Am J Physiol Regul Integr Comp Physiol 301: R402–R411, 2011.
First published May 4, 2011; doi:10.1152/ajpregu.00624.2010.
Environmental factors responsible for switching on the SO2⫺
excretory
4
system in the kidney of seawater eels
Taro Watanabe and Yoshio Takei
Laboratory of Physiology, Department of Marine Bioscience, Atmosphere and Ocean Research Institute, The University
of Tokyo, Kashiwa, Chiba, Japan
Submitted 16 September 2010; accepted in final form 29 April 2011
euryhaline teleost; sulfate transporter; solute carrier 26; kidney
(SO2⫺
4 ) play important roles in a variety of
metabolic and cellular processes. Possible physiological functions include production of chondrocytes and mucus as an
important component, detoxification of exogenous substances
by sulfation, elimination of waste compounds by sulfoconjugation, and biosynthesis of sulfated hormones, such as gastrin
and cholecystokinin (17). Accordingly, slight imbalance of
plasma SO2⫺
often leads to clinical syndromes (29). Despite
4
the importance of SO2⫺
4 regulation, the research on ion regulation has focused mainly on monovalent ions (Na⫹ and Cl⫺
for osmoregulation and H⫹ and HCO⫺
3 for pH regulation) and
divalent cations (Ca2⫹ and Mg2⫹ for regulation of muscular
contraction and others).
SULFATE IONS
Address for reprint requests and other correspondence: T. Watanabe, Center
for Cooperative Research Promotion, Atmosphere and Ocean Research Institute The Univ. of Tokyo, 5-1-5, Kashiwanoha, Kashiwa, Chiba, 277-8564,
Japan (e-mail: [email protected]).
R402
Vertebrate habitats can be divided largely into two categories in
2⫺
terms of SO2⫺
4 abundance: SO4 -poor [land and freshwater (FW)]
-rich
[seawater,
(SW)]
environment. As for aquatic
and SO2⫺
4
but SW
environments, FW contains only ⬃0.3 mM of SO2⫺
4
as
the
second
most-abundant
anion
(⬃30
mM).
contains SO2⫺
4
Therefore, teleost fish have opposite SO2⫺
4 regulation in FW and
SW, as their plasma typically contains ⬃1 mM of SO2⫺
4 regardless of FW and SW species. Among teleosts, euryhaline species
must reverse SO2⫺
4 regulation when they move between FW and
SW. It has been suggested that the gills and intestine are almost
2⫺
impermeable to SO2⫺
4 (6, 21), but obligatory influx of SO4 was
nullified by excretion via the kidney in marine teleosts (3, 7, 8,
28). We recently showed that the eel is unique in SO2⫺
4 regulation
compared with other euryhaline teleosts because it has much
higher plasma SO2⫺
4 in FW (⬃6 mM) than in SW (⬃1 mM) (35)
as reported previously (23). We also showed that 85% of the
2⫺
SO2⫺
4 in SW is taken up by the gills and 97% of SO4 that enter
the circulation is excreted by the kidney in SW eels (Watanabe T,
Takei Y, unpublished data).
Among members of the solute carrier (Slc) superfamily of
transporters, Slc4a1 (AE1), Slc13a1 (NaS-1), Slc26a1 (Sat-1),
Slc26a2 (DTDST), Slc26a3 (DRA), Slc26a6 (CFEX, PAT1),
Slc26a7, Slc26a8, Slc26a9, and Slc26a11 have been implicated in
transport in mammals (20). In teleost fish, Slc13a1,
SO2⫺
4
Slc26a1, Slc26a3, and Slc26a6a,b,c have been cloned in the eel
(23), Slc26a1 in the rainbow trout (14), Slc13a1 in the zebrafish
(19), and Slc26a6a,b,c in the pufferfish Takifugu obscurus (13).
Among them, Slc13a1 and Slc26a1 play key roles in SO2⫺
4
reabsorption at the renal proximal tubule of mammals (5) and FW
eels (23). On the other hand, Slc26a6 and Slc26a1 are suggested
to be involved in renal SO2⫺
4 secretion in mammals (16, 20). In
fishes, Slc26a1 seems to be involved in renal SO2⫺
4 secretion in
rainbow trout (14), Slc26a6a in pufferfish (13), and Slc26a6a,b,c
and Slc26a1 in SW eels (35). However, it is not yet known which
factor(s) in SW are responsible for changing the expression of the
transporter genes during the course of SW adaptation in euryhaline fishes.
High SO2⫺
4 diet induced a decrease in Slc13a1 expression in
the renal proximal tubule of mammals (18, 26) and injection of
Na2SO4 into the circulation increased Slc26a1 transcripts in the
kidney of rainbow trout (14), indicating the role of SO2⫺
4 in the
transporter regulation. It seems that major ions in SW are
responsible for changes in the transporter gene expression, but
it remains to be determined which ion(s) actually switch the
regulation to a SW type. We used the eel as a
renal SO2⫺
4
model species because it changes the SO2⫺
regulation most
4
drastically among euryhaline species (23, 35). Initially, we
examined the time course changes in SO2⫺
regulation after
4
transfer of FW eels to SW using plasma and urine SO2⫺
4
concentrations and expression of Slc13a1 and Slc26a6a genes
0363-6119/11 Copyright © 2011 the American Physiological Society
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Watanabe T, Takei Y. Environmental factors responsible for switching on the SO2⫺
excretory system in the kidney of seawater eels. Am J
4
Physiol Regul Integr Comp Physiol 301: R402–R411, 2011. First published May 4, 2011; doi:10.1152/ajpregu.00624.2010.—Eels are unique
in that they maintain lower plasma SO2⫺
concentration in SO2⫺
4
4 -rich
(⬃30 mM) seawater (SW) than in SO2⫺
-poor
(⬍0.3 mM) freshwater
4
(FW), showing drastic changes in SO2⫺
regulation between FW and
4
SW. We previously showed that the expression of renal SO2⫺
trans4
porter genes, FW-specific Slc13a1 and SW-specific Slc26a6a,
changes profoundly after transfer of FW eels to SW, which results in
the decrease in plasma SO2⫺
4 concentration after 3 days in SW. In this
study, we attempted to identify the environmental factor(s) that trigger
the switching of SO2⫺
regulation using changes in plasma and urine
4
SO2⫺
concentrations and expression of the transporter genes as
4
markers. Transfer of FW eels to 30 mM SO2⫺
or transfer of SW eels
4
2⫺
to SO2⫺
4 -free SW did not change the SO4 regulation. Major divalent
cations in SW, Mg2⫹ (50 mM) and Ca2⫹ (10 mM), were also
ineffective, but 50 mM NaCl was effective for switching the SO2⫺
4
regulation. Further analyses using choline-Cl and Na-gluconate
showed that Cl⫺ is a primary factor and Na⫹ is permissive for the Cl⫺
effect. Since plasma SO2⫺
and Cl⫺ concentrations were inversely
4
correlated, we injected various solutions into the blood and found that
Cl⫺ alone triggered the switching from FW to SW-type regulation.
Furthermore, the inhibitor of Na-Cl cotransporter (NCC) added to
media significantly impaired the expression of SW-specific Slc26a6a
in 150 mM NaCl. In summary, it appears that Cl⫺ ions in SW are
taken up into the circulation via the NCC together with Na⫹, and the
resultant increase in plasma Cl⫺ concentration enhances SO2⫺
excre4
tion by the kidney through downregulation of absorptive Slc13a1 and
upregulation of excretory Slc26a6a, resulting in low plasma SO2⫺
4
concentration in SW.
R403
RENAL SULFATE REGULATION IN EEL
in the kidney as markers. We then attempted to identify the
ions responsible for the switching by transferring fish to various ionic environments. As environmental Na⫹ and Cl⫺ were
suggested as responsible ions, we further examined how the
information of these ions is transmitted into the body to turn on
the switch using a specific inhibitor [hydrochlorochiazide
(HCTZ)] of Na-Cl cotransporter (NCC) that is present in the
gills and suggested to take up these ions from media.
MATERIALS AND METHODS
Animals
Transfer Experiment to Various Ionic Environments
Effect of changes in environmental SO2⫺
in FW eel. Six FW eels
4
were transferred either to SO2⫺
4 -enriched FW (30 mM Na2SO4 or 30
mM MgSO4), FW (control) or SW (positive control), and urine and
plasma samples were collected 1, 3, and 7 days after transfer. After 7
days, the kidney was dissected for gene expression analysis as
mentioned below. Details of ionic composition of experimental water
are described in Table 1.
Effect of changes in environmental SO2⫺
in SW eel. Six SW eels
4
were transferred either to SO2⫺
4 -enriched SW (added with 30 mM
Na2SO4 up to double strength of typical SW), SO2⫺
4 -free artificial
SW, or SW. The SO2⫺
4 -free SW was prepared by dissolving 1,502 g
NaCl, 407.0 g MgCl2-6H2O, and 44.4 g CaCl2 in 40 liters of FW (see
Table 1). Samples were collected 1 wk after the transfer as mentioned
below.
Effect of Intravascular Injection of Various Ions on Switching
in FW Eel
Eight FW eels were anesthetized in the tricaine methanesulfonate
for 15 min and cannulated with a polyethylene tube (0.5 mm ID, 0.8
mm OD) into the ventral aorta. After surgery, eels were placed in
plastic troughs with a circulating water system at 18°C. After ⬎ 18 h
of recovery, 100 ␮l of isotonic 0.15 M NaCl (control), 3 M NaCl, 1.5
M Na2SO4 (Na⫹ alone), or 1.5 M MgCl2 (Cl⫺ alone) were injected
(n ⫽ 5 in each group) in 1 min through the cannula into the circulation
followed by a flush with 100 ␮l of isotonic NaCl. Subsequently, blood
was collected 24 h after injection for measurement of ion concentrations, and the kidney was dissected out after anesthesia for measurement of Slc gene expression.
Table 1. Ionic composition of experimental media
SW
FW
Na⫹
Mg2⫹
Ca2⫹
CI⫺
SO2⫺
4
osmolality
450
⬍1.0
50
⬍0.5
10
⬍0.25
525
⬍0.5
30
⬍0.3
1080
⬍10
Effect of environmental SO2⫺
in FW eel
4
FW ⫹ Na2SO4
FW ⫹ MgSO4
60
⬍1.0
⬍0.5
30
⬍0.25
⬍0.25
⬍0.5
⬍0.5
30
30
75
50
Effect of environmental SO2⫺
in SW eel
4
SO4 free SW
SW ⫹ Na2SO4
450
510
50
50
10
10
570
525
⬍0.2
60
1070
1150
⬍0.2
⬍0.2
⬍0.2
⬍0.2
⬍0.2
35
40
100
300
900
⬍0.2
⬍0.2
⬍0.2
⬍0.2
⬍0.2
100
75
90
100
105
Effect of various concentrations of NaCl in FW eel
FW
FW
FW
FW
FW
⫹
⫹
⫹
⫹
⫹
CaCl2
20 mM NaCl
50 mM NaCl
150 mM NaCl
450 mM NaCl
⬍1.0
20
50
150
450
⬍0.5
⬍0.5
⬍0.5
⬍0.5
⬍0.5
10
⬍0.25
⬍0.25
⬍0.25
⬍0.25
20
20
50
150
450
Effect of Na⫹ and/or Cl⫺ in FW eel
FW
FW
FW
FW
FW
⫹
⫹
⫹
⫹
⫹
mannitol (osmolality)
Na-gluconate (Na⫹ alone)
choline-Cl (Cl⫺ alone)
50 mM Na⫹ ⫹ 10 mM Cl⫺
10 mM Na⫹ ⫹ 50 mM Cl⫺
⬍1.0
50
⬍1.0
50
10
⬍0.5
⬍0.5
⬍0.5
⬍0.5
⬍0.5
⬍0.25
⬍0.25
⬍0.25
⬍0.25
⬍0.25
⬍0.5
⬍0.5
50
10
50
Values are in micromoles. SW, saltwater; FW, freshwater.
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Cultured eels of ⬃200 g were purchased from a local dealer. All
eels were kept without feeding at 18°C in FW tanks for 2 to 4 wk until
experimentation. SW eels were transferred to SW and acclimated
to full SW for at least 2 wk before use. Ionic composition of FW
is 1.0 mM [Na⫹], 0.5 mM [Cl⫺], 0.25 mM [Ca2⫹], 0.5 mM
⫹
[Mg2⫹], 0.3 mM [SO2⫺
4 ], and that of SW is 450 mM [Na ]; 525
mM [Cl⫺]; 10 mM [Ca2⫹], 50 mM [Mg2⫹]; 30 mM [SO2⫺
4 ]. All
conditions for fish maintenance and experiments were approved by
the Committee for Animal Experiments at the University of Tokyo.
Effect of various concentrations of NaCl solution in FW eel. Six
FW eels were transferred either to FW containing 0 (control), 20, 50,
150 (isotonic), or 450 (SW level) mM NaCl (see Table 1), and
samples were collected for 1 wk after transfer as mentioned below. In
addition, six FW eels were transferred to 10 mM CaCl2 solution to
evaluate the role of Ca2⫹ ions in SW.
Effect of Na⫹ and/or Cl⫺ in FW eel. Five FW eels were transferred
either to 1) 50 mM Na-gluconate (Na⫹ alone), 2) 50 mM choline-Cl
(Cl⫺ alone), 3) 50 mM Na-gluconate⫹10 mM choline-Cl, or 4) 10
mM Na-gluconate⫹50 mM choline-Cl to evaluate relative importance
of Na⫹ and/or Cl⫺, or 5) 100 mM mannitol (equivalent to 50 mM
NaCl) to evaluate the role of osmolality in SO2⫺
regulation. Ionic
4
compositions of the solutions were described in Table 1. Samples
were collected for 1 wk after transfer to each solution.
For sampling, eels were lightly anesthetized in 0.1% (wt/vol)
tricaine methanesulfonate (Sigma, St. Louis, MO), and 100 ␮l of
blood was collected from the caudal vein into a chilled syringe
containing 2K-EDTA (20 ␮l/ml) and bladder urine was collected by
a syringe just before and 1, 3, and 7 days after transfer. Blood was
centrifuged at 10,000 g for 5 min at 4°C to prepare plasma. After
sampling at day 7, eels were euthanized with 2-phenoxy ethanol
(Wako Pure Chemical Industries, Osaka, Japan), and kidneys were
immediately dissected out, frozen in liquid nitrogen, and stored at
⫺80°C for later analysis.
R404
RENAL SULFATE REGULATION IN EEL
Effect of NCC Blocker in Media on Switching in FW Eel
⫹
⫺
Since it was shown that both Na and Cl are required for
switching of SO2⫺
4 regulation from FW type to SW type, we evaluated
the role of NCC in ion uptake from media. For this purpose, two
groups of FW eels were transferred to 150 mM NaCl solutions
containing 0 (control), 10⫺8 M, or 10⫺6 M HCTZ, an NCC blocker.
Since both HCTZ concentrations inhibited switching, 10 eels were
transferred either to 150 mM NaCl solution with or without 10⫺8 M
HCTZ for 3 days, and blood and the kidney were sampled for
subsequent analyses.
Measurement of Plasma Osmolality and Ion Concentrations
in Plasma and Urine
Expression Analysis of Renal SO2⫺
Transporter Genes
4
Since five Slc genes (Slc13a1, Slc26a1, and Slc26a6a,b,c) were shown
to be expressed abundantly in the kidney, changes in their expression
were examined after transfer to media with various ion concentrations.
One microgram of total RNA from kidney was reverse transcribed using
the SuperScript III First Strand Synthesis System for RT-PCR (Invitrogen, Carlsbad, CA). PCR was performed using gene-specific sense
and antisense primer combinations (Table 2) based on the sequences in
the database; Slc13a1 (AB111926), Slc26a1 (AB111927), Slc26a6a
(AB084425), Slc26a6b (AB111928), Slc26a6c (AB111929), and ␤-actin
(AB074846) 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.
Table 2. Primers for expression analysis
Gene
For RT-PCR
Primer Sequence
Sense
Slc13a1
Slc26a1
Slc26a6a
Slc26a6b
Slc26a6c
␤-actin
5=-CTGGATGACTGAAGCCTTGC-3=
5=-CCAGCCTGAATGACATGTGG-3=
5=-TCACTGAGATGCTCCGTGCC-3=
5=-AGCAACCGCGTGGACATGAT-3=
5=-TTGCAAAATTGACCTGCTGG-3=
5=-CTCCCTGGAGAAGAGCTACGAGC-3=
Slc13a1
Slc26a1
Slc26a6a
Slc26a6b
Slc26a6c
␤-actin
5=-GACGGAGACGACCAGCTTCA-3=
5=-GCCTTGGTGTCGAGGGATCT-3=
5=-ACTGTCTGGGGCTAAACGTTCT-3=
5=-GGAAAATGACCAGGCCAGGT-3=
5=-GCAGAGGAGCGGAAGATGGT-3=
5=-GACGGAGTATTTGCGCTCAGG-3=
Antisense
For realtime PCR
Sense
Slc13a1
Slc26a6a
GAPDH
5=-GGTCACTGTGCAGCGGAAA-3=
5=-AAGAGCTCAACTCGGCCTACAG-3=
5=-CGACTTCAATGGAGACACCCA-3=
Slc13a1
Slc26a6a
GAPDH
5=-TCAAGCTCGTCCAACTGAAGAG-3=
5=-AATCAGAGTCCCAATCACAATAACAA-3=
5=-CACAAAGTGGTCATTCAGTGCA-3=
Antisense
AJP-Regul Integr Comp Physiol • VOL
Quantitative analyses of gene expression were performed by realtime PCR for Slc26a6a and Slc13a1 in the ion injection experiment
and in the HCTZ experiment. The GAPDH gene expression was used
as an internal control. Reactions were performed with the SYBR
Green method using Kapa SYBR First qPCR Kit (Kapa Biosystems,
Cape Town, South Africa) in a ABI Prism 7900HT Sequence Detection System (PE Applied Biosystems, Foster City, CA). The primers
for Slc26a6a and Slc13a1 were designed using Primer Express software (PE Applied Biosystems), and those for eel GAPDH have been
described previously (24) (Table 2). The relative expression of the
transporters was normalized by the amount of GAPDH mRNA and
compared between experimental and control groups. All measurements were performed in duplicate.
Statistical Analyses
Changes in SO2⫺
4 concentration were analyzed by Dunnett’s test or
by Tukey-Kramer test. Correlations between plasma SO2⫺
and other
4
ion concentrations were analyzed by the linear regression analysis. All
analyses were performed using the statistical software, KyPlot 5.0
(Kyens, Tokyo, Japan). Significance was determined at *P ⬍ 0.05. All
results were expressed as means ⫾ SE.
RESULTS
Effect of Environmental SO2⫺
4 on Switching
Plasma SO2⫺
concentrations increased gradually after
4
transfer of FW eels to 30 mM Na2SO4 or MgSO4, and the
increase was significant after 3–7 days (Fig. 1A). However,
plasma SO2⫺
concentration decreased 3 days after transfer
4
2⫺
of FW eels to SW that contains 30 mM SO2⫺
4 . Urine SO 4
concentration continued to be low in eels transferred to 30
mM Na2SO4 or MgSO4 (Fig. 1B) even though plasma SO2⫺
4
concentration increased. Neither Slc13a1 nor Slc26a6a gene
expression changed after transfer to the Na2SO4 or MgSO4
solution (Fig. 1C). These results clearly show that the elevation
of SO2⫺
concentration in plasma or media is not a sufficient
4
stimulus to switch on the renal SO2⫺
4 excretory system in FW fish.
2⫺
Effect of SO2⫺
4 -Enriched or SO4 -Free SW on Switching
Plasma SO2⫺
4 concentration increased 1 day after transfer of
2⫺
SW eels to SO2⫺
4 -enriched SW (60 mM SO4 ), but gradually
decreased thereafter to the level of SW eels (Fig. 2A). Plasma
SO2⫺
4 concentration did not change 1 day after transfer of SW
eels to SO2⫺
4 -free SW, but decreased to a level lower than SW
eels thereafter. Urine SO2⫺
4 concentration tended to be higher
after transfer to SO2⫺
4 -enriched SW and lower after transfer to
SO2⫺
4 -free SW but the changes were not significant compared
with SW eels (Fig. 2B). The expression of Slc13a1 and
Slc26a6a did not change after transfer to SO2⫺
4 -enriched or
2⫺
SO2⫺
from
4 -free SW (Fig. 2C). Therefore, removal of SO4
2⫺
SW did not switch off the renal SO4 excretory system of SW
fish. The result of the experiment using SO2⫺
4 -enriched SW also
showed that the SW eel kidney has an extra capacity to extrude
2⫺
SO2⫺
4 and maintain plasma SO4 concentration at normal.
Effect of Environmental Na⫹ and/or Cl⫺ on Switching
Plasma SO2⫺
4 concentration gradually decreased after transfer of FW eels to 50 – 450 mM NaCl, and the decrease was
significant after 3–7 days, but 20 mM NaCl was not effective
(Fig. 3A). The degree of the decrease was concentration
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Plasma osmolality was measured using a vapor pressure osmometer (Type 5520; Wescor, Logan, UT). Anions (Cl⫺ and SO2⫺
4 ) and
cations (Na⫹ and Ca2⫹) in plasma and urine were measured using ion
chromatography (cat. no. AV10; Shimadzu, Kyoto, Japan) using
anion-exchange (IC-A3) and cation-exchange (IC-C3) column, respectively. The lower detection limit in this study is ⬃5 ␮M. Standard
curves for the anions and cations were prepared by commercial
standard solutions (Shimadzu).
Quantitative Analyses of Gene Expression
R405
RENAL SULFATE REGULATION IN EEL
dependent between 50 and 450 mM after 1–3 days, although
all fish did not survive for 7 days after transfer to 450 mM
NaCl. Plasma SO2⫺
concentration did not change after
4
transfer to 10 mM CaCl2 (Fig. 3A). Urine SO2⫺
concentra4
tion increased significantly 3 days after transfer to 150 mM
NaCl, although the increase was much smaller than after
transfer to SW (Fig. 3B). No change was detected in urine
SO2⫺
concentration after transfer to lower concentrations of
4
NaCl and 10 mM CaCl2. Urine could not be collected in a
sufficient volume for analysis after transfer to 450 mM NaCl
Fig. 2. Time course changes in plasma (A)
and urine (B) SO2⫺
4 concentration after transfer of saltwater (SW) eels to SO2⫺
4 -enriched
SW (Œ), SO2⫺
4 -free SW (double circle), and
normal SW (). C: gene expression of SO2⫺
4
transporters in the kidney 7 days after transfer to each medium. **P ⬍ 0.01.
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Fig. 1. Time course changes in plasma (A) and
urine (B) SO2⫺
concentrations after transfer
4
of freshwater (FW) eels to FW (Œ), FW⫹30
mM Na2SO4 (), FW⫹30 mM MgSO4 (Œ),
or SW (). C: gene expression of SO2⫺
4 transporters in the kidney 7 days after transfer to
each medium. SO2⫺
4 , sulfate ions. *P ⬍ 0.05;
**P ⬍ 0.01; ***P ⬍ 0.001.
R406
RENAL SULFATE REGULATION IN EEL
solution. Renal expression of Slc13a1 was downregulated 7
days after transferring FW eels to more than 50 mM NaCl,
while Slc26a6a was upregulated when fish were exposed to 20
mM NaCl (Fig. 3C). The result showed that Cl⫺ alone or Na
and/or Cl⫺ trigger the change in renal SO2⫺
regulatory sys4
tems, and its threshold was between 20 and 50 mM.
Relative Importance of Cl⫺ and Na⫹ on Switching
To determine which ion, Na⫹ and/or Cl⫺, is of primary
importance for the switching, we first transferred FW eels to 50
mM Na-gluconate (Na⫹ alone) or 50 mM choline-Cl (Cl⫺
alone) solutions, but plasma and urine SO2⫺
4 concentration and
transporter gene expression were not altered for 7 days (Fig. 4).
After transfer to solutions containing 50 mM Na⫹⫹10 mM
Cl⫺ or 10 mM Na⫹ ⫹ 50 mM Cl⫺, only the latter decreased
plasma SO2⫺
concentration as did transfer to 50 mM NaCl
4
solution (Fig. 4A). Downregulation of Slc13a1 and upregulation of Slc26a6a were apparent in the kidney of fish transferred
to solutions containing 50 mM Nacl or 10 mM Na⫹ ⫹ 50 mM
Cl⫺ (Fig. 4C), but the increase in urinary SO2⫺
4 concentration
was smaller than that after SW transfer (Fig. 4B). Therefore,
Cl⫺ is a primary ion in SW that triggers the switching of SO2⫺
4
excretory system in the kidney and Na⫹ has a permissive role.
Effect of Intravascular Injection of Ions on Switching
Intra-arterial injection of 1.5 M MgCl2 or 3 M NaCl upregulated Slc26a6a expression in the kidney of FW eels after 24 h,
but 1.5 M Na2SO4 or isotonic 0.15 M NaCl (control) was
without effect (Fig. 6A). Only 3 M NaCl solution significantly
downregulated the Slc13a1 expression (Fig. 6B). Plasma Cl⫺
concentration (n ⫽ 5 in each group) changed from 107.6 ⫾ 5.5
to 103.0 ⫾ 2.1 mM 24 h after isotonic NaCl injection, 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 mM after 1.5 M MgCl2, and
from 104.6 ⫾ 3.5 to 110.8 ⫾ 4.5 mM after 3 M NaCl. These
results strongly suggest that a transient increase in plasma
Cl⫺ concentration after injection of Cl⫺-containing solution
triggered switching of renal SO2⫺
regulation.
4
Effect of NCC Blocker in Media on Switching
Plasma concentrations of SO2⫺
and Cl⫺ were not signifi4
cantly different between HCTZ-treated fish and controls. The
upregulation of Slc26a6a expression observed significantly
after transfer to 150 mM NaCl (Fig. 7A). However, Slc13a1 did
not changed after transfer (Fig. 7B).
DISCUSSION
Relationship Between SO2⫺
4 and Other Ions in Plasma
When plasma SO2⫺
4 concentration was plotted against other
ion concentration in plasma, a significant negative correlation
was detected with Cl⫺ but not with Na⫹, Ca2⫹, and osmolarity
(Fig. 5). Plasma Cl⫺ showed positive correlation with the NaCl
concentration in media (data not shown). These results suggest an
intimate relationship between Cl⫺ and SO2⫺
concentrations in
4
plasma and between Cl⫺ concentrations in plasma and media.
AJP-Regul Integr Comp Physiol • VOL
Since SO2⫺
is excess in SW compared with body fluids,
4
marine fish must cope with SO2⫺
4 influx into the body across
concentration gradients. Using 35SO2⫺
4 , we found that in SWadapted eels, 85% of SO2⫺
uptake
occurs
via the gills and 15%
4
via digestive tracts, whereas 97% of SO2⫺
4 excretion occurs via
the kidney (Watanabe T, Takei Y, unpublished data). The role
of the kidney in SO2⫺
4 excretion has been reported in several
species of marine fish (3, 7, 8, 28). We also found that
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Fig. 3. Time course changes in plasma (A)
and urine (B) SO2⫺
4 concentration after transfer of FW eels to FW (Œ), FW⫹10 mM CaCl2
(□), FW⫹20 mM NaCl (), FW⫹50 mM
NaCl (), FW⫹150 mM NaCl (Œ), FW⫹450
mM NaCl () and SW (). C: gene expression of SO2⫺
transporters in the kidney 7
4
days after transfer to each medium. *P ⬍
0.05, **P ⬍ 0.01, ***P ⬍ 0.001.
RENAL SULFATE REGULATION IN EEL
R407
euryhaline tilapia and chum salmon maintain slightly higher
plasma SO2⫺
4 concentrations in SW than in FW, but the eel is
an exception with much lower plasma SO2⫺
4 concentration in
SW than in FW (Watanabe T, Takei Y, unpublished data). The
unique SO2⫺
accumulation in FW eel plasma was reported
4
previously by Nakada et al. (23). Thus the eel exhibits drastic
changes in SO2⫺
regulation in the kidney after transfer from
4
FW to SW and thus serves as an excellent model to investigate
the switching mechanism of SO2⫺
regulation. In mammals,
4
SO2⫺
filtered by the kidney easily reaches saturation and
4
excess SO2⫺
is excreted into the urine (15, 22). Therefore,
4
apparent switching does not seem to occur in mammals,
although the presence of a SO2⫺
excretory system is also
4
suggested in mammals (16, 20).
Environmental Ions Responsible for Switching
SO2⫺
4 Regulation
When euryhaline fish migrate between FW and SW, the
mechanisms for ion regulation change drastically to an opposite direction, from absorption to excretion or vice versa at the
osmoregulatory organs (gills, kidney, and intestine). However,
there has been no report on the factors in SW that trigger such
AJP-Regul Integr Comp Physiol • VOL
changes in ion regulation. For example, mitochondria-rich cells
in the gills are altered from an absorptive FW type to an
excretory SW type when euryhaline teleosts are transferred
from FW to SW (10). The environmental factors that induce
the change are most likely Na⫹ or Cl⫺ ions that exist at high
concentrations in SW and are the target of regulation by the
cells, but there is no report on the factor that triggers the
change. In this study, we investigated such factors that trigger
the change in SO2⫺
4 regulation using plasma and urine concentrations and renal SO2⫺
transporters as markers. As for the
4
transporters, we chose Slc13a1 and Slc26a6a because they are
known to be expressed only in FW and SW eels, respectively
(23, 35). The higher expression of the Slc26a6a gene in SW is
also reported very recently in the euryhaline pufferfish,
Takifugu obscurus (13). Since intravascular injection of SO2⫺
4
downregulates Slc26a1 in trout (14), we expected that SO2⫺
4
may be responsible for the change. However, neither addition
2⫺
of 30 mM SO2⫺
4 to FW nor removal of SO4 from SW affected
2⫺
the SO4 regulation in FW and SW eels, respectively. In
addition, injection of SO2⫺
4 directly into the blood of FW eels
did not trigger the change. Therefore, SO2⫺
4 is not responsible
for the switching of SO2⫺
regulation.
The
high osmolality or
4
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⫹
⫺
Fig. 4. Changes in plasma (A) and urine (B) SO2⫺
4 concentration after transfer of FW eels to media of different Na and Cl concentrations. Results are from
round-robin multivariate analyses (Tukey-Kramer test). Identical letters mean not significantly different; different letters mean significantly different. FWMan, 100
mM mannitol; FWNaG, 50 mM Na⫺ gluconate; FWChCl, 50 mM choline-Cl; FWNa50Cl10, 50 mM Na⫹⫹10 mM Cl⫺; FWNa10Cl50, 10 mM Na⫹⫹50 mM Cl⫺,
FWNaCl50, 50 mM NaCl.
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RENAL SULFATE REGULATION IN EEL
divalent cations (Ca2⫹ and Mg2⫹) also failed to induce the
change. However, 50 mM NaCl solution in media triggered the
change. Compared with SW transfer, eels transferred to NaCl
solutions decreased plasma SO2⫺
4 concentration more quickly
and the increase in urine SO2⫺
4 concentration was more slowly,
probably because of the absence of SO2⫺
in media. Since
4
transfer to 60 mM Na⫹ (30 mM Na2SO42) was without effect
on switching, Na⫹ alone may be insufficient for the trigger.
Therefore, Cl⫺ ions in SW may be responsible for switching
the SO2⫺
4 regulatory system in eels.
To analyze further the role of Na⫹ or Cl⫺, we transferred
FW eels to media that contained either Na⫹ or Cl⫺, but neither
50 mM choline-Cl nor 50 mM Na-gluconate had an effect on
switching. Since 10 mM Na⫹⫹50 mM Cl⫺ was effective but
50 mM Na⫹⫹10 mM Cl⫺ was without effect on switching, we
concluded that Cl⫺ is a primary factor and Na⫹ has a permissive role in the Cl⫺ effect. The threshold concentration of NaCl
for switching was ⬃50 mM, which is much lower than the Cl⫺
concentration in SW (⬃500 mM). Especially, Slc26a6a
showed a more sensitive and rapid response than Slc13a1 (35).
The high sensitivity to environmental Cl⫺ concentration indicates that the eel can prepare for future exposure to high SO2⫺
4
concentration during the course of downstream migration.
Environmental Cl⫺ ion as a Trigger for SO2⫺
4 Regulation
Cl⫺ is known as a trigger for elicitation of drinking in SW
(9). Teleosts drink copiously in SW to compensate for water
lost osmotically (21, 32), and eels start drinking within 1 min
after transfer from FW to SW (33). Using a similar analytical
technique, Hirano (9) showed that Cl⫺ alone (choline-Cl) is
sufficient for initiation of drinking without Na⫹ in the eel.
Ando and Nagashima (1) also showed that luminal Cl⫺ concentration in eel intestine is responsible for inhibition of
excessive drinking. Because intestinal lumen is, in effect, the
external environment, this also exemplifies environmental Cl⫺
sensing. These reports indicate that Cl⫺ sensing mechanisms
exist on the body surface and intestinal epithelia. In contrast to
Fig. 6. Expressions of the Slc26a6a (A) and
Slc13a1 (B) genes in the kidneys of FW eels 24
h after injection of each solution (on the x-axis)
into the circulation. *P ⬍ 0.05. All results were
expressed as means ⫾ SE.
AJP-Regul Integr Comp Physiol • VOL
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Fig. 5. Correlations between plasma SO2⫺
4
concentration and Cl⫺ (A), Na⫹ (B), Ca2⫹ (C)
concentration and osmolality (D) in eels 7
days after transfer to media of different ionic
conditions.
RENAL SULFATE REGULATION IN EEL
R409
Fig. 7. Expressions of the Slc26a6a (A) and Slc13a1 (B)
genes in the kidneys of FW eels 72 h after transfer to 150 mM
NaCl solution containing 10⫺8 M hydrochlorochiazide
(HCTZ), a blocker of Na-Cl cotransporter (NCC). *P ⬍ 0.05.
All results were expressed as means ⫾ SE.
NCC Responsible for Cl⫺ Uptake from the Environment
Only Cl⫺ and a small amount of Na⫹ in media are sufficient
for Cl⫺ uptake that initiates the switching of SO2⫺
4 regulation.
This indicates the involvement of NCC but not Na-K-2Cl
cotransporter (NKCC) that requires K⫹ in media. Anion exchanger that takes up Cl⫺ in exchange of other anion is also
unlikely as it functions without Na⫹ in media (27). Recently,
NCC was identified on the apical side of the gill cells and was
suggested to be responsible for Na⫹ and Cl⫺ uptake in zebrafish and tilapia in FW (11, 12). Consistent with the results
of zebrafish and tilapia, transfer of FW eels to 150 mM NaCl
solution containing HCTZ, a potent NCC blocker, significantly
inhibited Slc26a6a gene expression in the kidney compared
with controls. Therefore, NCC is the most likely candidate for
Cl⫺ uptake into the circulation in the presence of Na⫹. Since
injection of Cl⫺ ion into blood changed the renal SO2⫺
4
transporter, the increase may be detected by a Cl⫺ sensor in the
kidney, probably in the proximal tubular cells that are involved
in the SO2⫺
excretion (13, 33). However, the pathway of
4
information transfer that leads to changes in SO2⫺
4 transporter
genes has not yet been identified. In mammals, a Cl⫺ sensor is
localized in the macula densa of renal distal tubule, which is
thought to be NKCC2 and responsible for tubule-glomerular
feedback and renin release (25).
AJP-Regul Integr Comp Physiol • VOL
Mg2⫹ is the second most abundant cation in SW (⬃50 mM)
that is actively excreted by the kidney of marine teleosts (7, 8).
Because of the huge difference in Mg2⫹ concentration between
plasma and SW, Mg2⫹ regulation at the kidney is also thought
to be altered by the environmental salinity in fishes (2, 4).
Recently, transient receptor potential melastatin type channels
(TRPM6 and 7) were found to be involved in the Mg2⫹
reabsorption in the distal convoluted tubule of mammals (30).
The TRP channels may act also as a sensor because TRP
vanilloid type channels (TRPV2 and 4) are shown to be an
osmosensor for regulation of thirst and vasopressin secretion
(31). However, these transporters have not been identified in
teleosts, and much less is known about SO2⫺
4 regulation compared with Mg2⫹ regulation in fish (2, 4).
To summarize the data obtained in this study and from
previous studies, a model for switching the SO2⫺
4 regulation in
the eel in FW and SW is suggested in Fig. 8. After transfer to
SW, Cl⫺ is taken up from SW in parallel with Na⫹ probably
via the NCC, resulting in an transient increase in plasma Cl⫺
concentration. The increase is detected by an unknown sensor
in the circulatory system and turns on the switch for SO2⫺
4
excretion by the kidney through downregulating Slc13a1 and
upregulating Slc26a6a in the proximal tubules (13, 23, 35),
which facilitates SO2⫺
excretion into the tubular lumen and
4
decreases plasma SO2⫺
concentration. We showed that
4
Slc26a6a, Slc26a6b, and Slc26a6c are present on the apical
membrane and Slc26a1 on the basolateral membrane of the
epithelial cells of proximal tubule of SW eel kidney (35).
The Slc26a6a gene is expressed only after SW transfer,
while the Slc26a6b and Slc26a6c genes are expressed in
both media and are slightly upregulated after transfer to SW.
The upregulation of the Slc26a6a gene in SW has been
shown previously in the pufferfish (13). We also found that
Slc26a6b and Slc26a1 are localized in the same epithelial cells
in P2 segment of the proximal tubule and Slc26a6a and
Slc26a6c on the apical membrane of the same cells in P1
segment of the proximal tubule of SW eels (35). However, the
counterpart of Slc26a6a and Slc26a6c in the basolateral membrane has not been determined yet. In the pufferfish, Slc26a6a
and Slc26a1 are suggested to be in the same proximal tubular
cells, but this is not the case in SW eel kidney (13).
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the regulation of drinking, however, both Cl⫺ and Na⫹ are
required for switching the SO2⫺
regulation. Furthermore, the
4
change in SO2⫺
regulation occurs in 1–3 days after SW
4
transfer (35), which is in contrast to the immediate regulation
of drinking. We found that environmental Cl⫺ must be taken
up into the circulation to manifest its effect on SO2⫺
regula4
tion. This idea is supported further by the fact that injection of
Cl⫺ into the circulation triggered the change in SO2⫺
4 regulation and that the sum of Cl⫺ and SO2⫺
4 in plasma is maintained
at a certain level as confirmed by the inverse relationship of
⫺
plasma SO2⫺
4 and Cl concentration. The current data support
the notion that accumulation of high SO2⫺
in plasma of FW
4
eels is an energy-saving substitute for Cl⫺ as plasma anions
(23). In fact, eels are known to have poor ability to take up Cl⫺
from the environment among teleost species (34), which may
⫺
facilitate the use of SO2⫺
4 as a Cl substitute in starved fish.
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RENAL SULFATE REGULATION IN EEL
Perspectives and Significance
In this study, we found that Cl⫺ in SW, not SO2⫺
4 , is
responsible for switching the renal SO2⫺
regulation to an
4
excretory type to maintain low plasma SO2⫺
4 concentration in
SW by using eels that can survive direct transfer from FW to
SW. We further showed that Cl⫺ enters the circulation via
NCC with the help of Na⫹ and probably acts on the renal
proximal tubular cells to turn on the excretory SO2⫺
trans4
porter (Slc26a6a) and turn off the absorptive SO2⫺
transporter
4
(Slc13a1) through regulation of the gene expression. This is the
first in vertebrates to identify the environmental and internal
ions that reverse the SO2⫺
4 regulation and the route of uptake
that leads to the gene expression at the target tissue. The use of
eels made these findings possible because this fish changes the
SO2⫺
4 regulation most profoundly among euryhaline teleosts in
FW and SW environment. Thus, this study illustrates the
advantage of comparative fish studies for osmoregulation. In
addition, it is of interest to compare the switching factor with
other osmoregulatory organs such as the gills where mitochondria-rich cells also change from absorptive to excretory type in
FW and SW, respectively.
AJP-Regul Integr Comp Physiol • VOL
The next target of research seems to identify the sensor or
receptor for Cl⫺ ions located within the circulatory system. We
expect that the sensor may be the protein that transports Cl⫺
ion such as Cl channels or TRP-type receptors, as discussed
above. The location of the Cl⫺ sensor may be at the epithelial
cells of renal proximal tubule that express Slc26a6a and
Slc13a1 gene as these genes are expressed in SW and FW,
respectively, in eels. We have identified the cells that express
these genes in different segments of the proximal tubule of eel
kidney (35). Therefore, we may be able to examine the colocalization of the sensor and transporters in each epithelial cell
by immunocytochemical study after we can identify the sensor.
We are now searching for Cl⫺ sensor in the buccal cavity and
the intestinal lumen that regulates drinking in the eel (1, 9). We
are interested in the identity of the Cl⫺ sensor with the renal
Cl⫺ sensor for SO2⫺
4 regulation.
ACKNOWLEDGMNTS
We thank Drs. Susumu Hyodo, Makoto Kusakabe, and Jillian Healy and
Sanae Hasegawa of this laboratory for comments on the manuscript and
advice.
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Fig. 8. A schematic drawing of hypothetical pathway for
switching SO2⫺
4 regulation after transfer of eels from FW to
SW with emphasis on renal regulation.
RENAL SULFATE REGULATION IN EEL
GRANTS
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. Takei (13304063
and 16207004). T. Watanabe was supported by Global COE Program (Integrative Life Sciences Based on Study of Biosignaling Mechanisms), Ministry
of Education, Culture, Sports, Science, and Technology, Japan.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
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