by hypotonicity or AngII, lacking in these cells, may require reconstitution of a missing component. DK34316, DK062283.