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The aimof this study was to identify sex-dependent expression of renal transporter mRNA in lean and obese Zucker spontaneously hypertensive fatty (ZSF1) rats and to investigate the interaction of the most altered transporter, organic anion transporter 2 (Oat2), with diabetes-relevant metabolites and drugs. Higher incidence of glomerulosclerosis, tubulointerstitial fibrosis, and protein casts in Bowman’s space and tubular lumen was detected by PAS staining in obese male compared to female ZSF1 rats. Real-time PCR on RNA isolated fromkidney cortex revealed that Sglt1-2, Oat1-3, and Oct1 were higher expressed in kidneys of lean females. Oct2 and Mrp2 were higher expressed in obesemales. Renal mRNA levels of transporters were reduced with diabetic nephropathy in females and the expression of transcription factors Hnf1𝛽 andHnf4𝛼 in both sexes.Thehighest difference between lean and obese ZSF1 rats was found for Oat2. Therefore, we have tested the interaction of human OAT2 with various substances using tritium-labeled cGMP. Human OAT2 showed no interaction with diabetes-related metabolites, diabetic drugs, and ACE-inhibitors. However, OAT2- dependent uptake of cGMP was inhibited by furosemide. The strongly decreased expression of Oat2 and other transporters in female diabetic ZSF1 rats could possibly impair renal drug excretion, for example, of furosemide. Open-Access-Publikationsfonds 2015 peerReviewed

CFEX (chloride/formate exchanger ; Slc26a6) is an important anion exchanger of chloride, bicarbonate, oxalate (OX), formate, and hydroxyl ions in kidneys, liver, and the small intestine. Studies on CFEX-knockout mice indicated a possible role of CFEX in the development of hyperoxaluria and OX urolithiasis/nephrolithiasis, which in humans is more frequent in men. Here we studied the expression of the CFEX protein and mRNA in the organs of male and female rats, and employed a rat model of ethylene glycol (EG)-induced OX urolithiasis in order to correlate the expression of CFEX with sex-related hyperoxaluria. Rats drank EG in water (0.75 % vol/vol) or water (control) for 30 days. Tissue expressions of the CFEX protein and mRNA were analysed by immunochemical methods and qRT-PCR, respectively. The specificity of an anti-CFEX antibody, used in immunochemical studies, was confirmed in HEK293 cells transiently transfected with CFEX cDNA. In kidneys, the CFEX protein was immunolocalized to the proximal tubule brush-border membrane (BBM) with segmental (S3>>S1~S2) and sex (male>female) differences. Sex-unrelated expression was detected in the BBM of enterocytes (duodenum>jejunum) and in the hepatocyte canalicular membrane. In immunoblots, the CFEX protein band of ~120 kDa in various organs showed an expression pattern comparable to that in immunocytochemistry ; however, renal CFEX mRNA expression was not sex-dependent. Compared to controls and EG-treated females, the EG-treated male rats exhibited hyperoxalemia, hyperoxaluria and OX crystaluria, but the expression of CFEX mRNA and protein remained unaffected in the organs of both sexes. Thus, basic CFEX expression in both rat sexes was sufficient for OX handling even upon EG-treatment, indicating that in rats, CFEX plays no major role in generating EG-induced hyperoxaluria and nephrolithiasis.

In mice, the chloride-formate exchanger (CFEX, Slc26a6), also known as the putative anion transporter-1 (PAT-1), has been localized to the brush-border membrane (BBM) of renal proximal tubule (PT) and small intestine in mice. Its localization and protein expression in rat organs is unknown. Here, an anti-CFEX polyclonal antibody (CFEX-Ab) was used to investigate in Wistar rats the cell localization and abundance of CFEX protein in 1) kidneys, liver, gastrointestinal tract, and pancreas of adult intact males, 2) kidneys of adult male and female rats, and 3) kidneys of gonadectomised rats of both sexes. The studies were performed by immunocytochemistry in tissue cryosections and by Western blotting (WB) in isolated total cell membrane. In the kidneys, CFEX-Ab exclusively stained the BBM of PT with heterogeneous intensity (S1~S2>S3). In the liver, stained was the hepatocyte canalicular membrane, while the bile ducts were CFEX-negative. In the gastrointestinal tract, the antibody strongly stained the enterocyte BBM in duodenum and jejunum (duodenumjejunum) ; ileum, cecum, and colon remained unstained. In pancreas, CFEX protein was localized to the pancreatic duct apical membrane. A CFEX-related protein band of 100 kDa was detected in the membranes isolated from the above-mentioned organs, exhibiting zonal differences in the kidneys (cortexouter stripe) and gastrointestinal tract (duodenum>jejunum). Renal expression of CFEX protein was male- dominant, downregulated by castration, and unaffected by ovariectomy. We conclude that in rats, CFEX protein is expressed in the kidneys, liver, pancreas, and intestine, but only in the kidneys its expression exhibits stimulation by androgens.

Oxalate (Ox) urolithiasis is a systemic disorder that affects ~10 % of the adult human population with a male prevalence. It occurs ~2.5 times more often in men than in women. A trigger for urolithiasis is urine oversaturation with a crystal-forming material such as CaOx, where Ox originates from the liver metabolism and/or absorption from food. The Slc26 family of multifunctional anion exchangers, among which Sat-1 (Slc26a1) and chloride/formate exchanger CFEX (Slc26a6) are prime examples, is responsible for Ox trafficking in the organism. The aim of this work was to test the role of hepatic and renal Sat-1 in the generation of Ox urolithiasis in a rat model of this disease, following treatment with ethylene glycol (EG). Three-month-old male (M) and female (F) Wistar rats were divided into control (drank tap water) and EG-treated (drank 0.75 % EG in tap water for one month) groups. Urine and blood serums were sampled and tested for Ox crystals and concentration, while liver and kidney tissue samples were used in morphological, immunocytochemical, Western-blotting, and real-time RT-PCR studies. In EG-treated animals, M but not F exhibited hyperoxalemia, hyperoxaluria and large Ox crystals in their urine. However, the expression of Sat-1 protein was upregulated in both the liver and kidneys of EG treated F but not M. The expression of Sat-1 mRNA remained unchanged in all of the groups, indicating an involvement of a post- transcriptional regulation of protein expression. We conclude that Sat-1 does not contribute significantly to the generation of Ox urolithiasis in rats.

Question: The epidemiological incidence of nephrolithiasis is higher in men than in women. Most urinary stones consist of oxalate, which is largely synthesized in the liver and then released into the systemic circulation by the sulfate-oxalate exchanger, sat1. In the kidney proximal tubules, oxalate is excreted via a two-step process that includes uptake from the blood by the contraluminal sat1 and release into the urine by the luminal chloride-formate/oxalate exchanger, CFEX. The role of sat1 and CFEX in development of oxalate nephrolithiasis is unclear. Methodology: The ethylene glycol (EG) model of nephrolithiasis in male (M) and female (F) rats was used to induce oxaluric state, and to study the expression of sat1, CFEX, and rate-limiting enzymes of oxalate synthesis, alcohol dehydrogenase (Adh1) and hydroxyacid oxidase (Hao1), by real-time PCR and immunochemical methods. Result: As compared to controls, the EG-treated rats exhibited: (a) strong oxalosis and oxaluria (M>F), (b) increased abundance of sat1 protein, but not mRNA, in liver and kidneys of F rats only, (c) unchanged expression of CFEX mRNA, as well as (d) marginally changed expression of Adh1 and Hao1 mRNA in organs of both sexes. Conclusion: Despite hyperoxaluria, protein expression of sat1 in M and CFEX in both sexes was sufficient to handle the increased concentrations of oxalate induced by EG, indicating their limited role in oxalate nephrolithiasis.

Background: The epidemiological incidence of oxalate nephrolithiasis is higher in men than in women. Oxalate is predominantly produced in the liver and then released into the systemic circulation by sat1, a sulfate anion transporter, which is localized in the sinusoidal membrane of hepatocytes, where it mediates exit of oxalate in exchange for sulfate. In renal proximal tubules, sat1 is responsible for basolateral uptake of oxalate into the cell and release of sulfate into systemic circulation. Finally, oxalate is extruded into the urine by the luminal chloride-formate exchanger, CFEX, which also accepts oxalate as a substrate. Methods: The model of ethylene glycol (EG)-induced oxalate nephrolithiasis in adult male and female rats was used to monitor changes in sat1, CFEX, and the rate-limiting enzymes of oxalate synthesis, alcohol dehydrogenase (Adh1) and hydroxy acid oxidase (Hao1). Protein and mRNA expression was studied by immunochemistry and real time RT-PCR. Results: As compared to controls, EG-treated animals exhibited higher concentrations of oxalate in plasma and urine, and a higher abundance of oxalate crystals in urine with male dominance. Sat1 protein in liver and kidneys was male-dominant in controls, increased only in EG-treated female rats, while sat1 mRNA stayed unchanged. CFEX mRNA in both organs was sex-independent and unaffected by EG treatment. Adh1 and Hao1 mRNA in both organs exhibited distinct sex dependency which remained unchanged upon EG treatment. A general male-dominant Hao1 expression in kidneys of untreated rats was also shown by microarray analysis. Conclusions: In conclusion, despite hyperoxaluria in EG-treated animals, the expression of sat1 in males and of CFEX in both sexes was sufficient to handle the EG-induced production and secretion of oxalate.

INTRODUCTION. Sodium-independent sulfate anion transporter (sat-1 ; Slc26a1) plays a major role in transport of oxalate (OX) across the cell membrane by exchanging OX for sulfate or bicarbonate. Sat-1 mRNA has been detected strongly in liver and kidney and weakly in few other organs in rats, mice, and humans. By immunocytochemistry (IC), the protein has been localized in rats to the sinusoidal membrane of hepatocytes and basolateral membrane of proximal tubules (PT) in the kidney cortex, with the male (M)-dominant expression. In liver, which is a major OX producer, sat-1 mediates the extrusion of OX and uptake of sulfate, whereas in PT, it mediates the OX uptake, mainly in exchange for intracellular sulfate. In middle-aged humans, OX is a major cause of sex-related urolithiasis ; men excrete more OX and have higher incidence of OX stones then women. Accordingly, in studies of experimental urolithiasis in ethylene glycol (EG)-treated rats, testosterone stimulated, whereas female (F) sex hormones inhibited the urine excretion of OX and formation of OX stones. The role of OX transporters that in liver and kidneys exhibit the sex-related expression, such as sat-1, in the development of OX urolithiasis is not known. In the present study, we tested the expression of sat-1 at the protein and mRNA levels in the liver and kidneys of EG-treated rats. METHODS. Adult M and female (F) Wistar strain rats were treated with EG (0, 75% v/v in drinking water) for one month. Controls drunk water without EG. 24-h urine was collected in metabolic cages a day before sacrificing. OX in the blood plasma and urine were determined by ion-chromatography. Tissue morphology and OX crystals in the urine sediment were checked by light microscopy. Sat-1 protein expression was studied using a polyclonal antibody by IC in p-formaldehyde-fixed tissue cryosections, and by Western blotting of total cell membranes isolated from tissue homogenates. Tissue expression of sat-1 mRNA was studied by real time RT-PCR. RESULTS. In control M and F animals, the M-dominant sex differences in the urine excretion of OX, in the number and size of urine OX crystals, and in the expression of sat-1 protein (but not mRNA) in liver and kidneys, were confirmed. Compared to controls, the EG-treated animals exhibited: a) in plasma, 100% (F) and 4-fold (M) higher concentration of OX, b) in urine, 100% (F) and 17-fold (M) higher OX excretion, c) in urine sediment, similar and low abundance of small OX crystals in F, and increased abundance of large OX crystals in M, d) in tissues of both sexes, un-affected morphology in liver, and dilatated PT and distended peritubular spaces in kidneys, e) upregulated expression of sat-1 protein in the F liver and kidneys to the level in M organs, f) unchanged expression of sat-1 protein in the M liver and kidneys, and g) unchanged expression of sat-1 mRNA in both organs of F and M animals. CONCLUSION. Whereas the blood and urinary parameters in EG-treated rats indicate the low and high oxaluric state in F and M rats, respectively, the EG-induced elevated expression of sat-1 protein in the F liver and kidneys, and unchanged expression of this protein in M organs, indicate that sat-1 plays no significant role in generation of OX urolithiasis in EG-treated animals.

In the mammalian kidney, the first step in the elimination of endogenous and exogenous organic anions (OA) is the uptake of these anions across transporters that reside in the basolateral membrane (BLM) of epithelial cells in various nephron segments. In rodents, the active contributors to these processes are: a) primary-active Na/K-ATPase (EC 3.6.3.9), b) secondary-active sodium-dicarboxylate cotransporter 3 NaDC3 (Slc13a3), and c) tertiary-active OA exchangers Oat1 (Slc22a6) and Oat3 (Slc22a8). In order to investigate possible sex-related differences in cellular localization and protein expression of these transporters along the nephron, we have used specific polyclonal antibodies and studied the abundance of the above mentioned proteins by Western blotting (WB) in total cell membranes (TCM), isolated from the whole kidney homogenates, and by immunofluorescence cytochemistry (IC) in kidney cryosections from adult male (M) and female (F) mice. IC studies confirmed localization of these transporters in the BLM of various nephron segments: a) Na/K-ATPase ( -subunit) was stained with variable intensity in all nephron segments, b) NaDC3 was localized in the proximal tubule (PT ; S1S3 in the medullary rays>S3 in the outer stripe) and collecting duct (OMCDF). Finally, the staining intensity of Oat3 in various cortical tubules, as well as the abundance of its protein in isolated TCM, exhibited M-dominant sex differences. In conclusion, our results indicate that in the mouse kidney: a) the basolateral transporters of OA exhibit heterogeneous localization in epithelial cells along the nephron with largely M-dominant expression, and b) the expression of NaDC3 and Oats correlate well in the cortical PT, but not in the more distal nephron segments.

The sulfate anion transporter (sat-1, Slc26a1) has been cloned from rat liver, functionally characterized, and in rats localized to the basolateral cell membrane in renal proximal tubules and sinusoidal cell membrane in hepatocytes. In this work we confirm previously described localization in liver cells, and report on sex differences (SD) in sat-1 protein expression in the rat liver detected by immunocytochemistry in tissue cryosections, Western blotting, and transport studies in isolated total cell membranes. The sat-1-related ~85 kDa protein was localized exclusively to the hepatocyte sinusoidal membranes, where it showed a male (M)-dominant expression. In transport studies, isolated membrane vesicles from M liver exhibited a significantly higher uptake of radiolabeled sulfate in exchange for oxalate than the membranes from female (F) liver. However, the real-time RT-PCR data indicated an absence of SD at the level of sat-1 mRNA. In further studies, the expression of liver sat-1 protein was: (a) unaffected by castration, b) up-regulated by ovariectomy, and (c) down-regulated by estrogen- or progesterone-treatment in M. We conclude that SD (M>F) in the abundance of liver sat-1 transporter in rats are caused by the F sex hormones-driven inhibition of protein expression at the post-transcriptional level. A higher expression of sat-1 protein in the hepatocytes of M rats may conform to elevated uptake of sulfate and extrusion of oxalate. The extruded oxalate, being secreted by the renal proximal tubules, may contribute to the development of oxalate urolithiasis, which occurs more often in M.

It is assumed that the Na+-independent transport of sulfate (S) in the mammalian gastrointestinal tract (GIT) is like in the renal proximal tubule, e.g., mediated by CFEX/slc26a6 and sat-1/slc26a1 across the apical and serosal membrane, respectively. However, the exact characteristics of S transport and localization of these proteins in the GIT epithelium are not known. In this work we performed uptake studies in sat-1-expressing oocytes, to define the sat-1 functional properties, and PCR, Western blotting (WB) and immunocytochemical (IC) studies in the rat GIT, to reveal its expression in the epithelium. In oocytes, the transporter acted as an exchanger for S, bicarbonate, and oxalate with apparent affinities of 0.36+/-0.16, 3.5+/-2.0, and 0.049+/-0.026 mM, respectively. Glycolate and glyoxylate trans-stimulated the S uptake from 81+/-12 to 201+/-14 and 117+/-7 pmol/h/oocyte. By PCR, sat-1 mRNA was present in all parts of the GIT. In WB of isolated GIT cell membranes the anti-sat-1 antibody labeled a major ~80 kDa protein band, whereas IC studies revealed a granular pattern of intracellular staining in oxyntic cells of the stomach and in enterocytes of the small and large intestine. Endosomal localization of sat-1 was disproven. In rat GIT, sat-1 localization in peroxisomes is discussed.

The Na+-independent sulfate (S) transporter Sat-1 has been localized to the basolateral membrane (BLM) of proximal tubules in the rat kidney (K). The same transporter may also mediate S transport in the gastrointestinal tract (GIT) and liver (L), but its presence and the exact cellular localization in these organs has not been described. In this work we used a monoclonal antibody to Sat-1 protein (Sat-1-Ab) and performed localization studies by immunofluorescence cytochemistry (IC) and Western blotting (WB) in the L and in various GIT segments in rats. The Sat-1-Ab was verified in the K by achieving a positive staining of BLM in the proximal tubule S1 and S2 segments, and by labeling a single, ~85 kDa protein band in BLM isolated from renal cortical homogenate. In the L, the Sat-1-Ab strongly stained the sinusoidal membrane ; the canalicular membrane and other structures in hepatocytes remained unstained. However, the double staining in various GIT segments with the Sat-1-Ab and a polyclonal antibody to cell adhesion molecule 105 (CAM-105) indicated the localization of Sat-1 in an intracellular vesicular compartment. In the tested GIT segments, the intensity of intracellular Sat-1 staining showed the following pattern: esophagus < duodenum < jejunum < ileum < colon < stomach. The localization of Sat-1 in endocytic vesicles was disproven by an in vivo assay of FITC-dextran uptake in the colon cells, whereas the double-staining for Sat-1 and the mitochondrial marker cytochrome C in the colon and stomach revealed a complete colocalization of these proteins in mitochondria of enterocytes (colon) and oxyntic cells (stomach). However, in WB of total cell membranes, isolated from the L and various GIT segments, the Sat-1-Ab labeled weakly the ~40 kDa protein band, whereas the K-specific 85 kDa protein band was not labeled at all in these membranes. We conclude that in the rat L and GIT cells, the Sat-1 protein is different from that in the K and may exist as a shorter (truncated) isoform. In various GIT cells, the transporter may be localized in mitochondria.