FXYD proteins in renal medulla during sodium depletion 1 Locations, abundances and possible functions of FXYD ion transport regulators in rat renal medulla

The γ subunit of Na,K-ATPase (FXYD2) and CHIF (FXYD4) are considered pump regulators in kidney tubules. The aim of this study was to expand the information about their locations in kidney medulla and to evaluate their importance for electrolyte excretion in an animal model. The cellular and subcellular locations and abundances of γ and CHIF in medulla of control and sodium depleted rats were analyzed by immunofluorescence and immunoelectron microscopy, and semiquantitative Western blotting. The results showed that antibodies against γC-terminus and splice variant γa, but not splice variant γb, labeled intercalated cells, but not principal cells, in the initial part of the inner medullary collecting duct (IMCD1). In subsequent segments (IMCD2 and IMCD3) all principal cells exhibited distinct basolateral labeling for both γC–terminus, splice variant γa, and CHIF. Splice variant γb was abundant in inner stripe of outer medulla (ISOM) but absent in inner medulla (IM). Double labeling by high resolution immunoelectron microscopy showed close structural association between CHIF and Na,K-ATPase α1 subunit in basolateral membranes. The present observations provide new information about the cellular and subcellular locations of γ and CHIF in renal medulla and show a new γ variant in inner medulla. Extensive sodium chloride depletion did not induce significant changes in the locations or abundances of γC-terminus and CHIF in different kidney zones. We conclude that the unchanged levels of these two FXYD proteins suggest that they are not primary determinants for urine electrolyte composition during sodium chloride depletion.