Na(+)-D-glucose cotransporter SGLT1 is pivotal for intestinal glucose absorption and glucose-dependent incretin secretion.

To clarify the physiological role of Na(+)-D-glucose cotransporter SGLT1 in small intestine and kidney, Sglt1(-/-) mice were generated and characterized phenotypically. After gavage of d-glucose, small intestinal glucose absorption across the brush-border membrane (BBM) via SGLT1 and GLUT2 were analyzed. Glucose-induced secretion of insulinotropic hormone (GIP) and glucagon-like peptide 1 (GLP-1) in wild-type and Sglt1(-/-) mice were compared. The impact of SGLT1 on renal glucose handling was investigated by micropuncture studies. It was observed that Sglt1(-/-) mice developed a glucose-galactose malabsorption syndrome but thrive normally when fed a glucose-galactose-free diet. In wild-type mice, passage of D-glucose across the intestinal BBM was predominantly mediated by SGLT1, independent the glucose load. High glucose concentrations increased the amounts of SGLT1 and GLUT2 in the BBM, and SGLT1 was required for upregulation of GLUT2. SGLT1 was located in luminal membranes of cells immunopositive for GIP and GLP-1, and Sglt1(-/-) mice exhibited reduced glucose-triggered GIP and GLP-1 levels. In the kidney, SGLT1 reabsorbed ∼3% of the filtered glucose under normoglycemic conditions. The data indicate that SGLT1 is 1) pivotal for intestinal mass absorption of d-glucose, 2) triggers the glucose-induced secretion of GIP and GLP-1, and 3) triggers the upregulation of GLUT2.

Renal expression of organic anion transporter Oat5 in rats and mice exhibits the female-dominant sex differences.

The organic anion transporter 5 (Oat5, Slc22a19) was previously localized to the brush-border of proximal tubule (PT) S3 segment in rat and mouse kidneys. Here we report on sex hormone-regulated expression of Oat5 in rat kidneys, after reinvestigating: a) expression of its mRNA by end-point and real time RT-PCR in the tissue, b) abundance of its protein by Western blotting (WB) in isolated membranes, and c) immunolocalization in tissue cryosections. In untreated male (M) and female (F) adult rats, the expression of Oat5 mRNA was predominant in the outer stripe (OS), exhibiting sex differences (M

Low doses of ochratoxin A upregulate the protein expression of organic anion transporters Oat1, Oat2, Oat3 and Oat5 in rat kidney cortex.

Mycotoxin ochratoxin A (OTA) is nephrotoxic in various animal species. In rodents, OTA intoxication impairs various proximal tubule (PT) functions, including secretion of p-aminohippurate (PAH), possibly via affecting the renal organic anion (OA) transporters (Oat). However, an effect of OTA on the activity/expression of specific Oats in the mammalian kidney has not been reported. In this work, male rats were gavaged various doses of OTA every 2nd day for 10 days, and in their kidneys we studied: tubule integrity by microscopy, abundance of basolateral (rOat1, rOat3) and brush-border (rOat2, rOat5) rOat proteins by immunochemical methods, and expression of rOats mRNA by RT-PCR. The OTA treatment caused: a) dose-dependent damage of the cells in S3 segments of medullary rays, b) dual effect upon rOats in PT: low doses (50-250 microg OTA/kg b.m.) upregulated the abundance of all rOats, while a high dose (500 microg OTA/kg b.m.) downregulated the abundance of rOat1, and c) unchanged mRNA expression for all rOats at low OTA doses, and its downregulation at high OTA dose. Changes in the expression of renal Oats were associated with enhanced OTA accumulation in tissue and excretion in urine, whereas the indicators of oxidative stress either remained unchanged (malondialdehyde, glutathione, 8-hydroxydeoxyguanosine) or became deranged (microtubules). While OTA accumulation and downregulation of rOats in the kidney are consistent with the previously reported impaired renal PAH secretion in rodents intoxicated with high OTA doses, the post-transcriptional upregulation of Oats at low OTA doses may contribute to OTA accumulation and development of nephrotoxicity.

Optimal methods of antigen retrieval for organic anion transporters in cryosections of the rat kidney.

To localise antigens by immunocytochemistry (IC), the samples of tissues or cells are usually denatured by fixation, and either frozen and cryosectioned, or embedded in paraffin before sectioning. p-Formaldehyde (PFA; formalin) is a common fixative, which preserves antigenicity of proteins, but damages the tissue/cell morphology and "masks" the antibody binding sites (epitopes). In order to "unmask" epitopes, some kind of antigen retrieval (AR) is used. The aim of this study was: a) to find an optimal AR method in cryosections of in vivo PFA-fixed kidneys for organic anion transporters (Oat) that reside in the basolateral (Oat1, Oat3) and brush-border membrane (Oat2, Oat5) of the rat renal proximal tubules, and b) using optimal method, to compare IC staining of Oats in kidneys that had been PFA-fixed in vivo or in vitro. IC staining in untreated cryosections was compared with that following detergent treatment or microwave heating in citrate buffer of pH 3, pH 6, or pH 8, with or without alcohol pre-treatment. The preferred AR method for Oat1, Oat2, and Oat5 was heating of cryosections at pH 6, and for Oat3 heating at pH 3, without alcohol pre-treatment. Compared with tissue fixed in vivo, tissue fixed in vitro exhibited damaged tubule morphology, similar staining intensity of Oat1 and Oat3, and higher staining intensity of Oat2 and Oat5. We conclude that for optimal IC presentation, each Oat in the rat kidney has to be treated individually, with different fixation and AR approach.

Revised immunolocalization of the Na+-D-glucose cotransporter SGLT1 in rat organs with an improved antibody.

Previously, we characterized localization of Na(+)-glucose cotransporter SGLT1 (Slc5a1) in the rat kidney using a polyclonal antibody against the synthetic COOH-terminal peptide of the rat protein (Sabolic I, Skarica M, Gorboulev V, Ljubojevic M, Balen D, Herak-Kramberger CM, Koepsell H. Am J Physiol Renal Physiol 290: 913-926, 2006). However, the antibody gave some false-positive reactions in immunochemical studies. Using a shortened peptide for immunization, we have presently generated an improved, more specific anti-rat SGLT1 antibody (rSGLT1-ab), which in immunochemical studies with isolated membranes and tissue cryosections from male (M) and female (F) rats exhibited 1) in kidneys and small intestine, labeling of a major protein band of approximately 75 kDa; 2) in kidneys of adult animals, localization of rSGLT1 to the proximal tubule (PT) brush-border membrane (S1 < S2 < S3) and intracellular organelles (S1 > S2 > S3), with zonal (cortex < outer stripe) and sex differences (M < F) in the protein expression, which correlated well with the tissue expression of its mRNA in RT-PCR studies; 3) in kidneys of castrated adult M rats, upregulation of the protein expression; 4) in kidneys of prepubertal rats, weak and sex-independent labeling of the 75-kDa protein band and immunostaining intensity; 5) in small intestine, sex-independent regional differences in protein abundance (jejunum > duodenum = ileum); and 6) thus far unrecognized localization of the transporter in cortical thick ascending limbs of Henle and macula densa in kidney, bile ducts in liver, enteroendocrine cells and myenteric plexus in the small intestine, and initial ducts in the submandibular gland. Our improved rSGLT1-ab may be used to identify novel sites of SGLT1 localization and thus unravel additional physiological functions of this transporter in rat organs.

Identification of a new urate and high affinity nicotinate transporter, hOAT10 (SLC22A13).

The orphan transporter hORCTL3 (human organic cation transporter like 3; SLC22A13) is highly expressed in kidneys and to a weaker extent in brain, heart, and intestine. hORCTL3-expressing Xenopus laevis oocytes showed uptake of [(3)H]nicotinate, [(3)H]p-aminohippurate, and [(14)C]urate. Hence, hORCTL3 is an organic anion transporter, and we renamed it hOAT10. [(3)H]Nicotinate transport by hOAT10 into X. laevis oocytes and into Caco-2 cells was saturable with Michaelis constants (K(m)) of 22 and 44 microm, respectively, suggesting that hOAT10 may be the molecular equivalent of the postulated high affinity nicotinate transporter in kidneys and intestine. The pH dependence of hOAT10 suggests p-aminohippurate(-)/OH(-), urate(-)/OH(-), and nicotinate(-)/OH(-) exchange as possible transport modes. Urate inhibited [(3)H]nicotinate transport by hOAT10 with an IC(50) value of 759 microm, assuming that hOAT10 represents a low affinity urate transporter. hOAT10-mediated [(14)C]urate uptake was elevated by an exchange with l -lactate, pyrazinoate, and nicotinate. Surprisingly, we have detected urate(-)/glutathione exchange by hOAT10, consistent with an involvement of hOAT10 in the renal glutathione cycle. Uricosurics, diuretics, and cyclosporine A showed substantial interactions with hOAT10, of which cyclosporine A enhanced [(14)C]urate uptake, providing the first molecular evidence for cyclosporine A-induced hyperuricemia.

Renal expression of organic anion transporter OAT2 in rats and mice is regulated by sex hormones.

The renal reabsorption and/or excretion of various organic anions is mediated by specific organic anion transporters (OATs). OAT2 (Slc22a7) has been identified in rat kidney, where its mRNA expression exhibits gender differences [females (F) > males (M)]. The exact localization of OAT2 protein in the mammalian kidney has not been reported. Here we studied the expression of OAT2 mRNA by RT-PCR and its protein by Western blotting (WB) and immunocytochemistry (IC) in kidneys of adult intact and gonadectomized M and F, sex hormone-treated castrated M, and prepubertal M and F rats, and the protein in adult M and F mice. In adult rats, the expression of OAT2 mRNA was predominant in the outer stripe (OS) tissue, exhibiting 1) gender dependency (F > M), 2) upregulation by castration and downregulation by ovariectomy, and 3) strong downregulation by testosterone and weak upregulation by estradiol and progesterone treatment. A polyclonal antibody against rat OAT2 on WB of isolated renal membranes labeled a approximately 66-kDa protein band that was stronger in F. By IC, the antibody exclusively stained brush border (BB) of the proximal tubule S3 segment (S3) in the OS and medullary rays (F > M). In variously treated rats, the pattern of 66-kDa band density in the OS membranes and the staining intensity of BB in S3 matched the mRNA expression. The expression of OAT2 protein in prepubertal rats was low and gender independent. In mice, the expression pattern largely resembled that in rats. Therefore, OAT2 in rat (and mouse) kidney is localized to the BB of S3, exhibiting gender differences (F > M) that appear in puberty and are caused by strong androgen inhibition and weak estrogen and progesterone stimulation.

Rat renal glucose transporter SGLT1 exhibits zonal distribution and androgen-dependent gender differences.

SGLT1 (SLC5A1) mediates a part of glucose and galactose reabsorption in the mammalian proximal tubule (PT), but the detailed localization of the transporter along the tubule is still disputable. Here, we used several methods to localize rat SGLT1 (rSGLT1) in the kidneys of intact and variously treated male (M) and female (F) rats. In immunoblots of isolated cortical (C) and outer stripe (OS) brush-border membranes (BBM), a peptide-specific polyclonal antibody for rSGLT1 labeled a sharp inzone-, and gender-dependent approximately 40-kDa protein and a broad approximately 75-kDa band that exhibited strong zonal (OS > C) and gender differences (F > M). In tissue cryosections, the antibody strongly stained BBM of the S3 PT segments in the OS and medullary rays (F > M) and smooth muscles of the blood vessels and renal capsule (F approximately M) and weakly stained the apical domain of other PT segments in the C (F approximately M). The phlorizin-sensitive uptake of d-[(3)H]galactose in BBM vesicles, as well as the tissue abundance of rSGLT1-specific mRNA, matched the immunoblotting data related to the 75-kDa protein and the immunostaining in S3, proving zonal and gender differences in the functional transporter. Ovariectomy had no effect, castration upregulated, whereas treatment of castrated rats with testosterone, but not with estradiol or progesterone, downregulated the 75-kDa protein and the immunostaining in S3. We conclude that in the rat kidney, the expression of SGLT1 is represented by a 75-kDa protein localized largely in the PT S3 segments, where it exhibits gender differences (F > M) at both the protein and mRNA levels that are caused by androgen inhibition.