Cell localization and sex-dependent expression of chloride/formate exchanger CFEX (Slc26a6) in rat kidneys

The chloride/formate exchanger CFEX, a member of the solute carrier family SLC26A6 in humans/Slc26a6 in rodents, in various mammalian organs mediates transport of anions including chloride, bicarbonate, oxalate, formate and hydroxyl ion. Except in mice, its distribution and expression in the kidneys of other species are poorly documented. Here we used a commercial polyclonal anti-peptide antibody (CFEX-Ab) to study distribution of protein (rCFEX) along the nephron, and its possible sex-related expression in adult male (M) and female (F) Wistar rats. In order to validate specificity of the CFEX-Ab, we transiently transfected the HEK293 cells with the rCFEX cDNA ; total RNA was isolated from the rat kidney, cDNA was synthesized and amplified by PCR using specific primers, a full-length rCFEX cDNA was inserted into the pcDNA3.1/His C and used for transformation of the competent DH5α E. coli, and the HEK293 cells were transfected with this vector using polyethyleneimine. Specificity of the CFEX-Ab was verified by immunofluorescence cytochemistry (IFC) ; the antibody strongly stained the plasma membrane of rCFEX-transfected cells, whereas the staining was not observed in mock-transfected cells and in the rCFEX-transfected cells incubated with the peptide-blocked antibody. The CFEX-Ab was further characterized by IFC on tissue cryosections and by Western blotting (WB) of isolated total cell (TCM) or brush-border (BBM) membranes. By IFC, the CFEX-Ab stained the BBM of proximal tubules (PT) with heterogeneous intensity (S1 ~ S2 > S3). By WB of TCM or BBM, the CFEX-Ab labeled a single protein band of 120 kDa. The rCFEX-related staining of PT BBM and labeling of the protein band were abolished with the immunizing peptide-blocked antibody. The intensity of rCFEX-related staining in PT BBM, as well as the density of rCFEX-related protein band exhibited strong sex differences, M > F. The observed expression of rCFEX protein was downregulated by castration and unaffected by ovariectomy, whereas in the sex hormone-treated gonadectomized males, testosterone upregulated, while estrogen and progesterone had no effect on the renal expression of protein. Collectively, in the rat kidneys the rCFEX protein is localized to the PT BBM showing zonal differences (cortex  outer stripe) and M-dominant expression due to androgen stimulation.