Articles in PresS. Am J Physiol Regul Integr Comp Physiol (May 4, 2011). doi:10.1152/ajpregu.00624.2010
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Environmental factors responsible for switching on the SO42- excretory
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system in the kidney of seawater eels
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Taro Watanabe1 and Yoshio Takei
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Laboratory of Physiology, Department of Marine Bioscience, Atmosphere and Ocean
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Research Institute, The University of Tokyo, Kashiwa, Chiba 277-8564, Japan
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Running title: RENNAL SULFATE REGULATION IN EEL
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Center for Cooperative Research Promotion, Atmosphere and Ocean Research Institute
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The University of Tokyo, 5-1-5, Kashiwanoha, Kashiwa, Chiba, 277-8564, Japan
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Tel: +81-4-7136-6458, Fax: +81-4-7136-6459, E-mail: [email protected]
Present address: Dr. Taro Watanabe
Copyright © 2011 by the American Physiological Society.
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ABSTRACT
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Eels are unique in that they maintain lower plasma SO42- concentration in
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SO42--rich (~30 mM) seawater (SW) than in SO42--poor (<0.3 mM) fresh water (FW),
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showing drastic changes in SO42- regulation between FW and SW. We previously
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showed that the expression of renal SO42- transporter genes, FW-specific Slc13a1 and
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SW-specific Slc26a6a, changes profoundly after transfer of FW eels to SW, which
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results in the decrease in plasma SO42- concentration after 3 days in SW. In this study,
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we attempted to identify the environmental factor(s) that trigger the switching of SO42-
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regulation using changes in plasma and urine SO42- concentrations and expression of the
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transporter genes as markers. Transfer of FW eels to 30 mM SO42- or transfer of SW
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eels to SO42--free SW did not change the SO42- regulation. Major divalent cations in SW,
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Mg2+ (50 mM) and Ca2+ (10 mM), was also ineffective but 50 mM NaCl was effective
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for switching the SO42- regulation. Further analyses using choline-Cl and Na-gluconate
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showed that Cl- is a primary factor and Na+ is permissive for the Cl- effect. Since plasma
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SO42- and Cl- concentrations were inversely correlated, we injected various solutions
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into the blood and found that Cl- alone triggered the switching from FW to SW-type
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regulation. Further, the inhibitor of Na-Cl cotransporter (NCC) added to media
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significantly impaired the expression of SW-specific Slc26a6a in 150 mM NaCl. In
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summary, it appears that Cl- ions in SW are taken up into the circulation via the NCC
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together with Na+, and the resultant increase in plasma Cl- concentration enhances SO42-
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excretion by the kidney through down-regulation of absorptive Slc13a1 and
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up-regulation of excretory Slc26a6a, resulting in low plasma SO42- concentration in SW.
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Keywords: euryhaline teleost, sulfate transporter, solute carrier 26, kidney,
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INTRODUCTION
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Sulfate ions (SO42-) play important roles in a variety of metabolic and cellular
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processes. Possible physiological functions include production of chondrocytes and
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mucus as important component, detoxification of exogenous substances by sulfation,
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elimination of waste compounds by sulfoconjugation, and biosynthesis of sulfated
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hormones such as gastrin and cholecystokinin (17). Accordingly, slight imbalance of
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plasma SO42- often leads to clinical syndromes (29). Despite the importance of SO42-
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regulation, the research on ion regulation has focused mainly on monovalent ions (Na+
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and Cl- for osmoregulation and H+ and HCO3- for pH regulation) and divalent cations
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(Ca2+ and Mg2+ for regulation of muscular contraction and others).
Vertebrate habitats can be divided largely into two categories in terms of SO42-
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abundance; SO42--poor (land and freshwater, FW) and SO42--rich (seawater, SW)
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environment. As for aquatic environments, FW contains only ~0.3 mM of SO42- but SW
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contains SO42- as the second most abundant anion (~30 mM). Therefore, teleost fish
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have opposite SO42- regulation in FW and SW as their plasma typically contains ~1 mM
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of SO42- irrespective of FW and SW species. Among teleosts, euryhaline species must
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reverse SO42- regulation when they move between FW and SW. It has been suggested
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that the gills and intestine are almost impermeable to SO42- (6, 21), but obligatory influx
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of SO42- was nullified by excretion via the kidney in marine teleosts (3, 7, 8, 28). We
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recently showed that the eel is unique in SO42- regulation compared with other
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euryhaline teleosts because it has much higher plasma SO42- in FW (~6 mM) than in
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SW (~1 mM) (35) as reported previously (23). We also showed that 85% of the SO42- in
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SW is taken up by the gills and 97% of SO42- that enter the circulation is excreted by the
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kidney in SW eels (unpublished data).
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Among members of the solute carrier (Slc) superfamily of transporters, Slc4a1
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(AE1), Slc13a1 (NaS-1), Slc26a1 (Sat-1), Slc26a2 (DTDST), Slc26a3 (DRA), Slc26a6
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(CFEX, PAT1), Slc26a7, Slc26a8, Slc26a9 and Slc26a11 have been implicated in SO42-
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transport in mammals (20). In teleost fish, Slc13a1, Slc26a1, Slc26a3, and Slc26a6a,b,c
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have been cloned in the eel (23), Slc26a1 in the rainbow trout (14), Slc13a1 in the
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zebrafish (19), and Slc26a6a,b,c in the pufferfish, Takifugu obscurus (13). Among them,
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Slc13a1 and Slc26a1 play key roles in SO42- reabsorption at the renal proximal tubule of
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mammals (5) and FW eels (23). On the other hand, Slc26a6 and Slc26a1 are suggested
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to be involved in renal SO42- secretion in mammals (16, 20). In fishes, Slc26a1 seems to
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be involved in renal SO42- secretion in rainbow trout (14), Slc26a6a in pufferfish (13),
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and Slc26a6a,b,c and Slc26a1 in SW eels (35). However, it is not yet known which
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factor(s) in SW are responsible for changing the expression of the transporter genes
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during the course of SW adaptation in euryhaline fishes.
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High SO42- diet induced a decrease in Slc13a1 expression in the renal proximal
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tubule of mammals (18, 26) and injection of Na2SO4 into the circulation increased
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Slc26a1 transcripts in the kidney of rainbow trout (14), indicating the role of SO42- in
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the transporter regulation. It seems that major ions in SW are responsible for changes in
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the transporter gene expression, but it remains to be determined which ion(s) actually
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switch the renal SO42- regulation to a SW type. We used the eel as a model species
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because it changes the SO42- regulation most drastically among euryhaline species (23,
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35). Initially, we examined the time-course changes in SO42- regulation after transfer of
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FW eels to SW using plasma and urine SO42- concentrations and expression of Slc13a1
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and Slc26a6a genes in the kidney as markers. Then, we attempted to identify the ions
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responsible for the switching by transferring fish to various ionic environments. As
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environmental Na+ and Cl- were suggested as responsible ions, we further examined
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how the information of these ions is transmitted into the body to turn on the switch
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using a specific inhibitor (hydrochlorochiazide) of Na-Cl cotransporter that is present in
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the gills and suggested to take up these ions from media.
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MATERIALS AND METHODS
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Animals
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Cultured eels of ca. 200 g were purchased from a local dealer. All eels were kept
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without feeding at 18°C in FW tanks for 2 to 4 weeks until experimentation. SW eels
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were transferred to SW and ad acclimated to full SW for at least 2 weeks before use.
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Ionic composition of FW is [Na+], 1.0 mM; [Cl-], 0.5 mM; [Ca2+], 0.25 mM, [Mg2+], 0.5
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mM; [SO42-], 0.3 mM, and that of SW is [Na+], 450 mM; [Cl-], 525 mM; [Ca2+], 10 mM,
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[Mg2+], 50 mM; [SO42-], 30 mM. All conditions for fish maintenance and experiments
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were approved by the Committee for Animal Experiments at the University of Tokyo.
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Transfer experiment to various ionic environments
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1. Effect of changes in environmental SO42- in FW eel Six FW eels were transferred
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either to SO42--enriched FW (30 mM Na2SO4 or 30 mM MgSO4), FW (control) or SW
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(positive control), and urine and plasma samples were collected 1, 3 and 7 days after
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transfer. After 7 days, the kidney was dissected for gene expression analysis as
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mentioned below. Details of ionic composition of experimental water are described in
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Table 1.
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2. Effect of changes in environmental SO42- in SW eel Six SW eels were transferred
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either to SO42--enriched SW (added with 30 mM Na2SO4 up to double strength of
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typical SW), SO42--free artificial SW, or SW. The SO42- free SW was prepared by
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dissolving 1,502 g NaCl, 407.0 g MgCl2-6H2O and 44.4 g CaCl2 in 40 liters of FW (see
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Table 1). Samples were collected one week after the transfer as mentioned below.
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3. Effect of various concentrations of NaCl solution in FW eel Six FW eels were
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transferred either to FW containing 0 (control), 20, 50, 150 (isotonic) or 450 (SW level)
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mM NaCl (see Table 1), and samples were collected for one week after transfer as
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mentioned below. In addition, six FW eels were transferred to10 mM CaCl2 solution to
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evaluate the role of Ca2+ ions in SW.
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4. Effect of Na+ and/or Cl- in FW eel Five FW eels were transferred either to (i) 50
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mM Na-gluconate (Na+ alone), (ii) 50 mM choline-Cl (Cl- alone), (iii) 50 mM
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Na-gluconate + 10 mM choline-Cl or (iv) 10 mM Na-gluconate + 50 mM choline-Cl to
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evaluate relative importance of Na+ and/or Cl-, or (v) 100 mM mannitol (equivalent to
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50 mM NaCl) to evaluate the role of osmolality in SO42- regulation. Ionic compositions
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of the solutions were described in Table 1. Samples were collected for one week after
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transfer to each solution.
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For sampling, eels were lightly anesthetized in 0.1% (w/v) tricaine
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methanesulfonate (Sigma, USA), and 100 μl of blood was collected from the caudal
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vein into a chilled syringe containing 2K-EDTA (20 μl/ ml) and bladder urine was
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collected by a syringe just before and 1, 3 and 7 days after transfer. Blood was
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centrifuged at 10,000 xg for 5 min at 4oC to prepare plasma. After sampling at day 7,
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eels were euthanized with 2-phenoxy ethanol (Wako Pure Chemical Industries, Osaka,
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Japan), and kidneys were immediately dissected out, frozen in liquid nitrogen, and
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stored at -80°C for later analysis.
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Effect of intravascular injection of various ions on switching in FW eel.
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Eight FW eels were anesthetized in the tricaine methanesulfonate for 15 min and
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cannulated with a polyethylene tube (0.5 mm I. D., 0.8 mm O. D.) into the ventral aorta.
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After surgery, eels were placed in plastic troughs with a circulating water system at
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18°C. After more than 18 h of recovery, 100 μl of isotonic 0.15 M NaCl (control), 3 M
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NaCl, 1.5 M Na2SO4 (Na+ alone) or 1.5 M MgCl2 (Cl- alone) were injected (n=5 in each
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group) in 1 min through the cannula into the circulation followed by a flush with 100 μl
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of isotonic NaCl. Subsequently, blood was collected 24 h after injection for
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measurement of ion concentrations, and the kidney was dissected out after anesthesia
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for measurement of Slc gene expression.
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Effect of Na-Cl cotransporter blocker in media on switching in FW eel
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Since it was shown that both Na+ and Cl- are required for switching of SO42-
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regulation from FW-type to SW-type, we evaluated the role of Na-Cl cotransporter
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(NCC) in ion uptake from media. For this purpose, two groups of FW eels were
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transferred to 150 mM NaCl solutions containing 0 (conrol), 10-8 M, or 10-6 M
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hydrochlorochiazide (HCTZ), an NCC blocker. Since both HCTZ concentrations
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inhibited switching, 10 eels were transferred either to 150 mM NaCl solution with or
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without 10-8 M HCTZ for 3 days, and blood and the kidney were sampled for
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subsequent analyses.
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Measurement of plasma osmolality and ion concentrations in plasma and urine
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Plasma osmolality was measured using a vapor pressure osmometer (Type 5520,
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Wescor Inc, Logan, USA). Anions (Cl- and SO42-) and cations (Na+ and Ca2+) in plasma
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and urine were measured using ion chromatography (AV10, Shimadzu, Kyoto, Japan)
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using anion-exchange (IC-A3) and cation-exchange (IC-C3) column, respectively. The
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lower detection limit in this study is ca. 5μM. Standard curves for the anions and
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cations were prepared by commercial standard solutions (Shimadzu).
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Expression analysis of renal SO42- transporter genes
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Since five Slc genes (Slc13a1, Slc26a1, and Slc26a6a,b and c) were shown to be
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expressed abundantly in the kidney, changes in their expression were examined after
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transfer to media with various ion concentrations. One μg of total RNA from kidney
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was reverse-transcribed using the SuperScript III First Strand Synthesis System for
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RT-PCR (Invitrogen, Carlsbad, CA). PCR was performed using gene specific sense and
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antisense primer combinations (Table 2) based on the sequences in the database;
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Slc13a1 (AB111926), Slc26a1 (AB111927), Slc26a6a (AB084425), Slc26a6b
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(AB111928), Slc26a6c (AB111929) and β-actin (AB074846) used as an internal control.
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PCR amplification was performed under the following conditions: 94oC for 1 min, then
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30 cycles of 94oC for 30 s, 60oC for 30 s, and 72oC for 1 min, and finally 72oC for 5
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min.
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Quantitative analyses of gene expression
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Quantitative analyses of gene expression were performed by real-time PCR for
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Slc26a6a and Slc13a1 in the ion injection experiment and in the HCTZ experiment. The
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GAPDH gene expression was used as an internal control. Reactions were performed
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with the SYBR Green method using KAPA SYBR First qPCR Kit (Kapa Biosystems,
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Cape Town, South Africa) in a ABI Prism 7900HT Sequence Detection System (PE
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Applied Biosystems, Foster City, CA). The primers for Slc26a6a and Slc13a1 were
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designed using Primer Express software (PE Applied Biosystems), and those for eel
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GAPDH have been described previously (24) (Table 2). The relative expression of the
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transporters was normalized by the amount of GAPDH mRNA and compared between
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experimental and control groups. All measurements were performed in duplicate.
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Statistical analyses
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Changes in SO42- concentration were analyzed by Dunnett’s test or by
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Tukey-Kramer test. Correlations between plasma SO42- and other ion concentrations
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were analyzed by the linear regression analysis. All analyses were performed using the
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statistical software, KyPlot 5.0 (Kyens, Tokyo, Japan). Significance was determined at
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*p<0.05. All results were expressed as means ± S.E.M.
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RESULTS
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Effect of environmental SO42- on switching
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Plasma SO42- concentrations increased gradually after transfer of FW eels to 30
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mM Na2SO4 or MgSO4, and the increase was significant after 3 - 7 days (Fig. 1a).
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However, plasma SO42- concentration decreased 3 days after transfer of FW eels to SW
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that contains 30 mM SO42-. Urine SO42- concentration continued to be low in eels
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transferred to 30 mM Na2SO4 or MgSO4 (Fig. 1b) even though plasma SO42-
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concentration increased. Neither Slc13a1 nor Slc26a6a gene expression changed after
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transfer to the Na2SO4 or MgSO4 solution (Fig. 1c). These results clearly show that the
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elevation of SO42- concentration in plasma or media is not a sufficient stimulus to
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switch on the renal SO42- excretory system in FW fish.
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Effect of SO42--enriched or SO42--free SW on switching
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Plasma SO42- concentration increased 1 day after transfer of SW eels to
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SO42--enriched SW (60 mM SO42-), but gradually decreased thereafter to the level of
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SW eels (Fig. 2a). Plasma SO42- concentration did not change 1 day after transfer of SW
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eels to SO42--free SW, but decreased to a level lower than SW eels thereafter. Urine
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SO42- concentration tended to be higher after transfer to SO42--enriched SW and lower
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after transfer to SO42--free SW but the changes were not significant compared with SW
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eels (Fig. 2b). The expression of Slc13a1 and Slc26a6a did not change after transfer to
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SO42--enriched or SO42--free SW (Fig. 2c). Therefore, removal of SO42- from SW did not
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switch off the renal SO42- excretory system of SW fish. The result of the experiment
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using SO42--enriched SW also showed that the SW eel kidney has an extra capacity to
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extrude SO42 and maintain plasma SO42 concentration normal.
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Effect of environmental Na+ and/or Cl- on switching
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Plasma SO42- concentration gradually decreased after transfer of FW eels to 50 -
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450 mM NaCl and the decrease was significant after 3 - 7 days, but 20 mM NaCl was
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not effective (Fig. 3a). The degree of the decrease was concentration-dependent
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between 50 and 450 mM after 1 - 3 days, although all fish did not survive for 7 days
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after transfer to 450 mM NaCl. Plasma SO42- concentration did not change after transfer
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to 10 mM CaCl2 (Fig. 3a). Urine SO42- concentration increased significantly 3 days after
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transfer to 150 mM NaCl, although the increase was much smaller than after transfer to
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SW (Fig. 3b). No change was detected in urine SO42- concentration after transfer to
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lower concentrations of NaCl and 10 mM CaCl2. Urine could not be collected in a
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sufficient volume for analysis after transfer to 450 mM NaCl solution. Renal expression
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of Slc13a1 was down-regulated 7 days after transferring FW eels to more than 50 mM
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NaCl, while Slc26a6a was up-regulated when fish were exposed to 20 mM NaCl (Fig.
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3c). The result showed that Cl- alone or Na and/or Cl- trigger the change in renal SO42-
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regulatory systems, and its threshold was between 20 and 50 mM.
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Relative importance of Cl- and Na+ on switching
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In order to determine which ion, Na+ and/or Cl-, is of primary importance for the
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switching, we first transferred FW eels to 50 mM Na-gluconate (Na+ alone) or 50 mM
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choline-Cl (Cl- alone) solutions, but plasma and urine SO42- concentration and
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transporter gene expression were not altered for 7 days (Fig. 4). After transfer to
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solutions containing 50 mM Na++10 mM Cl- or 10 mM Na+ + 50 mM Cl-, only the
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latter decreased plasma SO42- concentration as did transfer to 50 mM NaCl solution (Fig.
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4a). Down-regulation of Slc13a1 and up-regulation of Slc26a6a were apparent in the
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kidney of these fish (Fig. 4c), but the increase in urinary SO42- concentration was
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smaller than that after SW transfer (Fig. 4b). Therefore, Cl- is a primary ion in SW that
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triggers the switching of SO42- excretory system in the kidney and Na+ has a permissive
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role.
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Relationship between SO42- and other ions in plasma
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When plasma SO42- concentration was plotted against other ion concentration in
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plasma, a significant negative correlation was detected with Cl- but not with Na+, Ca2+
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and osmolality (Fig. 5). Plasma Cl- showed positive correlation with the NaCl
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concentration in media (data not shown). These results suggest an intimate relationship
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between Cl- and SO42- concentrations in plasma and between Cl- concentrations in
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plasma and media.
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Effect of intravascular injection of ions on switching
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Intra-arterial injection of 1.5 M MgCl2 or 3 M NaCl up-regulated Slc26a6a
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expression in the kidney of FW eels after 24 h, but 1.5 M Na2SO4 or isotonic 0.15 M
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NaCl (control) was without effect (Fig. 6a). Only 3 M NaCl solution significantly
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down-regulated the Slc13a1 expression (Fig. 6b). Plasma Cl- concentration (n=5 in each
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group) changed from 107.6 + 5.5 to 103.0 + 2.1 mM 24 h after isotonic NaCl injection,
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from 96.8 + 7.7 to 98.4 + 6.7 mM after 1.5 M Na2SO4, from 100.0 + 6.6 to 104.8 + 6.6
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mM after 1.5 M MgCl2, and from 104.6 + 3.5 to 110.8 + 4.5 mM after 3 M NaCl. These
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results strongly suggest that a transient increase in plasma Cl- concentration after
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injection of Cl--containing solution triggered switching of renal SO42- regulation.
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Effect of NCC blocker in media on switching
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Plasma concentrations of SO42- and Cl- were not significantly different between
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HCTZ-treated fish and controls. The up-regulation of Slc26a6a expression observed
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significantly after transfer to 150 mM NaCl (Fig. 7a). However, Slc13a1 did not
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changed after transfer (Fig. 7b).
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DISCUSSION
Since SO42- is excess in seawater compared with body fluids, marine fish must
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cope with SO42- influx into the body across concentration gradients. Using 35SO42-, we
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found that in SW-adapted eels, 85% of SO42- uptake occurs via the gills and 15% via
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digestive tracts, whereas 97% of SO42- excretion occurs via the kidney (unpublished
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data). The role of the kidney in SO42- excretion has been reported in several species of
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marine fish (3, 7, 8, 28). We also found that euryhaline tilapia and chum salmon
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maintain slightly higher plasma SO42- concentrations in SW than in FW, but the eel is an
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exception with much lower plasma SO42- concentration in SW than in FW (unpublished
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data). The unique SO42- accumulation in FW eel plasma was reported previously by
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Nakada et al. (23). Thus the eel exhibits drastic changes in SO42- regulation in the
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kidney after transfer from FW to SW and thus serves as an excellent model to
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investigate the switching mechanism of SO42- regulation. In mammals, SO42- filtered by
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the kidney easily reaches saturation and excess SO42- is excreted into the urine (15, 22).
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Therefore, apparent switching does not seem to occur in mammals, although the
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presence of a SO42- excretory system is also suggested in mammals (16, 20).
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Environmental ions responsible for switching SO42- regulation
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When euryhaline fish migrate between FW and SW, the mechanisms for ion
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regulation change drastically to an opposite direction, from absorption to excretion or
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vice versa at the osmoregulatory organs (gills, kidney and intestine). However, there has
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been no report on the factors in SW that trigger such changes in ion regulation. For
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example, mitochondria-rich cells in the gills are altered from an absorptive FW type to
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an excretory SW type when euryhaline teleosts are transferred from FW to SW (10).
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The environmental factors that induce the change are most likely Na+ or Cl- ions that
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exist at high concentrations in SW and are the target of regulation by the cells, but there
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is no report on the factor that triggers the change. In this study, we investigated such
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factors that trigger the change in SO42- regulation using plasma and urine concentrations
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and renal SO42- transporters as markers. As for the transporters, we chose Slc13a1 and
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Slc26a6a because they are known to be expressed only in FW and SW eels, respectively
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(23, 35). The higher expression of the Slc26a6a gene in SW is also reported very
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recently in the euryhaline pufferfish, Takifugu obscurus (13). Since intravascular
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injection of SO42- down-regulates Slc26a1 in trout (14), we expected that SO42- may be
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responsible for the change. However, neither addition of 30 mM SO42- to FW nor
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removal of SO42- from SW affected the SO42- regulation in FW and SW eels,
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respectively. In addition, injection of SO42- directly into the blood of FW eels did not
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trigger the change. Therefore, SO42- is not responsible for the switching of SO42-
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regulation. The high osmolality or divalent cations (Ca2+ and Mg2+) also failed to induce
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the change. However, 50 mM NaCl solution in media triggered the change. Compared
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with SW transfer, eels transferred to NaCl solutions decreased plasma SO42-
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concentration more quickly and the increase in urine SO42- concentration was more
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slowly, probably because of the absence of SO42- in media. Since transfer to 60 mM Na+
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(30 mM Na2SO42) was without effect on switching, Na+ alone may be insufficient for
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the trigger. Therefore, Cl- ions in SW may be responsible for switching the SO42-
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regulatory system in eels.
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In order to analyze further the role of Na+ or Cl-, we transferred FW eels to media
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that contained either Na+ or Cl-, but 50 mM choline-Cl or 50 mM Na-gluconate had no
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effect on switching. Since10 mM Na++50 mM Cl- was effective but 50 mM Na++10
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mM Cl- was without effect on switching, we concluded that Cl- is a primary factor and
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Na+ has a permissive role in the Cl- effect. The threshold concentration of NaCl for
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switching was ca. 50 mM, which is much lower than the Cl- concentration in SW (~500
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mM). Especially, Slc26a6a showed a more sensitive and rapid response than Slc13a1
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(35). The high sensitivity to environmental Cl- concentration indicates that the eel can
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prepare for future exposure to high SO42- concentration during the course of
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downstream migration.
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Environmental Cl- ion as a trigger for SO42- regulation
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Cl- is known as a trigger for elicitation of drinking in SW (9). Teleosts drink
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copiously in SW to compensate for water lost osmotically (21, 32), and eels start
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drinking within one minute after transfer from FW to SW (33). Using a similar
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analytical technique, Hirano showed that Cl- alone (choline-Cl) is sufficient for
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initiation of drinking without Na+ in the eel (9). Ando and Nagashima also showed that
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luminal Cl- concentration in eel intestine is responsible for inhibition of excess drinking
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(1). As intestinal lumen is in effect the external environment, this also exemplifies
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environmental Cl- sensing. These reports indicate that Cl- sensing mechanisms exist on
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the body surface and intestinal epithelia. In contrast to the regulation of drinking,
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however, both Cl- and Na+ are required for switching the SO42- regulation. Furthermore,
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the change in SO42- regulation occurs in 1 - 3 days after SW transfer (35), which is in
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contrast to the immediate regulation of drinking. We found that environmental Cl- must
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be taken up into the circulation to manifest its effect on SO42- regulation. This idea is
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supported further by the fact that injection of Cl- into the circulation triggered the
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change in SO42- regulation, and that the sum of Cl- and SO42- in plasma is maintained at
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a certain level as confirmed by the inverse relationship of plasma SO42- and Cl-
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concentration. The current data support the notion that accumulation of high SO42- in
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plasma of FW eels is an energy-saving substitute for Cl- as plasma anions (23). In fact,
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eels are known to have poor ability to take up Cl- from the environment among teleost
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species (34), which may facilitate the use of SO42- as a Cl- substitute in starved fish.
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Na-Cl cotransporter responsible for Cl- uptake from the environment
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Only Cl- and a small amount of Na+ in media are sufficient for Cl- uptake that
13
initiates the switching of SO42- regulation. This indicates the involvement of Na-Cl
14
cotransporter (NCC) but not Na-K-2Cl cotransporter (NKCC) that requires K+ in media.
15
Anion exchanger that takes up Cl- in exchange of other anion is also unlikely as it
16
functions without Na+ in media (27). Recently, NCC was identified on the apical side of
17
the gill cells and suggested to be responsible for Na+ and Cl- uptake in zebrafish and
18
tilapia in FW (11, 12). Consistent with the results of zebrafish and tilapia, transfer of
19
FW eels to 150 mM NaCl solution containing HCTZ, a potent NCC blocker,
20
significantly inhibited Slc26a6a gene expression in the kidney compared with controls.
21
Therefore, NCC is the most likely candidate for Cl- uptake into the circulation in the
22
presence of Na+. Since injection of Cl- ion into blood changed the renal SO42-
23
transporter, the increase may be detected by a Cl- sensor in the kidney, probably in the
24
proximal tubular cells that are involved in the SO42- excretion (13, 33). However, the
16
1
pathway of information transfer that leads to changes in SO42- transporter genes has not
2
yet been identified. In mammals, a Cl- sensor is localized in the macula densa of renal
3
distal tubule, which is thought to be NKCC2 and responsible for tubule-glomerular
4
feedback and renin release (25).
5
Mg2+ is the second most abundant cation in SW (~50 mM) that is actively
6
excreted by the kidney of marine teleosts (7, 8). Because of the huge difference in Mg2+
7
concentration between plasma and SW, Mg2+ regulation at the kidney is also thought to
8
be altered by the environmental salinity in fishes (2, 4). Recently, transient receptor
9
potential melastatin type channels (TRPM6 and 7) were found to be involved in the
10
Mg2+ reabsorption in the distal convoluted tubule of mammals (30). The TRP channels
11
may act also as a sensor, because TRP vanilloid type channels (TRPV2 and 4) are
12
shown to be an osmosensor for regulation of thirst and vasopressin secretion (31).
13
However, these transporters have not been identified in teleosts, and much less is known
14
about SO42- regulation compared with Mg2+ regulation in fish (2, 4).
15
To summarize the data obtained in this study and from previous studies, a model
16
for switching the SO42- regulation in the eel in FW and SW is suggested in Fig. 8. After
17
transfer to SW, Cl- is taken up from SW in parallel with Na+ probably via the NCC,
18
resulting in an transient increase in plasma Cl- concentration. The increase is detected
19
by an unknown sensor in the circulatory system and turns on the switch for SO42-
20
excretion by the kidney through down-regulating Slc13a1 and up-regulating Slc26a6a
21
in the proximal tubules (13, 23, 35), which facilitates SO42- excretion into the tubular
22
lumen and decreases plasma SO42- concentration. We showed that Slc26a6a, b and c are
23
present on the apical membrane and Slc26a1 on the basolateral membrane of the
24
epithelial cells of proximal tubule of SW eel kidney (35). The Slc26a6a gene is
17
1
expressed only after SW transfer, while the Slc26a6b and Slc26a6c genes are expressed
2
in both media and slightly up-regulated after transfer to SW. The up-regulation of the
3
Slc26a6a gene in SW has been shown previously in the pufferfish (13). We also found
4
that Slc26a6b and Slc26a1 are localized in the same epithelial cells in P2 segment of the
5
proximal tubule and Slc26a6a and c on the apical membrane of the same cells in P1
6
segment of the proximal tubule of SW eels (35). However, the counterpart of Slc26a6a
7
and Slc26a6c in the basolateral membrane has not been determined yet. In the pufferfish,
8
Slc26a6a and Slc26a1 are suggested to be in the same proximal tubular cells, but this is
9
not the case in SW eel kidney (13).
10
11
Significance and Perspectives
12
In this study, we found that Cl- in SW, not SO42-, is responsible for switching the renal
13
SO42- regulation to an excretory type to maintain low plasma SO42- concentration in SW
14
using eels that can survive direct transfer from FW to SW. We further showed that Cl-
15
enters the circulation via NCC with the help of Na+ and probably acts on the renal
16
proximal tubular cells to turn on the excretory SO42- transporter (Slc26a6a) and turn off
17
the absorptive SO42- transporter (Slc13a1) through regulation of the gene expression.
18
This is the first in vertebrates to identify the environmental and internal ions that reverse
19
the SO42- regulation and the route of uptake that leads to the gene expression at the
20
target tissue. The use of eels made these findings possible because this fish changes the
21
SO42- regulation most profoundly among euryhaline teleosts in FW and SW
22
environment. Thus, this study illustrates the advantage of comparative fish studies for
23
osmoregulation. In addition, it is of interest to compare the switching factor with other
24
osmoregulatory organs such as the gills where mitochondria-rich cells also changes
18
1
2
from absorptive to excretory type in FW and SW, respectively.
Next target of research seems to identify the sensor or receptor for Cl- ions
3
located within the circulatory system. We expect that the sensor may be the protein that
4
transport Cl- ion such as Cl channels, or TRP-type receptors as discussed above. The
5
location of the Cl- sensor may be at the epithelial cells of renal proximal tubule that
6
express Slc26a6a and Slc13a1 gene as these genes are expressed in SW and FW,
7
respectively, in eels. We have identified the cells that express these genes in different
8
segments of the proximal tubule of eel kidney (35). Therefore, we may be able to
9
examine the co-localization of the sensor and transporters in each epithelial cell by
10
immunocytochemical study after we can identify the sensor. We are now searching for
11
Cl- sensor in the buccal cavity and the intestinal lumen that regulates drinking in the eel
12
(1, 9). We are interested in the identity of the Cl- sensor with the renal Cl- sensor for
13
SO42- regulation.
14
15
16
17
ACKNOWLEDGEMENTS
We thank Dr. Susumu Hyodo, Dr. Makoto Kusakabe, Dr. Jillian Healy and Ms.
Sanae Hasegawa of this laboratory for comments on the manuscript and advice.
18
19
20
GRANTS
This research was supported in part by Grant-in-Aid for Basic Research (A) from
21
Japan Society for the Promotion of Science to Y. T. (13304063 and16207004). T. W.
22
was supported by Global COE Program (Integrative Life Sciences Based on Study of
23
Biosignaling Mechanisms), MEXT, Japan.
24
19
1
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35. Watanabe T, and Takei Y. Molecular physiology and functional morphology of
2
SO42- excretion by the kidney of seawater-adapted eels. J Exp Biol 214: (in press)
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1
Figure legends
2
Fig. 1 Time-course changes in a) plasma and b) urine SO42- concentration after transfer
3
of FW eels to FW (open circle), 30 mM Na2SO4 (open triangle), 30 mM MgSO4
4
(closed triangle), or SW (closed circle). c) Gene expression of SO42- transporters in
5
the kidney 7 days after transfer to each medium.
6
Fig. 2 Time-course changes in a) plasma and b) urine SO42- concentration after transfer
7
of SW eels to SO42--enriched SW (open circle), SO42--free SW (double circle) and
8
normal SW (closed circle). c) Gene expression of SO42- transporters in the kidney
9
7 days after transfer to each medium.
10
Fig. 3 Time-course changes in a) plasma and b) urine SO42- concentration after transfer
11
of FW eels to FW (open circle), 10 mM CaCl2 (open square), 20 mM NaCl (open
12
triangle), 50 mM NaCl (open inverted triangle), 150 mM NaCl (closed triangle),
13
450 mM NaCl (closed inverted triangle) and SW (closed circle). c) Gene
14
expression of SO42- transporters in the kidney 7 days after transfer to each
15
medium.
16
Fig. 4 Changes in a) plasma and b) urine SO42- concentration after transfer of FW eels to
17
media of different Na+ and Cl- concentrations. FWMan, 100 mM mannitol; FWNaG,
18
50 mM sodium gluconate; FWChCl, 50 mM choline chloride; FWNa50Cl10, 50 mM
19
Na+ + 10 mM Cl-; FWNa10Cl50, 10 mM Na+ + 50 mM Cl-.
20
Fig. 5 Correlations between plasma SO42- concentration and a) Cl-, b) Na+ c) Ca2+
21
concentration and d) osmolality in eels 7 days after transfer to media of different
22
ionic conditions.
23
Fig. 6 Expression of the (a) Slc26a6a, (b) Slc13a1 gene in the kidney of FW eels 24 h
24
after injection of each solution (on the abscissa) into the circulation. *p<0.05 All
25
1
2
results were expressed as means ± S.E.M.
Fig. 7 Expression of the (a) Slc26a6a, (b) Slc13a1 gene in the kidney of FW eels 72 h
3
after transfer to 150 mM NaCl solution containing 10-8 M hydrochlorochiazide, a
4
blocker of Na-Cl cotransporter. *p<0.05 All results were expressed as means ±
5
S.E.M.
6
7
Fig. 8 A schematic drawing of hypothetical pathway for switching SO42- regulation after
transfer of eels from FW to SW with emphasis on renal regulation.
26
Table 1 Ionic composition of experimental media.
27
Table 2 Primers for expression analysis.
Fig. 1 Watanabe and Takei
Fig. 2 Watanabe and Takei
Fig. 3 Watanabe and Takei
Fig. 4 Watanabe and Takei
Fig. 5 Watanabe and Takei
1000
120
800
80
600
400
40
200
relative expression (%)
1200
3
Cl
4
SO
l2
gC
2
Na
M
Na
M
M
M
5
1.
5
1.
Cl
l2
gC
Cl
Na
Na
M
4
SO
Fig. 6 Watanabe and Takei
9%
0.
M
M
2
Cl
Na
Na
M
9%
0.
5
1.
5
1.
3
0
0
160
relative expression (%)
*
*
b
*
a
0
80
Cl
Na
Cl
Na
Cl
Cl
a
TZ M N
HC m
M 50
-8
1
10 +
M
m
M
m
a
TZ M N
HC m
M 50
-8
1
10 +
0
15
Fig. 7 Watanabe and Takei
40
40
0
15
0
80
relative expression (%)
120
120
relative expression (%)
b
*
a
Fig. 8 Watanabe and Takei