engleski

Immunocharacterization of the sodium-glucose cotransporter SGLT2 in rat kidneys

A bulk of D-glucose reabsorption in the mammalian kidney (K) is mediated by the low affinity, high capacity Na+-glucose cotransporter SGLT2. In various transport and hybridization studies, this transporter has been localized in the cells of proximal tubule segments S1 and S2. The transporter exhibits the 1:1 Na+:glucose coupling ratio, and unlike SGLT1, a high affinity Na+-glucose cotransporter localized mainly to the proximal tubule S3 segments, it does not transport D-galactose. cDNA for SGLT2 has been identified first in humans and later in rats and mice. The expression of its mRNA has been detected in most human tissues, the highest levels being present in kidney. A convincing immunolocalization of SGLT2 along the mammalian nephron has not been reported. In this work we generated a novel polyclonal antibody (Ab) specific for peptide sequence in the rat SGTL2 protein, and performed detailed Western blotting (WB) and immunocytochemical (IC) studies of the expression of SGLT2 protein along the nephron in variously-treated adult (3 months) and prepubertal (3 weeks) male (M) and female (F) rats. WB experiments were performed with brush-border (BBM) and total cell membranes isolated from tissue homogenates, whereas the IC studies were performed in tissue cryosections. In addition, the expression of SGLT2 mRNA was determined by RT-PCR using RNA isolated from various tissue zones. In WB of isolated membranes, the Ab labeled a single protein band at ~75 kDa. The protein band and the immunostaining was absent in isolated membranes and tissue cryosections, respectively, from the rat small intestine, indicating a specific localization of SGLT2 in the K. In membranes from the K cortex (C) and outer stripe (OS) of intact adult rats, the density of 75 kDa protein band: a) exhibited strong zonal (C>OS) and gender differences (F>M), and b) increased weakly by castration and remained unaffected by ovariectomy. In castrated rats, the protein band was downregulated by testosterone and upregulated by estradiol treatment. By IC, the Ab stained BBM of S1 and S2 (S1>S2) with gender-dependent manner (F>M). The staining intensity in gonadectomized animals, and in sex hormone-treated castrated M matched the pattern of WB data. In prepubertal rats, the respective protein band in isolated K membranes and the staining intensity in PT were weaker than in adult animals, and gender-independent. The expression of SGLT2 mRNA was found to be high in the C and low in the OS, but gender-independent. Our data indicate that in the rat K: 1) SGLT2 is localized to the BBM of proximal tubule S1 and S2 segments (S1>S2) in the C, 2) at the protein level, SGLT2 expression exhibits zonal (C>OS) and gender differences (F>M) that occur after puberty, and 3) at the mRNA level, the expression of SGLT2 exhibits no gender differences, thus indicating the involvement of posttranslational mechanisms in sex hormone-regulated expression of SGLT2 protein.

kidney; glucose cotransporter; gender differences

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