engleski

Immunolocalization of the Na+-Glucose Cotransporter SGLT1 in Male and Female Rats with an Improved Antibody

Previously we described the localization and gender differences (GD) of the high affinity SGLT1 (Slc5a1) in rat kidney (K) and small intestine (SI) using an affinity purified antibody (Ab) for the rat SGLT1-specific peptide (rSGLT1-Ab), which, however, cross-reacted with a 40 kDa polypeptide in Western blots (WB) (Sabolic et al., Am J Physiol Renal Physiol 290:913-926, 2006). Using a shortened peptide for immunization, we generated an improved, more specific rSGLT1-Ab. In WB of isolated brush-border membranes from rat K and SI, the Ab labeled a single protein band of ~75 kDa. The K expression of rSGLT1 protein was correlated with the expression of rSGLT1 mRNA in RT-PCR studies. In K of adult male (M) and female (F) rats, WB and RT-PCR data, and immunolocalization data in tissue cryosections (IC) revealed: 1) major localization of rSGLT1 protein in the proximal tubule (PT) S2 (weakly) and S3 segments (strongly), 2) GD in both tissue zones (M<F), and 3) upregulation after castration but no change after ovariectomy. In prepubertal rats, labeling of the 75 kDa protein band in renal membranes and the IC staining of PT were weak and gender independent. At variance to our previous Ab, the new Ab did not react with large vessels in K. In SI, WB and IC studies with the novel Ab revealed regional (jejunum &#61502; duodenum = ileum) but no GD. With the new Ab the rSGLT1 protein was observed in locations that have not been described earlier, i.e., in the luminal cell membrane of thick ascending limbs of Henle, macula densa, and distal tubules in K, bile ducts in liver, and the initial ducts in salivary gland. In brain and uterus the rSGLT1-Ab stained smooth muscles of large arteries, but the staining of capillaries in brain, heart and skeletal muscle, that has previously been observed with our old Ab crossreacting with the 40 kDa polypeptide, was not detected with the novel Ab. Whereas our old Ab is apparently not suited to identify novel sites of SGLT1 localization, the new improved Ab may be used for this purpose and to unravel additional physiological functions of SGLT1.

SGLT1; gender differences; immunolocalization; proximal tubule

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