Protein localization of SLC26A2 (DTDST) in rat kidney

The SLC26 family represents a group of integral membrane anion transport proteins. Mutations in one member of this protein family, SLC26A2 (DTDST or diastrophic dysplasia sulfate transporter), result in various chondrodysplasias due to undersulfation of proteoglycans in chondrocytes, a major site of DTDST protein expression. DTDST mRNA has been detected in the kidney, but protein expression has not been characterized. Our objective for this study was to determine the protein localization of this sulfate transporter in the kidney. We used immunofluorescence (IMF) techniques with an anti-DTDST monoclonal antibody to examine kidneys harvested from adult rats. Double labeling was performed with antibodies directed against megalin, which is found in the microvillus membrane and coated pits of the proximal tubule. IMF analysis indicated that DTDST protein expression was limited to the microvillus membrane of proximal tubule cells in the renal cortex but absent in glomeruli and other nephron segments. DTDST was also detected in isolated rat kidney proximal tubule microvillus membranes by Western blot analysis, confirming the immunofluorescent localization of the DTDST transporter to this nephron segment. The functional role of the DTDST protein in the kidney is unknown, but it may play a role in proximal tubule sulfate transport.     

The congenital chloride diarrhea gene is expressed in seminal vesicle, sweat gland, inflammatory colon epithelium, and in some dysplastic colon cells

Congenital chloride diarrhea (CLD) is an autosomal recessive disorder of intestinal electrolyte transportation caused by mutations in the anion transporter protein encoded by the down-regulated in adenoma (DRA), or CLD, gene. In this study, in situ hybridization and immunohistochemistry were performed to investigate the expression of CLD in extraintestinal normal epithelia and in intestinal inflammatory and neoplastic epithelia. The expression of the closely related anion transporter diastrophic dysplasia sulfate transporter, DTDST, was also examined and compared with that of CLD in colon. The only extraintestinal tissues showing CLD expression were eccrine sweat glands and seminal vesicles. In inflammatory bowel disease and ischemic colitis, expression of CLD mRNA in colon epithelium was similar to histologically normal colon epithelium, but the protein was found deeper in crypts, including proliferative epithelial cells. In intestinal tumors, the expression pattern of CLD was dependent on the differentiation status of the tissue studied: epithelial polyps with no or minor dysplasia showed abundant expression, whereas adenocarcinomas were negative. The DTDST gene was abundantly expressed in the upper crypt epithelium of colonic mucosa.     

Functional characterization of three novel tissue-specific anion exchangers SLC26A7, -A8, and -A9

A second distinct family of anion exchangers, SLC26, in addition to the classical SLC4 (or anion exchanger) family, has recently been delineated. Particular interest in this gene family is stimulated by the fact that the SLC26A2, SLC26A3, and SLC26A4 genes have been recognized as the disease genes mutated in diastrophic dysplasia, congenital chloride diarrhea, and Pendred syndrome, respectively. We report the expansion of the SLC26 gene family by characterizing three novel tissue-specific members, named SLC26A7, SLC26A8, and SLC26A9, on chromosomes 8, 6, and 1, respectively. The SLC26A7-A9 proteins are structurally very similar at the amino acid level to the previous family members and show tissue-specific expression in kidney, testis, and lung, respectively. More detailed characterization by immunohistochemistry and/or in situ hybridization localized SLC26A7 to distal segments of nephrons, SLC26A8 to developing spermatocytes, and SLC26A9 to the lumenal side of the bronchiolar and alveolar epithelium of lung. Expression of SLC26A7-A9 proteins in Xenopus oocytes demonstrated chloride, sulfate, and oxalate transport activity, suggesting that they encode functional anion exchangers. The functional characterization of the novel tissue-specific members may provide new insights to anion transport physiology in different parts of body.     

Nucleotide-sugar transporter SLC35D1 is critical to chondroitin sulfate synthesis in cartilage and skeletal development in mouse and human

Proteoglycans are a family of extracellular macromolecules comprised of glycosaminoglycan chains of a repeated disaccharide linked to a central core protein. Proteoglycans have critical roles in chondrogenesis and skeletal development. The glycosaminoglycan chains found in cartilage proteoglycans are primarily composed of chondroitin sulfate. The integrity of chondroitin sulfate chains is important to cartilage proteoglycan function; however, chondroitin sulfate metabolism in mammals remains poorly understood. The solute carrier-35 D1 (SLC35D1) gene (SLC35D1) encodes an endoplasmic reticulum nucleotide-sugar transporter (NST) that might transport substrates needed for chondroitin sulfate biosynthesis. Here we created Slc35d1-deficient mice that develop a lethal form of skeletal dysplasia with severe shortening of limbs and facial structures. Epiphyseal cartilage in homozygous mutant mice showed a decreased proliferating zone with round chondrocytes, scarce matrices and reduced proteoglycan aggregates. These mice had short, sparse chondroitin sulfate chains caused by a defect in chondroitin sulfate biosynthesis. We also identified that loss-of-function mutations in human SLC35D1 cause Schneckenbecken dysplasia, a severe skeletal dysplasia. Our findings highlight the crucial role of NSTs in proteoglycan function and cartilage metabolism, thus revealing a new paradigm for skeletal disease and glycobiology.     

Molecular cloning of SLC26A7, a novel member of the SLC26 sulfate/anion transporter family, from high endothelial venules and kidney

A unique characteristic of endothelial cells from high endothelial venules (HEVEC) in lymphoid organs and chronically inflamed tissues is their capacity to incorporate large amounts of sulfate into sialomucin-type counter-receptors for the lymphocyte homing receptor L-selectin. We have previously shown that HEVEC express two functional classes of sulfate transporters: sodium/sulfate cotransporters and sulfate/anion exchangers. Here, we report the molecular cloning from human HEVEC of a 2.9-kb cDNA encoding SLC26A7, a novel member of the SLC26 (solute carrier 26) sulfate/anion exchanger family. SLC26A7 exhibits 30% identity with three known sulfate transporters from the SLC26 family: SLC26A2 (also known as DTDST), SLC26A1 (also known as SAT1), and SLC26A3 (also known as DRA). Northern blot analysis revealed specific expression of SLC26A7 mRNA in kidney. Alternative splicing and polyadenylation of SLC26A7 pre-mRNA in kidney suggest the existence of two protein isoforms, SLC26A7.1 and SLC26A7.2, differing in their carboxy termini.     

Phenotypic and genotypic overlap between atelosteogenesis type 2 and diastrophic dysplasia

Mutations in the diastrophic dysplasia sulfate transporter gene DTDST have been associated with a family of chondrodysplasias that comprises, in order of increasing severity, diastrophic dysplasia (DTD), atelosteogenesis type 2 (AO2), and achondrogenesis type 1B (ACG1B). To learn more about the molecular basis of DTDST chondrodysplasias and about genotype-phenotype correlations, we studied fibroblast cultures of three new patients: one with AO-2, one with DTD, and one with an intermediate phenotype (AO2/DTD). Reduced incorporation of inorganic sulfate into macromolecules was found in all three. Each of the three patients was found to be heterozygous for a c862t transition predicting a R279W substitution in the third extracellular loop of DTDST. In two patients (DTD and AO2/DTD), no other structural mutation was found, but polymerase chain reaction amplification and single-strand conformation polymorphism analysis of fibroblast cDNA showed reduced mRNA levels of the wild-type DTDST allele: these two patients may be compound heterozygotes for the “Finnish” mutation (as yet uncharacterized at the DNA level), which causes reduced expression of DTDST. The third patient (with AO2) had the R279W mutation compounded with a novel mutation, the deletion of cytosine 418 (Δc418), predicting a frameshift with premature termination. Also the Δc418 allele was underrepresented in the cDNA, in accordance with previous observations that premature stop codons reduce mRNA levels. The presence of the DTDST R279W mutation in a total of 11 patients with AO2 or DTD emphasizes the overlap between these conditions. This mutation has not been found so far in 8 analyzed ACG1B patients, suggesting that it allows some residual activity of the sulfate transporter. Received: 14 June 1996 / Revised: 8 August 1996     

Atelosteogenesis type II is caused by mutations in the diastrophic dysplasia sulfate-transporter gene (DTDST): evidence for a phenotypic series involving three chondrodysplasias.

Atelosteogenesis type II (AO II) is a neonatally lethal chondrodysplasia whose clinical and histological characteristics resemble those of another chondrodysplasia, the much less severe diastrophic dysplasia (DTD). The similarity suggests a shared pathogenesis involving lesions in the same biochemical pathway and perhaps the same gene. DTD is caused by mutations in the recently identified diastrophic dysplasia sulfate-transporter gene (DTDST). Here, we report that AOII patients also have DTDST mutations, which lead to defective uptake of inorganic sulfate and insufficient sulfation of macromolecules by patient mesenchymal cells in vitro. Together with our recent observation that a third even more severe chondrodysplasia, achondrogenesis type IB, is also caused by mutations in DTDST, these results demonstrate a phenotypic series of three chondrodysplasias of increasing severity caused by lesions in a single sulfate-transporter gene. The severity of the phenotype appears to be correlated with the predicted effect of the mutations on the residual activity of the DTDST protein.     

SLC26A9 is expressed in gastric surface epithelial cells, mediates Cl-/HCO3- exchange, and is inhibited by NH4+

HCO3- secretion by gastric mucous cells is essential for protection against acidic injury and peptic ulcer. Herein we report the identification of an apical HCO3- transporter in gastric surface epithelial cells. Northern hybridization and RT-PCR demonstrate the expression of this transporter, also known as SLC26A9, in mouse and rat stomach and trachea (but not kidney). In situ hybridization in mouse stomach showed abundant expression of SLC26A9 in surface epithelial cells with apical localization on immunofluorescence labeling. Functional studies in HEK-293 cells demonstrated that SLC26A9 mediates Cl-/HCO3- exchange and is also capable of Cl--independent HCO3- extrusion. Unlike other anion exchangers or transport proteins reported to date, SLC26A9 activity is inhibited by ammonium (NH4+). The inhibitory effect of NH4+ on gastric HCO3- secretion was also indicated by reduced gastric juxtamucosal pH (pHjm) in rat stomach in vivo. This report is the first to describe the inhibition of HCO3- transport in vitro and the reduction of pHjm in stomach in vivo by NH4+. Given its critical localization on the apical membrane of surface epithelial cells, its ability to transport HCO3-, and its inhibition by NH4+, we propose that SLC26A9 mediates HCO3- secretion in surface epithelial cells and is essential for protection against acidic injury in the stomach. Disease states that are associated with increased ammonia (NH3)/NH4+ generation (e.g., Helicobacter pylori) may impair gastric HCO3- secretion and therefore predispose patients to peptic ulcer by inhibiting SLC26A9.     

The cyanobacterial bicarbonate transporter BicA: its physiological role and the implications of structural similarities with human SLC26 transporters

The cyanobacterial Na+-dependent HCO3- transporter BicA is a member of the ubiquitous and important SulP/SLC26 family of anion transporters found in eukaryotes and prokaryotes. BicA is an important component of the cyanobacterial CO2 concentrating mechanism, an adaptation that contributes to cyanobacteria being able to achieve an estimated 25% of global primary productivity, largely in the oceans. The human SLC26 members are involved in a range of key cellular functions involving a diverse range of anion transport activities including Cl-/HCO3-, I-/HCO3-, and SO42-/HCO3- exchange; mutations in SLC26 members are known to be associated with debilitating diseases such as Pendred syndrome, chondrodysplasias, and congenital chloride diarrhoea. We have recently experimentally determined the membrane topology of BicA using the phoA-lacZ reporter system and here consider some of the extrapolated implications for topology of the human SLC26 family and the Sultr plant sulphate transporters.     

Matrix disruptions, growth, and degradation of cartilage with impaired sulfation

Diastrophic dysplasia (DTD) is an incurable recessive chondrodysplasia caused by mutations in the SLC26A2 transporter responsible for sulfate uptake by chondrocytes. The mutations cause undersulfation of glycosaminoglycans in cartilage. Studies of dtd mice with a knock-in Slc26a2 mutation showed an unusual progression of the disorder: net undersulfation is mild and normalizing with age, but the articular cartilage degrades with age and bones develop abnormally. To understand underlying mechanisms, we studied newborn dtd mice. We developed, verified and used high-definition infrared hyperspectral imaging of cartilage sections at physiological conditions, to quantify collagen and its orientation, noncollagenous proteins, and chondroitin chains, and their sulfation with 6-μm spatial resolution and without labeling. We found that chondroitin sulfation across the proximal femur cartilage varied dramatically in dtd, but not in the wild type. Corresponding undersulfation of dtd was mild in most regions, but strong in narrow articular and growth plate regions crucial for bone development. This undersulfation correlated with the chondroitin synthesis rate measured via radioactive sulfate incorporation, explaining the sulfation normalization with age. Collagen orientation was reduced, and the reduction correlated with chondroitin undersulfation. Such disorientation involved the layer of collagen covering the articular surface and protecting cartilage from degradation. Malformation of this layer may contribute to the degradation progression with age and to collagen and proteoglycan depletion from the articular region, which we observed in mice already at birth. The results provide clues to in vivo sulfation, DTD treatment, and cartilage growth.     

Functional role of specific amino acid residues in human thiamine transporter SLC19A2: mutational analysis

SLC19A2 is a membrane thiamine transporter expressed in a variety of human tissues, including the gastrointestinal tract. Little is currently known about the structure/function relationship of SLC19A2. We examined the effect of introducing mutations in SLC19A2 identical to those found in thiamine-responsive megaloblastic anemia syndrome (TRMA), on functional activity and membrane expression of the transporter. We also examined the effect of mutating the only conserved anionic residue (E138) in the transmembrane (TM) domains of the SLC19A2 and that of the putative glycosylation sites (N63, N314). Northern blot analysis showed SLC19A2 mRNA was expressed at the same level in HeLa cells transfected with wild-type or mutated SLC19A2. Introducing the clinically relevant mutations (D93H, S143F, G172D) or mutation at the conserved anionic residue (E138A) of SLC19A2 led to a significant (P < 0.01) inhibition of thiamine uptake. Mutations of the two potential N-linked glycosylation sites (N63Q, N314Q) of SLC19A2 did not affect functional activity; they did, however, lead to a noticeable reduction in apparent molecular weight of protein. Western blot analysis showed all proteins (except D93H) were expressed in the membrane (not the cytoplasmic) fraction of HeLa cells. These results provide direct confirmation that clinically relevant mutations in SLC19A2 observed in TRMA cause malfunctioning of the transporter and/or a defect in its translation/stability. Results also show conserved TM anionic residue of the SLC19A2 protein is critical for its function. Furthermore, native SLC19A2 is glycosylated, but this is not important for its function.     

In vivo contribution of amino acid sulfur to cartilage proteoglycan sulfation

Cytoplasmic sulfate for sulfation reactions may be derived either from extracellular fluids or from catabolism of sulfur-containing amino acids and other thiols. In vitro studies have pointed out the potential relevance of sulfur-containing amino acids as sources for sulfation when extracellular sulfate concentration is low or when its transport is impaired such as in DTDST [DTD (diastrophic dysplasia) sulfate transporter] chondrodysplasias. In the present study, we have considered the contribution of cysteine and cysteine derivatives to in vivo macromolecular sulfation of cartilage by using the mouse model of DTD we have recently generated [Forlino, Piazza, Tiveron, Della Torre, Tatangelo, Bonafe, Gualeni, Romano, Pecora, Superti-Furga et al. (2005) Hum. Mol. Genet. 14, 859-871]. By intraperitoneal injection of [35S]cysteine in wild-type and mutant mice and determination of the specific activity of the chondroitin 4-sulfated disaccharide in cartilage, we demonstrated that the pathway by which sulfate is recruited from the intracellular oxidation of thiols is active in vivo. To check whether cysteine derivatives play a role, sulfation of cartilage proteoglycans was measured after treatment for 1 week of newborn mutant and wild-type mice with hypodermic NAC (N-acetyl-L-cysteine). The relative amount of sulfated disaccharides increased in mutant mice treated with NAC compared with the placebo group, indicating an increase in proteoglycan sulfation due to NAC catabolism, although pharmacokinetic studies demonstrated that the drug was rapidly removed from the bloodstream. In conclusion, cysteine contribution to cartilage proteoglycan sulfation in vivo is minimal under physiological conditions even if extracellular sulfate availability is low; however, the contribution of thiols to sulfation becomes significant by increasing their plasma concentration.     

Characterization and expression pattern analysis of the facilitative glucose transporter 10 gene (slc2a10) in Danio rerio

The SLC2A10 gene located on chromosome 20q13.1 encodes the facilitative glucose transporter 10 (GLUT10), a class III member of the SLC2A facilitative glucose transporter family. Mutations in the human SLC2A10 gene cause arterial tortuosity syndrome (ATS), a rare autosomal recessive connective tissue disorder. In this work, we report the characterization of the slc2a10 ortholog gene in zebrafish (Danio rerio) and its expression pattern during embryonic development and in adult tissues. The slc2a10 gene consists of 5 exons, spanning 8 kb and mapping to a region on chromosome 11 that exhibits conserved synteny with human chromosome 20. The gene encodes Glut10, a 513 amino acid protein that maintains the 12 transmembrane domain structure typical of the GLUTs family, and shares the specific functional motifs involved in sugar transport with the vertebrate GLUT10. RT-PCR analysis showed that two specific splice variants, both including the 5’-UTR region, were expressed during embryogenesis and in different adult zebrafish tissues and organs. In situ hybridization analyses demonstrated a maternal origin of the total slc2a10 mRNA and its ubiquitous distribution until the early somitogenesis stage. In later embryonic stages, slc2a10 mRNA was detected in the otic vesicles, hatching gland cells, pectoral fin, posterior tectum and swim bladder. Overall, these results suggest a wide role of slc2a10 during zebrafish development.     

Enhanced expression of TGF-beta and c-fos mRNAs in the growth plates of developing human long bones

The expression of mRNAs for type I and type II procollagens, transforming growth factor-beta (TGF-beta) and c-fos was studied in developing human long bones by Northern blotting and in situ hybridization. The cells producing bone and cartilage matrix were identified by hybridizations using cDNA probes for types I and II collagen, respectively. Northern blotting revealed that the highest levels of TGF-beta mRNA were associated with the growth plates. By in situ hybridization, this mRNA was localized predominantly in the osteoblasts and osteoclasts of the developing bone, in periosteal fibroblasts and in individual bone marrow cells. These findings are consistent with the view that TGF-beta may have a role in stimulation of type I collagen production and bone formation. Only a low level of TGF-beta mRNA was detected in cartilage where type II collagen mRNA is abundant. In Northern hybridization, the highest levels of c-fos mRNA were detected in epiphyseal cartilage. In situ hybridization revealed two cell types with high levels of c-fos expression: the chondrocytes bordering the joint space and the osteoclasts of developing bone. These differential expression patterns suggest specific roles for TGF-beta and c-fos in osseochondral development.     

The chicken alpha 1 (XI) collagen gene is widely expressed in embryonic tissues.

Complementary DNA and genomic DNA clones corresponding to the chicken alpha 1 (XI) collagen gene were isolated and characterized. These recombinant DNA clones covered 2667 base pairs of the mRNA and encode 624 amino acids of the triple helical region plus the entire carboxyl-terminal propeptide. Northern blot analysis showed a major band of approximately 6.5 kilobases and a minor band of approximately 7.5 kilobases. A combination of Northern blot and in situ hybridization analyses showed that, in addition to its presence in cartilage, this mRNA also is present in a wide variety of chicken noncartilaginous embryonic tissues including brain, heart, skeletal muscle, calvaria, and skin, but was not detected in liver. Type II collagen mRNA has also been detected at low levels in these same tissues. Also, similar to the mRNA for the alpha 1 chain for type II collagen, the alpha 1 (XI) collagen mRNA is detected in limb mesenchyme prior to condensation and differentiation of the core mesenchyme into cartilage.     

Identification, characterization and cloning of SLC6A8C, a novel splice variant of the creatine transporter gene

SLC6A8 deficiency is caused by mutations in the X-linked creatine transporter gene (SLC6A8), which leads to cerebral creatine deficiency, mental retardation, speech and language delay, autistic-like behaviour and epilepsy. Insight in the mechanism of how the transporter is regulated is largely unknown and it is of importance for the development of successful treatment strategies of cerebral creatine deficient syndromes. Our goal was to characterize CRT2 (SLC6A8B), a published splice variant of the creatine transporter. Surprisingly, using RT-PCR we found a novel splice variant, SLC6A8C, which is predominantly found in human tissues with a high energy requirement such as brain, kidney, heart, small intestines and skeletal muscle, where SLC6A8 transporter is most required. The 5' untranslated region (UTR) of the SLC6A8C mRNA was identified using the Smart Race cDNA amplification kit. The SLC6A8C mRNA contains intron 4 and exons 5 through 13 of SLC6A8, including part of the 3' UTR. An open reading frame was found, which predicts a truncated protein identical to the SLC6A8 transporter, comprising the five last C-terminal transmembrane domains of the SLC6A8 transporter. SLC6A8C open reading frame was cloned as a fusion protein with EGFP and the SLC6A8C protein expression was detected by Western Blot. RT-PCR and sequence analysis showed that this splice variant is conserved in evolution, since we also detected it in mouse. This study reveals the presence of a novel SLC6A8 splice variant, SLC6A8C in human and mouse.     

Requirements for sulfate transport and the diastrophic dysplasia sulfate transporter in fibronectin matrix assembly

Diastrophic dysplasia sulfate transporter (DTDST) is a sulfate/chloride antiporter whose function is impaired in several human chondrodysplasias. We show that DTDST is upregulated by dexamethasone stimulation of HT1080 fibrosarcoma cells and is required for fibronectin (FN) extracellular matrix deposition by these cells. DTDST imports sulfate for the modification of glycosaminoglycans. We find that N-sulfation of these chains is important for FN matrix assembly and that sulfation of cell surface proteoglycans is reduced in the absence of DTDST. Of the candidate HT1080 cell surface proteoglycans, only loss of syndecan-2 compromises FN assembly, as shown by syndecan-2 small interfering RNA knockdown. DTDST is both necessary and sufficient to induce FN matrix assembly in HT1080 cells. Knockdown of DTDST ablates FN matrix, whereas its overexpression increases assembly without dexamethasone stimulation. These results identify a previously unrecognized regulatory pathway for matrix assembly via modulation of a sulfate transporter and proteoglycan sulfation. These data raise the possibility that FN assembly defects contribute to chondrodysplasias.     

Molecular cloning, expression, and chromosomal mapping of human chondroitin 4-sulfotransferase, whose expression pattern in human tissues is different from that of chondroitin 6-sulfotransferase

Chondroitin 4-sulfotransferase (C4ST) catalyzes the transfer of sulfate from 3'-phosphoadenosine 5'-phosphosulfate to position 4 of the N-acetylgalactosamine residues of chondroitin. We previously reported the cloning of C4ST cDNA from mouse brain. We here report the cloning and expression of human C4ST cDNA. The cDNA was isolated from a human fetal brain cDNA library by hybridization with a DNA probe prepared from rat poly(A)(+) RNA used for the cloning of mouse C4ST cDNA. The cDNA comprises a single open reading frame that predicts a Type II transmembrane protein composed of 352 amino acids. The protein has an amino acid sequence homology of 96% with mouse C4ST. When the cDNA was introduced into a eukaryotic expression vector and transfected in COS-7 cells, the sulfotransferase activity that transfers sulfate to both chondroitin and desulfated dermatan sulfate was overexpressed. Northern blot analysis indicated that human C4ST mRNAs (6.0 and 1.9 kb) are expressed ubiquitously in various adult human tissues. Dot blot analysis has shown that human C4ST is strongly expressed in colorectal adenocarcinoma and peripheral blood leukocytes, whereas strong expression of human chondroitin 6-sulfotransferase (C6ST) is observed in aorta and testis. These observations suggest that the expression of C4ST and C6ST may be controlled differently in human tissues. The C4ST gene was localized to chromosome 12q23.2-q23.3 by fluorescence in situ hybridization.     

Cloning of rainbow trout SLC26A1: involvement in renal sulfate secretion

The kidney plays an important role in ion regulation in both freshwater and seawater fish. However, ion transport mechanisms in the teleost kidney are poorly understood, especially at the molecular level. We have cloned a kidney-specific SLC26 sulfate/anion exchanger from rainbow trout (Oncorhynchus mykiss) that is homologous to the mammalian SLC26A1 (Sat-1). Excretion of excess plasma sulfate concentration after Na2SO4 injection corresponded to significantly higher expression of the cloned SLC26A1 mRNA. Detailed morphological observation of rainbow trout renal tubules was also performed by light microscopy and transmission electron microscopy. According to the structure of brush border and tubular system in the cytoplasm, renal tubules of rainbow trout were classified into proximal tubule first and second (PI and PII) segments and distal tubules. In situ hybridization revealed that SLC26A1 anion exchanger mRNA is specifically localized in the PI segment of kidneys from both seawater- and freshwater-adapted rainbow trout. With immunocytochemistry, Na+-K+-ATPase and vacuolar-type H+-ATPase were colocalized to the same cells and distributed in the basolateral and the apical membranes, respectively, of the cells where the SLC26A1 mRNA expressed. These findings suggest that the cloned kidney-specific SLC26A1 is located in kidney proximal tubules and is involved in excretion of excess plasma sulfate in rainbow trout.