Marine teleosts actively excrete SO42– and keep the plasma concentration of this ion much lower than that of environmental seawater (SW). We used the eel as a model to study the excretory mechanism of SO42– because this euryhaline species changes SO42– regulation drastically after transfer from freshwater (FW) to SW. Time-course studies showed that plasma SO42– concentration decreased 3 days after transfer of eels from FW to SW, while urine SO42– concentration increased on 1 day. Detailed analyses showed that urine SO42– concentration increased linearly from 6 h after SW transfer; however, this did not immediately translate to increased SO42– excretion because the volume of urine was decreased. We identified five SO42– transporters in the eel kidney. Three of these (Slc26a1, Slc26a6b and Slc26a6c) are expressed in both SW- and FW-acclimated eels while Slc26a6a and Slc13a1 are expressed in SW-acclimated eels and FW-acclimated eels, respectively. We showed that changes in Slc26a6a and Slc13a1 gene expression occurred 1–3 days after SW transfer. In SW eel kidneys, immunohistochemistry using specific antisera against each transporter protein showed that Slc26a6a and Slc26a6c are localized on the apical membrane of the P1 segment of the proximal tubule, while Slc26a6b is localized on the apical membrane and Slc26a1 on the basolateral membrane of the P2 segment. The current study revealed complex molecular mechanisms of SO42– excretion in the SW eel kidney that involve segment-specific localization of multiple Slc transporters in proximal tubules and modulation of their expression in different SO42– environments. This precise regulatory mechanism may endow the eel with euryhalinity.
The SO42– content of seawater (SW) is >40-fold higher than the SO42– concentration in the plasma of marine teleosts. Thus, marine teleosts are continuously exposed to a risk of excess SO42–, which enters the gills and the digestive tracts from the ingested SW. To avoid hypersulfatemia, teleosts actively excrete SO42–, together with Mg2+ and Ca2+, primarily via the kidney (Berglund and Forster, 1958; Hickman, 1968; Marshall and Grosell, 2006). The urine of marine teleosts is isotonic to their plasma but contains concentrations of SO42–, Mg2+ and Ca2+ that are higher than those of SW. Renal SO42– excretion is essential for SO42– homeostasis in marine teleosts (Renfro, 1999), as shown in the flounder (Beyenbach et al., 1986; Dickman and Renfro, 1986; Pelis and Renfro, 2004; Renfro et al., 1999; Renfro and Pritchard, 1982; Renfro and Pritchard, 1983), goosefish (Berglund and Forster, 1958) and killifish (Cliff and Beyenbach, 1992).
In humans, plasma SO42– concentration has been used as a marker of renal failure in conditions such as uremia (Holmes et al., 1960). Because of increasing clinical significance in renal failure, the mammalian kidney has been extensively studied in terms of SO42– regulation as well as the regulation of other ions (Berglund and Sorbo, 1960). The SO42–-retaining system is dominant in mammals to enable them to survive in the SO42–-deficient, terrestrial environment. However, the filtered load of SO42– exceeds the capacity of maximal tubular reabsorption and the excess is excreted into the urine (Mudge et al., 1973; Lin and Levy, 1983). Recently, several SO42– transporters have been identified in mammals, and all belong to the solute carrier (Slc) superfamily, which consists of 47 families (He et al., 2009). Two SO42– transporters, named NaSi-1 (Slc13a1) and Sat-1 (Slc26a1), have been well studied and are suggested to be involved in SO42– reabsorption. Slc13a1 is known to be a Na+/SO42– cotransporter located on the apical or brush border membrane of the epithelial cells lining the renal proximal tubule and the small and large intestine of mammals (Besseghir and Roch-Ramel, 1987; Silberg et al., 1995; Byeon et al., 1998; Markovich, 2001). Slc26a1 is known to be a multifunctional anion exchanger located on the basolateral membrane of epithelial cells that have Slc13a1 on the apical side (Bissig et al., 1994; Karniski et al., 1998; Krick et al., 2009). Slc26a6 has also been localized to the apical membrane of the renal proximal tubule in mammals, which exchanges several anions: oxalate/SO42–, Cl–/formate, Cl–/oxalate, oxalate/formate, oxalate/oxalate, Cl–/HCO3– and Cl–/OH– (Knauf et al., 2001; Jiang et al., 2002; Xie et al., 2002; Thomson et al., 2005).
The renal proximal tubule has been suggested as an important site for SO42– regulation in marine teleosts (Renfro and Pritchard, 1983; Dickman and Renfro, 1986; Cliff and Beyenbach, 1992). Though the information about SO42– transporters in marine teleosts is limited, Katoh and colleagues identified Slc26a1 in the renal proximal tubule of SW-acclimated rainbow trout (Katoh et al., 2006). More recently, Kato and coworkers identified Slc26a6a in the renal proximal tubule of pufferfish (Takifugu obscurus) and showed that the expressed protein transports SO42– (Kato et al., 2009). In freshwater (FW) fish, Nakada and colleagues showed that Slc26a1 and Slc13a1 are located in the proximal tubule of the eel kidney and play important roles in renal SO42– reabsorption (Nakada et al., 2005).
We recently showed that the kidney is a major site of SO42– excretion in SW eels using 35SO42– as a tracer (T.W. and Y.T., unpublished). We also found that SO42– regulation changes dramatically in eels after transfer from FW to SW, more so than in other euryhaline teleosts (Mozambique tilapia and chum salmon), a finding previously noted by Nakada and colleagues (Nakada et al., 2005). In this paper, therefore, we used the eel as an experimental animal and examined the time course of changes in renal SO42– excretion after transfer of eels from FW to SW. We then attempted to identify the transporters responsible for the regulation of SO42– excretion and examined their gene expression in the kidney after SW transfer. Finally, in an attempt to delineate the whole regulatory system of SO42– excretion in the SW eel kidney, SO42– transporters were localized using specific antisera raised against the transporter proteins.
MATERIALS AND METHODS
Japanese eels, Anguilla japonica (Temminck and Schlegel 1847), weighing 192±5 g (N=45), were purchased from a local dealer. They were kept in a FW tank at 18°C for at least 2 weeks before use (FW eel). Some eels were transferred to a SW tank and acclimated at 18°C for at least 2 weeks before use (SW eel). The major ionic composition of FW (tap water) and SW were as follows: [Na+], 1.0 mmol l–1; [Cl–], 0.5 mmol l–1; [Ca2+], 0.25 mmol l–1; [Mg2+], 0.5 mmol l–1; and [SO42–], 0.3 mmol l–1 in FW; and [Na+], 450 mmol l–1; [Cl–], 525 mmol l–1; [Ca2+], 10 mmol l–1; [Mg2+], 50 mmol l–1; and [SO42–], 30 mmol l–1 in SW. All experiments including fish maintenance were approved by the Committee of Animal Experiments at the University of Tokyo.
Time-course changes in SO42– concentration in body fluids after transfer to SW
To examine the time course changes in SO42– concentration in plasma, urine and rectal fluid, FW eels (N=9) were directly transferred to full-strength SW. On days 0, 1, 3, 7 and 14 after transfer, eels were lightly anesthetized in 0.1% (w/v) tricaine methanesulfonate (Sigma, St Louis, MO, USA), and 100 μl of blood was collected from the caudal vein into a chilled syringe; bladder urine and rectal fluid were collected by syringe via inserted tubes (PE10, Clay Adams, Franklin Lakes, NJ, USA). Blood was transferred to a chilled tube and centrifuged at 10,000 g for 5 min at 4°C to separate plasma. After sampling on day 14, eels were killed by excess exposure to saturated 2-phenoxyethanol (Wako Pure Chemical Industries, Osaka, Japan), and the kidneys were immediately dissected out, frozen in liquid nitrogen and stored at –80°C for later analysis.
Short-term changes in urine volume and SO42– concentration
FW eels (N=5) were anesthetized in 0.1% (w/v) tricaine methanesulfonate for 15 min and cannulated with polyethylene tubes (0.5 mm i.d., 0.8 mm o.d.) into the ventral aorta and urinary bladder. After surgery, eels were placed in plastic troughs with circulating, aerated water at 18°C. After more than 18 h of recovery, circulating water in the trough was changed from FW to SW by a three-way stopcock. Total bladder urine, and 30 μl of blood via the aortic tube, were collected for plasma analysis at 0, 1, 3, 6, 12 and 24 h after the salinity change.
SO42– concentrations in plasma and urine were measured using ion chromatography (AV10, Shimadzu, Kyoto, Japan). An anion-exchange column (IC-A3, Shimadzu), was used for the measurement of SO42–. Standard curves for SO42– were made using commercial standard solutions (Shimadzu). Concentration was estimated from a regression line calculation using Class-VP version 6.12 software (Shimadzu). Inter- and intra-assay coefficients of variation were 2.53% and 3.89%, respectively.
Identification of SO42– transporters in the kidney
To examine the tissue distribution of SO42– transporters, the gills, esophagus, stomach, anterior intestine, posterior intestine, rectum, kidney and urinary bladder were collected from FW and SW eels (N=3 each). The kidney was also collected from eels 1, 3, 7 and 14 days after SW transfer (N=4 each). Tissue samples were immediately frozen in liquid nitrogen and kept at –80°C until use. For immunohistochemical analyses, a sample of kidney from both FW and SW fish (N=3 each) was fixed in 4% paraformaldehyde in 0.1 mol l–1 phosphate-buffered saline (PBS), pH 7.4 for 24 h.
Total RNA was isolated from various tissues using acid guanidinium thiocyanate–phenol–chloroform extraction with Isogen (Nippon Gene, Tokyo, Japan). Tissues were homogenized in Isogen (100 mg ml–1) by Micro smash™ MA-100 (TOMY, Tokyo, Japan), followed by chloroform extraction, isopropanol precipitation, and 75% (v/v) ethanol washing of precipitated RNA. The RNA obtained was dissolved in diethyl pyrocarbonate (DEPC)-treated water, and its concentration was measured spectrophotometrically at 260, 280 and 300 nm using a spectrophotometer (DU-640, Beckman, Fullerton, CA, USA).
Sampled tissues were used to identify the Slc genes that were expressed abundantly in the kidney and other tissues and to compare gene expression between FW and SW fish. Time-course analysis of gene expression (1, 3, 7 and 14 days following SW transfer) was also performed using cDNA obtained from the kidney. A 1 μg sample of total RNA from each tissue was reverse-transcribed using the SuperScript III First Strand Synthesis System for RT-PCR (Invitrogen, Carlsbad, CA, USA). PCR was performed using gene-specific sense and antisense primer combinations (Table 1) based on the Slc sequences deposited by Nakada and colleagues in the DDBJ/EMBL/GenBank database (Nakada et al., 2005); Slc13a1 (AB111926), Slc26a1 (AB111927), Slc26a3 (AB111930), Slc26a6a (AB084425), Slc26a6b (AB111928), Slc26a6c (AB111929). β-Actin (AB074846) was used as an internal control. PCR amplification was performed under the following conditions: 94°C for 1 min, then 30 cycles of 94°C for 30 s, 60°C for 30 s and 72°C for 1 min, and finally 72°C for 5 min.
Fixed kidney samples were washed twice in 70% ethanol and then embedded in Paraplast (Kendall, Mansfield, MA, USA). Sections (6 μm) were sequentially rehydrated through a graded ethanol series and non-specific signal was blocked with 0.6% hydrogen peroxide (H2O2) in methanol for 30 min at room temperature and 2% normal goat serum in PBS (NGS-PBS for Slc13a1 and Slc26 proteins) or 2% normal horse serum (NHS-PBS for β-actin) containing 0.01% sodium azide for 30 min at room temperature. Sections were then incubated with antiserum against Slc13a1, Slc26a1, Slc26a6a, Slc26a6b and Slc26a6c diluted 1:10000 with NGS-PBS or against β-actin diluted 1:500 with NHS-PBS for 48 h at 4°C, and stained with elite avidin–biotin–peroxidase complex kit (Vector Laboratories, Burlingame, CA, USA) according to the manufacturer's instructions. The signal was visualized by exposure to 0.05% diaminobenzidine tetrahydrochloride and 0.01% H2O2 in 50 mmol l–1 Tris buffer (pH 7.2), and counterstained with hematoxylin. The antisera for Slc13a1 and Slc26a1 (kindly provided by Dr Shigehisa Hirose of Tokyo Institute of Technology, Japan) were raised in rabbits against recombinant proteins (Nakada et al., 2005). Antisera for Slc26a6a, Slc26a6b and Slc26a6c were raised in rabbits against synthetic peptides (Slc26a6a, NH2-KRLHSNKPVLVDR-COOH; Slc26a6b, NH2-CQRDASSETTQAMNGTYF-COOH; Slc26a6c, NH2-CAVTMRGNTRL-COOH). Antiserum for β-actin (C4, sc-47778) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Specificity of the antisera for Slc13a1 and Slc26a1 was confirmed previously by immunohistochemistry and western blot analysis (Nakada et al., 2005). The specificity of antisera for Slc26a6a, Slc26a6b and Slc26a6c was confirmed by preimmune serum and antibody preadsorption, as follows: 1 ml primary antibody solution (antiserum:NGS-PBS of 1:10,000) was incubated with antigen (10–7 mol l–1) for 18 h at 4°C before being applied to tissue sections. After antibody preadsorption, there was no immunoreactive signal (data not shown). For periodic acid–Schiff (PAS) staining, kidney sections (6 μm) were rehydrated and incubated in 0.5% periodic acid solution for 10 min and Schiff's reagent for 5 min at 4°C. Sections were then rinsed 3 times with H2SO3 solution (10% NaHSO3:HCl:water of 6:5:100) for 3 min, and then counterstained with hematoxylin.
Changes in SO42– concentration after SW transfer were analyzed by Dunnett's test. All analyses were performed using the statistical software KyPlot 5.0 (Kyens, Tokyo, Japan). Significance was defined at *P<0.05, **P<0.01 and***P<0.001. All results are expressed as means ± s.e.m.
Time-course changes in SO42– regulation after SW transfer
Plasma SO42– concentration tended to increase 1 day after SW transfer but decreased abruptly after day 3, suggesting an activation of SO42– excretory mechanisms after this time point (Fig. 1A). The decrease in plasma SO42– concentration continued until 7 days after transfer, when levels appeared to stabilize. Urine SO42– concentration increased rapidly at first before decreasing. Despite this decrease, urine SO42– levels after SW transfer remained significantly higher than in FW (Fig. 1B). Rectal fluid SO42– concentration increased 1 day after SW transfer but to a smaller extent than the change in urine SO42– concentration (Fig. 1C). Urinary SO42– excretion showed a pattern similar to the change in urine SO42– concentration, but the levels 3 days after transfer were smaller because of decreased urine volume (Fig. 1D).
Short-term changes in plasma and renal SO42– regulation
As shown in Fig. 1, plasma SO42– concentration did not change significantly 1 day after SW transfer, but urine SO42– concentration increased profoundly. In order to examine the changes occurring within 1 day of transfer, we measured plasma and urine SO42– concentration and urine volume for 24 h. Plasma SO42– concentration increased slightly after 1 h, but returned to the FW level, where it remained thereafter (Fig. 2A). In contrast, urine SO42– concentration stayed low for 6 h and increased linearly thereafter, reaching a 100-fold increase after 24 h, suggesting that the reabsorptive mechanism of the FW-type kidney was turned off after 6 h (Fig. 2B). Urine volume decreased immediately to 1/10 that of FW eels within 2 h of SW transfer and was maintained at the low level for 24 h (Fig. 2B). Urinary SO42– excretion decreased slightly after 1 h and increased linearly thereafter (Fig. 2C).
Tissue distribution of Slc transporter genes
Eel Slc genes, except for Slc26a3, were abundantly expressed in the kidney, followed by the intestine, with low levels of expression in the urinary bladder (Fig. 3). The Slc26a3 gene was expressed almost exclusively in the digestive tract. The gills did not express any of the Slc genes abundantly. The Slc13a1 gene was expressed abundantly in the FW eel kidney while Slc26a6a was expressed abundantly in the SW eel kidney. Even after an additional 10 cycles, no signal was detected in the negative tissues shown in Fig. 3 (data not shown).
Time-course analyses of Slc gene expression after SW transfer
Expression of Slc13a1 decreased slightly 1 day after SW transfer, and the signal disappeared after day 3 (Fig. 4). Conversely, Slc26a6a transcript was detectable after 1 day, and increased gradually for 2 weeks. The expression of other Slc genes that were detected in the eel kidney consistently did not show changes for 14 days after SW transfer (Fig. 4).
Morphological identification of proximal tubule segments in eel kidney
It has been suggested that the renal tubules of European eel (Olivereau and Olivereau, 1977) and of Japanese eel (Teranishi and Kaneko, 2010) can be divided into the proximal tubule and distal tubule based on luminal staining by PAS. Proximal tubules were further divided into the P1 and P2 segments by the signal intensity derived from the length and density of the brush border membrane. In this study, the proximal P1 and P2 segments and distal tubule were identifiable by PAS staining (Fig. 5A). The P1 and P2 segments were also distinguishable by the density of the luminal immunohistochemical signal using an antiserum against β-actin (Fig. 5B). PAS staining and β-actin immunoreactivity were stronger in P1 segments than in P2 segements. A schematic drawing of the eel proximal nephron is shown in Fig. 5C.
Immnohistochemical identification of Slc proteins in eel kidney
We used the relative abundance of immunoreactive β-actin in the luminal brush border membrane to distinguish the P1 and P2 segments of proximal tubules (Fig. 6A). In serial sections of SW eel kidney, immunoreactive Slc26a6a and Slc26a6c were localized on the apical membrane of the P1 segment of proximal tubules (Fig. 6C,E). In contrast, immunoreactive Slc26a6b was located in the apical membrane (Fig. 6D) and Slc26a1 in the basolateral membrane of the P2 segment of the same tubules (Fig. 6B). No immunoreactivity was detected with Slc13a1 antiserum in the SW eel kidney (Fig. 6F).
Because SO42– is an important anion for body functions such as mucus formation and regulation of enzyme activity, its regulation has been the focus of intensive research particularly in the field of mammalian physiology (for review, see Markovich, 2001). In these studies, the mechanisms for SO42– absorption and retention have been investigated but little is known about how animals get rid of excess SO42–. This is partly because the land is generally a SO42–-deficient environment. Therefore, marine teleosts, which live in an environment with high levels of SO42–, provide a unique opportunity to investigate the mechanism of SO42– excretion. In particular, euryhaline teleosts are excellent models for analyzing the mechanism of SO42– regulation because they maintain SO42– homeostasis in both SO42–-deficient FW and SO42–-rich SW by reversing regulation from absorption/retention to excretion. In the present study, we showed that the eel, a euryhaline teleost, switches on excretory mechanisms and switches off reabsorptive mechanisms in the kidney within 3 days of SW transfer. This process involves regulation of multiple SO42– transporters localized on different (apical or basolateral) sides of epithelial cells in different segments of renal proximal tubules.
SO42– regulation by the kidney after SW transfer
Eels are unique among euryhaline teleosts because they exhibit a higher plasma SO42– concentration in FW than in SW, which is the opposite situation to that for other euryhaline teleosts (Nakada et al., 2005) (T.W. and Y.T., unpublished). In the initial part of this study, therefore, we examined the time course of changes in SO42– regulation after transfer of eels from FW to SW. We found that at 3 days after SW transfer SO42– regulation had switched from absorption/retention to excretion, with plasma and urine SO42– concentration stabilizing (Fig. 1). SO42– ions are actively secreted from the renal proximal tubules of marine teleosts (Renfro and Pritchard, 1983; Dickman and Renfro, 1986; Cliff and Beyenbach, 1992); therefore, the lower plasma SO42– concentration in SW eels may be due to up-regulated SO42– excretion by the kidney.
Analysis of short-term changes after SW transfer showed that plasma SO42– concentration slightly increased after 1 h but returned to FW levels after 3 h (Fig. 2). This transient increase may be due to increased SO42– uptake by the gills, because 35SO42– uptake from the environment was accelerated in FW eels compared with that in SW eels (T.W. and Y.T., unpublished). The intestine may also contribute to the transient increase in SO42– plasma concentration as vigorous drinking occurs immediately after SW transfer in eels, which could lead to increased intestinal SO42– absorption (Hirano, 1974). The urine SO42– concentration increased after 6 h, but total excretion did not increase because of the immediate decrease in urine volume after SW transfer (Fig. 2C).
SO42– transporters involved in SO42– excretion by the kidney
Among eel homologs of SO42– transporters, four genes (Slc26a6a, Slc26a6b, Slc26a6c and Slc26a1) were shown to be expressed significantly in the kidney of SW eels. Further, Slc26a6a was expressed only in the SW eel kidney, which indicates a role in SO42– excretion in SW. The Slc26a6a gene has also been identified in the euryhaline pufferfish and its expression is up-regulated in SW (Kato et al., 2009). Slc26a6a has the highest SO42– transport activity among the Slc26a6 family members, and it excretes SO42– in exchange for Cl– in the kidney (Kato et al., 2009) but secretes HCO –3 in exchange for Cl– in the intestine (Kurita et al., 2008). In contrast, Slc13a1 was expressed only in the FW eel kidney as shown previously by Nakada and colleagues (Nakada et al., 2005). The time-course study of the transporter genes revealed that the disappearance of Slc13a1 transcripts and appearance of Slc26a6a transcripts occurred 3 days after SW transfer, which is synchronous with the time course of changes in plasma and urine SO42– concentration after SW transfer. Expression of the Slc26a1 gene was unchanged in the eel kidney after SW transfer, but in rainbowtrout expression of the gene was slightly increased 3 h after SO42– injection (Katoh et al., 2006). Slc26a1 is responsible for both excretion (current study) and reabsorption (Nakada et al., 2005) of SO42– in SW and FW kidney of eels, respectively. It seems that the Slc26a6 genes have diversified into three types (a–c) in the teleost and all are involved in the excretion of SO42– in the marine teleost kidney.
In contrast to the obvious down-regulation of the Slc13a1 gene in the eel kidney after SW transfer, as shown in a previous study (Nakada et al., 2005), the expression of the Slc13a1 gene is unaltered in the kidney of SW-adapted pufferfish (Kato et al., 2009). This result suggests physiological regulation such as phosphorylation of transporter activity in addition to transcriptional regulation. In mammals, phosphorylation inhibitors reduced SO42– transport activity of these transporters (Markovich, 2000). Tunicamycin (an N-glycosylation inhibitor) also reduced SO42– uptake by Slc13a1 and Slc26a1 in Xenopus oocytes (Markovich, 2001) and site-directed mutagenesis of the putative N-glycosylation sites of rat Slc13a1 and Slc26a1 reduced SO42– transport activity in Xenopus oocytes (Li and Pajor, 2003).
Kato and colleagues suggested that Na+/K+-ATPase located at the basolateral membrane of tubular cells may serve as a driving force for the electrogenic Cl–/SO42– exchanger (Slc26a6a) by increasing the intracellular negative membrane potential (Kato et al., 2009). Ura and colleagues (Ura et al., 1996) and Teranishi and Kaneko (Teranishi and Kaneko, 2010) observed immunoreactive Na+/K+-ATPase at the basolateral membrane of renal proximal tubular cells in SW-acclimated eels. Thus, a similar driving force of SO42– transport may function in the proximal tubule of SW eels.
Localization of SO42– transporters in SW eel kidney
The teleosts, which make up more than half of the total vertebrate species, are an incredibly diverse group (Nelson, 2006). This variation is reflected in the kidney morphology, with some marine fish lacking distal tubules and even glomeruli (Berglund and Forster, 1958). However, generally, the kidney of euryhaline fish is well developed and possesses all nephron segments. It has been shown that the eel possesses two different segments in the proximal tubule in addition to the neck segment, which exhibit different cellular morphology (Olivereau and Lemoine, 1968; Olivereau and Olivereau, 1977; Teranishi and Kaneko, 2010). The most distinct among them is the density of the brush border membrane, which can be visualized by β-actin immunostaining (Fig. 5B). The more dense the brush border the greater the amount of glycoprotein, which can be visualized by PAS staining (Fig. 5A).
Our immunohistochemical study showed that the SO42– transporters Slc26a6a, Slc26a6b, Slc26a6c and Slc26a1 exist in the proximal tubules of the SW eel kidney, indicating their role in the active excretion of SO42–. Slc26a6a may be particularly important because (1) the P1 segment is thought to be the site of divalent ion secretion in marine teleosts (Beyenbach, 1995), (2) up-regulation of the Slc26a6a gene occurred after transfer of eels to SW and (3) Slc26a6a possesses the greatest SO42–-transporting activity of in pufferfish (Kato et al., 2009). We also showed that Slc26a6c co-localizes with Slc26a6a on the apical membrane of the cells in the P1 segment. However, we could not detect a counterpart transporter on the basolateral side that transports SO42– from the blood into the tubular cells. In the P2 segment, however, Slc26a6b and Slc26a1 are localized on the apical and basolateral side, respectively, in the SW eel kidney (Fig. 6). Therefore, these transporters are most likely to be involved in SO42– excretion in this segment. Slc26a6a is also localized on the apical side of the renal proximal tubular cells in the pufferfish (Kato et al., 2009), but a counterpart on the basolateral side has not yet been determined.
In mammals, immunohistochemical studies have shown that Slc26a6 is localized on the apical side of the renal proximal tubule (Knauf et al., 2001; Thomson et al., 2005), while Slc26a1 is localized on the basolateral membrane (Karniski et al., 1998). They are assumed to work together to excrete SO42– in the same proximal tubular cells (Markovich and Aronson, 2007), but colocalization in the same cells has not yet been demonstrated. The apical location of Slc26a6a and basolateral location of Slc26a1 have been reported in the renal proximal tubule of two teleost species [rainbow trout (Katoh et al., 2006) and pufferfish (Kato et al., 2009)], but co-localization of the two transporters in the same cells has not been demonstrated in these studies. Therefore, the current immunohistochemical study is the first to show the detailed localization of the four possible SO42– transporters most likely to be responsible for SO42– excretion in the SW eel kidney.
From the data obtained in this study, we propose a model for renal SO42– excretion (Fig. 7). The apical Slc26a6a is most likely to be responsible for active renal SO42– excretion in the SW eel kidney as this gene is expressed only in SW eels. Basolateral Slc26a1 may also be involved, but it is localized in a different segment of the proximal tubule. As a counterpart of Slc26a1 to achieve SO42– transport from the blood into the tubular lumen, we identified Slc26a6b in the P2 segment. However, we could not identify the counterpart of Slc26a6a and Slc26a6c, which must be located on the basolateral side of the P1 segment. Slc26a2, Slc26a3, Slc26a7, Slc26a8, Slc26a9 and Slc26a11 are possible SO42– transporters in mammals (Mount and Romero, 2004), and we could isolate Slc26a2 cDNA in the eel. However, the expression level of Slc26a2 was too low for it to have a significant function in the SW eel kidney (data not shown). In the eel, Slc26a3 was not expressed in the kidney (Figs 3 and 4). Other Slc26 genes have not yet been cloned in eels. Once their counterparts are identified, we can delineate the whole picture of the mechanism of SO42– excretion in SW eel kidney, which hopefully represents the system of marine teleost fish in general.
We would like to thank Dr Susumu Hyodo, Dr Makoto Kusakabe and Ms Sanae Hasegawa from our laboratory for encouragement and advice. We also thank Dr Jillian Healy from our laboratory for editing English of this manuscript and Dr Shigehisa Hirose of Tokyo Institute of Technology for kindly providing antisera for Slc13a1 and Slc26a1.
This research was supported in part by Grant-in-Aid for Basic Research (A) from the Japan Society for the Promotion of Science to Y.T. (13304063 and 16207004). T.W. was supported by Global COE Program (Integrative Life Sciences Based on Study of Biosignaling Mechanisms), MEXT, Japan.