Comparative Biochemistry and Physiology, Part B 202 (2016) 23–30
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Comparative Biochemistry and Physiology, Part B
journal homepage: www.elsevier.com/locate/cbpb
Different expression patterns of renal Na+/K+-ATPase α-isoform-like
proteins between tilapia and milkfish following salinity challenges
Wen-Kai Yang a, Chang-Hung Chung b,c, Hui Chen Cheng a, Cheng-Hao Tang d,⁎, Tsung-Han Lee a,e,⁎⁎
a
Department of Life Sciences, National Chung Hsing University, Taichung 40227, Taiwan
Graduate Institute of Science Education, National Changhua University of Education, Changhua 50007, Taiwan
c
Taichung Municipal Kuang Rong Junior High School, Taichung 41265, Taiwan
d
Department of Oceanography, National Sun Yat-sen University, Kaohsiung 80424, Taiwan
e
Agricultural Biotechnology Center, National Chung Hsing University, Taichung 40227, Taiwan
b
a r t i c l e
i n f o
Article history:
Received 21 March 2016
Received in revised form 28 July 2016
Accepted 30 July 2016
Available online 03 August 2016
Keywords:
α-isoform
Kidney
Milkfish
Na+/K+-ATPase
Salinity
Tilapia
a b s t r a c t
Euryhaline teleosts can survive in a broad range of salinity via alteration of the molecular mechanisms in certain
osmoregulatory organs, including in the gill and kidney. Among these mechanisms, Na+/K+-ATPase (NKA) plays
a crucial role in triggering ion-transporting systems. The switch of NKA isoforms in euryhaline fish gills substantially contributes to salinity adaptation. However, there is little information about switches in the kidneys of
euryhaline teleosts. Therefore, the responses of the renal NKA α-isoform protein switch to salinity challenge in
euryhaline tilapia (Oreochromis mossambicus) and milkfish (Chanos chanos) with different salinity preferences
were examined and compared in this study. Immunohistochemical staining in tilapia kidneys revealed the localization of NKA in renal tubules rather than in the glomeruli, similar to our previous findings in milkfish kidneys.
Protein abundance in the renal NKA pan α-subunit-like, α1-, and α3-isoform-like proteins in seawateracclimated tilapia was significantly higher than in the freshwater group, whereas the α2-isoform-like protein
exhibited the opposite pattern of expression. In the milkfish, higher protein abundance in the renal NKA pan
α-subunit-like and α1-isoform-like proteins was found in freshwater-acclimated fish, whereas no difference
was found in the protein abundance of α2- and α3-isoform-like proteins between groups. These findings suggested that switches for renal NKA α-isoforms, especially the α1-isoform, were involved in renal osmoregulatory
mechanisms of euryhaline teleosts. Moreover, differences in regulatory responses of the renal NKA α-subunit to
salinity acclimation between tilapia and milkfish revealed that divergent mechanisms for maintaining osmotic
balance might be employed by euryhaline teleosts with different salinity preferences.
© 2016 Elsevier Inc. All rights reserved.
1. Introduction
Maintaining an internal environment with stable conditions is
essential for animals to survive in a variety of habitats. Euryhaline teleosts can adapt to a broad range of ambient salinities, although salinity
adaptation is a complex process involving a set of physiological
responses to environments with differing ionoregulatory requirements
(Marshall and Grosell, 2006; Hwang and Lee, 2007). The osmoregulation of teleosts is regulated by a group of structures, including the
Abbreviations: A.u., Arbitrary units; GAPDH, Glyceraldehyde-3-phosphate
dehydrogenase; FW, Freshwater/fresh water; NKA, Na+/K+-ATPase; SE, Standard error;
SW, Seawater.
⁎ Correspondence to: C.-H. Tang, Department of Oceanography, National Sun Yat-sen
University, 70 Lien-hai Road, Kaohsiung 80424, Taiwan.
⁎⁎ Correspondence to: T.-H. Lee, Department of Life Sciences, National Chung Hsing
University, 145 Xingda Road, Taichung 40227, Taiwan.
E-mail addresses: [email protected] (C.-H. Tang),
[email protected] (T.-H. Lee).
http://dx.doi.org/10.1016/j.cbpb.2016.07.008
1096-4959/© 2016 Elsevier Inc. All rights reserved.
gills, kidney, and gut (Evans and Claiborne, 2009; Whittamore, 2012).
The kidney is involved in osmoregulatory mechanisms of freshwater
(FW) and seawater (SW) teleosts, although its function is entirely
different in these two environments (Beyenbach, 2004; Takei et al.,
2014). To maintain osmolality and ionic balance within narrow physiological ranges under different salinity conditions, the primary function
of FW teleost kidneys is to excrete excess water and reabsorb filtered
solutes to minimize salt loss and produce dilute urine. Conversely, in
the kidney of marine teleosts, the principal role is to conserve water,
and urine production is very low. Furthermore, the osmolality of
urine, in which the predominant electrolytes are Mg2 +, SO24 −, and
Ca2 +, rather than Na+ and Cl−, is similar to that of the body fluid
(Beyenbach, 2004; Marshall and Grosell, 2006).
Previous studies that extensively reviewed the functional morphology of teleostean nephrons (the structural unit of kidney) stated that
the nephron generally consisted of a well-developed glomerulus
(although some teleosts are aglomerular), a ciliated neck segment, a
proximal tubule, a distal tubule, and a collecting tubule (Beyenbach,
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W.-K. Yang et al. / Comparative Biochemistry and Physiology, Part B 202 (2016) 23–30
2004; Marshall and Grosell, 2006). Because the teleost kidney lacks
the loop of Henle and exhibits neither zonation nor a countercurrent system of tubular elements, they are not able to excrete
hypertonic urine (Marshall and Grosell, 2006; Whittamore, 2012).
Moreover, various adaptive changes in fish kidneys, including morphology, excretion, and absorption of ions, glomerular filtration
rate, urine production, and metabolism, in response to salinity acclimation have been reported (Beyenbach, 2004; Marshall and Grosell,
2006).
Na+/K+-ATPase (NKA) is a crucial primary enzyme expressed in
animal cells to create an electrochemical gradient providing the driving
force for ion transports in osmoregulatory organs, including fish gills
and kidneys (Marshall and Grosell, 2006; Whittamore, 2012). Some
review articles stated that modulation of NKA activity or kinetics in
osmoregulatory organs of fish was essential for salinity acclimation
(Marshall and Grosell, 2006; Hwang and Lee, 2007; Whittamore, 2012).
Several mechanisms were reported to participate in regulation of NKA
activity, including changes in isoform composition (Scheiner-Bobis,
2002; Blanco, 2005; Suhail, 2010). Pagliarani et al. (1991) demonstrated
that NKA proteins isolated from FW- and SW-acclimated fish gills had
different biochemical properties and suggested that different NKA
isoforms might be correlated with ion regulation in different environmental salinities. Because α-subunit is the catalytic subunit of NKA, it
plays a crucial role in osmoregulation (Evans and Claiborne, 2009).
Four α-isoforms of NKA with highly tissue-dependent distributions
have been reported (Scheiner-Bobis, 2002; Blanco, 2005; Suhail,
2010). In birds and mammals, in which the α1-isoform expressed
ubiquitously, the α2-isoform is present primarily in muscle, heart, and
brain. The α3-isoform is mainly found in neurons and ovaries. In addition, the α4-isoform is expressed only in the testis. Among these
isoforms, the α1-isoform is predominantly expressed in transporting
epithelia to maintain osmotic balance and cell volume regulation in a
housekeeping capacity. Furthermore, specialized requirements of
differentiated cell-specific functions for cation transport were fulfilled
by the other α-isoforms (Takeyasu et al., 1990; Blanco, 2005). The
tissue-specific and development-dependent expression of the αsubunit was suggested to extend to all vertebrate classes, including
teleosts (Takeyasu et al., 1990; Pressley, 1992).
In fish, only NKA α1-, α2-, and α3-isoforms have been identified
(Armesto et al., 2014). In fish gills, alteration of NKA α-isoform expression correlating to salinity acclimation has been examined at mRNA and
protein levels in detail (Richards et al., 2003; Tang et al., 2009a; Armesto
et al., 2014). However, responses of renal NKA α-isoforms to environmental salinities have been poorly addressed. It is well known that the
protein is the function executor of a specific gene, although recent studies on mRNA expression patterns of NKA α-isoforms showed their
diverse responses to salinity changes (Feng et al., 2002; Richards et al.,
2003; Ip et al., 2012; Armesto et al., 2014). Furthermore, previous studies have reported changes in protein abundance but not mRNA levels of
NKA α-subunit isoforms in brains and gills of teleosts with different
treatments (Morrison et al., 2006; Hiong et al., 2014). These findings
were the motivation for the present study, in which we explored the
protein expression and potential roles of NKA α-isoforms in kidneys
of euryhaline teleosts during salinity acclimation. Tilapia (Oreochromis
mossambicus) and milkfish (Chanos chanos) can maintain their blood
osmolality, chloride concentrations, and muscle water contents within
narrow physiological ranges under different salinity conditions (Lin
et al., 2003; Inokuchi et al., 2008; Tang et al., 2009b). These results
indicated that both species are excellent osmoregulators, which make
them good experimental species for studies on osmoregulation. In
addition, because tilapia and milkfish are euryhaline teleosts with FW
and SW preferences, respectively, differential regulatory responses
may be used to maintain physiological homeostasis. The present study
documented protein expression patterns of the NKA α-isoforms, as
well as the NKA distribution in kidneys of tilapia and milkfish acclimated to FW and SW, illustrating and comparing the regulatory roles of
NKA α-isoforms in kidneys of two euryhaline teleosts following salinity
challenges.
2. Materials and methods
2.1. Experimental animals and environments
Tilapia and milkfish were used in the present study. Adult tilapia of
12.1 ± 4.7 g body mass and 7.0 ± 0.8 cm in total length were obtained
from laboratory stock. Juvenile milkfish of 41.4 ± 25.1 g body mass and
14.7 ± 2.5 cm in total length were obtained from a fish farm in Tainan,
Taiwan. For the following experiments, the two fish species were acclimated to FW (aerated dechlorinated local tap water) or SW (35‰) for at
least 2 weeks at 28 ± 1 °C under a 12-h light: 12-h dark cycle. SW was
prepared from FW by adding standardized amounts of the synthetic sea
salt “Instant Ocean” (Aquarium Systems Co., Mentor, OH, USA). The
details of water parameters were identical to that of our previous
study (Kang et al., 2010; Yang et al., 2011). Water was continuously
circulated through fabric-floss filters. Fish were fed a daily diet of commercial fodder. In the following experiments, the fish were not fed and
were anesthetized with MS-222 (100–200 mg L−1) before sampling.
The protocol employed for the experimental fish was reviewed and
approved by the Institutional Animal Care and Use Committee of the
National Chung Hsing University (IACUC approval no. 96–48).
2.2. Antiserum/antibody
Six primary antibodies were used in the present study: (1) NKA pan
α-subunit: a mouse monoclonal antibody (α5; Developmental Studies
Hybridoma Bank, Iowa City, IA, USA) raised against the α-subunit of
the avian NKA was employed for immunohistochemical staining and
immunoblotting (200 × and 5000 × dilution, respectively); (2) NKA
α1-isoform: a mouse monoclonal antibody (a6F; Developmental
Studies Hybridoma Bank) raised against the α1-isoform of the avian
NKA was employed for immunoblotting (10,000× dilution); (3) NKA
α2-isoform: a rabbit polyclonal antibody (ab107033; Abcam, Cambridge, UK) raised against the α2-isoform of the human NKA was
employed for immunoblotting (1000× dilution); (4) NKA α3-isoform:
a goat polyclonal antibody (sc-16052; Santa Cruz Biotechnology, Santa
Cruz, CA, USA) raised against the α3-isoform of the human NKA was
employed for immunoblotting (1000 × dilution); (5) actin: a mouse
monoclonal antibody (MAB1501; Millipore, Bedford, MA, USA) raised
against chicken actin was applied in immunoblotting assays (10,000×
dilution) as the loading control for tilapia kidneys; and (6) GAPDH
(glyceraldehyde-3-phosphate dehydrogenase): a rabbit polyclonal
antibody (sc-25778; Santa Cruz Biotechnology) raised against human
GAPDH was applied in immunoblotting assays (5000× dilution) as the
loading control for milkfish kidneys.
Due to the higher sequence homology of individual NKA α-isoforms
among vertebrates than those of different isoforms within one species
(Blanco and Mercer, 1998; Guynn et al., 2002; Armesto et al., 2014)
including tilapia (Feng et al., 2002), we detected the individual NKA
α-isoforms by the heterologous antibodies for specific isoforms.
Among the isoform-specific heterologous antibodies used in this
study, α5 (pan α-subunit) is known to react comprehensively with
NKA α-subunits in fish and other animals (Ip et al., 2012; Chen et al.,
2013; Hiong et al., 2014; Ching et al., 2015), while the a6F antibody is
known to react specifically with NKA α1-isoform in fish (Lee et al.,
1998; Tang et al., 2009a; Hiong et al., 2014). Moreover, to confirm the
specificity of these anti-NKA antibodies, a preliminary immunoblotting
was carried out (Supplementary Fig. S1).
The secondary antibodies employed for immunoblotting were
horseradish peroxidase-conjugated goat anti-mouse IgG and goat antirabbit IgG (GTX213112-01 and GTX213110-01, respectively; GeneTex,
Hsinchu, Taiwan), or rabbit anti-goat IgG (305-035-003; Jackson
ImmunoResearch, West Grove, PA, USA).
W.-K. Yang et al. / Comparative Biochemistry and Physiology, Part B 202 (2016) 23–30
2.3. Preparation of tissues and homogenates
The methods used in this study to prepare tissues and homogenates
were modified from Tang et al. (2009b, 2010). All subsequent operations were carried out on ice. Following anesthesia, kidneys from the
two fish species were dissected and immediately fixed in Bouin's fluid
(Sigma, St. Louis, MO, USA) for immunohistochemical staining or stored
at −80 °C for NKA activity assay or immunoblotting.
For tissue homogenates, kidneys were suspended in a mixture of
homogenization medium (100 mM imidazole–HCl, 5 mM Na2EDTA,
200 mM sucrose, and 0.1% sodium deoxycholate, pH 7.6) and proteinase
inhibitors (10 mg antipain, 5 mg leupeptin, and 50 mg benzamidine
dissolved in 5 mL aprotinin) (v/v, 20:1). Homogenization was
performed in 2 mL microtubes with a Polytron PT1200E (Kinematica,
Lucerne, Switzerland) at maximal speed for 30 s on ice. The homogenate
was then centrifuged at 13,000 × g at 4 °C for 20 min. The sample
supernatants were collected and used for protein concentration measurements, NKA activity assay, and immunoblotting. Protein concentrations were determined with the BCA Protein Assay (Pierce, Rockford, IL,
USA) using bovine serum albumin (Pierce) as a standard.
25
700 nm. NKA activity was expressed as μmol inorganic phosphate released per mg of protein per hour.
2.6. Immunoblotting
The immunoblotting procedures were modified from Yang et al.
(2011, 2013). Briefly, the tissue supernatants were heated with denaturing buffer at 37 °C for 30 min, followed by electrophoresis on an 8%
sodium dodecyl sulfate–polyacrylamide gel. After electrophoresis and
transfer of the proteins from the gels to PVDF membranes (Millipore),
the blots were pre-incubated in wash buffer (phosphate-buffered saline
with 0.2% Tween 20) containing 5% (wt/vol) nonfat dried milk to minimize non-specific binding. Then, the blots were incubated sequentially
in primary antibodies and secondary antibodies. The blots were finally
developed using the Immobilon™ Western (Millipore) and observed
in a Universal hood with a cooling-CCD (charge-coupled device) camera
(ChemiDoc XRS+; Bio-Rad, Hercules, CA, USA) and associated software
(Quantity One v4.6.8; Bio-Rad). Immunoreactive bands were analyzed
using Image Lab v3.0 (Bio-Rad). The results were converted to numerical values to compare the relative protein abundances of the immunoreactive bands.
2.4. Paraffin section preparation and immunohistochemical staining
2.7. Statistical analysis
The protocols used herein were modified from Yang et al. (2009)
and Tang et al. (2010). Briefly, after fixation and dehydration, tissues
were embedded in paraffin blocks and then cut at 5-μm thickness. The
deparaffinized sections were rinsed with phosphate-buffered saline
(137 mM NaCl, 3 mM KCl, 10 mM Na2HPO4, and 2 mM KH2PO4, pH
7.4). Endogenous peroxidase was inactivated by incubating the sections
with 3% hydrogen peroxide. Afterward, the sections were stained with
primary antibody before being processed with a commercial kit
(PicTure; Zymed, South San Francisco, CA, USA). After staining, the sections were stained with hematoxylin (1.05175.0500; Merck, Darmstadt,
Germany) for histological evaluation. Finally, the immunostained sections were observed under an optical microscope (BX50; Olympus,
Tokyo, Japan) equipped with a digital camera (COOLPIX 5000; Nikon,
Tokyo, Japan).
To identify different segments of nephrons, the other sets of sections
were subjected to hematoxylin and eosin staining or periodic acid-Schiff
staining. The sections were observed as described above (data not
shown). Moreover, the characteristics of the segments were identified
based on the descriptions in previous studies (Katoh et al., 2008; Tang
et al., 2010; Teranishi and Kaneko, 2010; Yang et al., 2016).
All values are expressed as the mean ± standard error (SE).
Differences between the groups were analyzed via Mann–Whitney U
tests using SPSS 12.0 (SPSS, Chicago, IL, USA), and P values of b 0.05
were considered significant.
3. Results
3.1. Renal NKA localization in tilapia and milkfish
To investigate the distribution of NKA α-subunit in the kidneys of
the two euryhaline species, NKA was immunohistochemically stained
using monoclonal antibody α5 to label all the NKA α-subunits. The
immunoreactive results revealed that the NKA distribution in the tilapia
kidneys was similar between FW- and SW-acclimated individuals and
that NKA was distributed in the renal tubules including the proximal
and distal, as well as collecting tubules and ducts, but not in the glomeruli (Fig. 1). Moreover, NKA immunoreaction was detected in the
basolateral membrane of epithelial cells of all observed renal tubules.
NKA distribution was also similar between the kidneys of milkfish and
tilapia (Table 1).
2.5. Tilapia renal NKA activity assay
3.2. Salinity effects on renal NKA expression in tilapia
The assay method was used to measure the inorganic phosphate
concentrations for determining NKA activity (Lin et al., 2004; Yang
et al., 2009). In this study, the reaction medium (100 mM imidazole–
HCl, 125 mM NaCl, 75 mM KCl, and 7.5 mM MgCl2, pH 7.6) was prepared
according to Hwang et al. (1988). The reaction was initiated by mixing
500 μL of the tilapia renal supernatant, 100 μL of 10 mM Na2ATP,
350 μL of the reaction medium, and 50 μL of 3.75 mM ouabain (specific
inhibitor of NKA) or deionized water per microtube; each sample was
assayed in triplicate. NKA enzyme activity was defined as the difference
between the amount of inorganic phosphates liberated in the presence
and absence of ouabain in the reaction mixture. Then, the reaction
mixture was incubated at 37 °C for 30 min, followed by the addition of
200 μL ice-cold 30% trichloroacetic acid to stop the reaction. The formation of unreduced phosphomolybdate is directly proportional to the
amount of inorganic phosphate. The reaction mixtures and color reagent (560 mM H2SO4, 8.1 mM ammonium molybdate, and 175.5 mM
FeSO4) were mixed at a 1:2 (v/v) ratio and incubated at 20 °C for
30 min prior to detection. Finally, the reaction tube was centrifuged
and the inorganic phosphate concentration in the supernatant was determined with a spectrophotometer (U-2001; Hitachi, Tokyo, Japan) at
All immunoblotting of kidney tissues from tilapia acclimated to
environments of different salinities (FW and SW) resulted in a single
immunoreactive band (about 100 kDa for all NKA α-subunits/isoformlike proteins; Fig. 2a). Quantifications of immunoreactive bands of
NKA pan α-subunit, α1-, and α3-isoform-like proteins in SWacclimated fish were higher than those of FW-acclimated individuals
(approximately 4.8-, 7.5-, and 4.7-fold, respectively; Fig. 2b,c,e). Furthermore, in the NKA α2-isoform-like protein, the relative protein
abundance of immunoreactive bands in the SW group was lower (approximately 0.6-fold) than that of the FW group (Fig. 2d). Although
the protein expression of all NKA α-isoform-like proteins was salinitydependent in the tilapia kidneys (Fig. 2), renal NKA activity was not significantly different between FW- and SW-acclimated individuals
(Table 1).
3.3. Salinity effects on renal NKA expression in milkfish
In milkfish kidneys, renal tissues of different salinity groups (FW and
SW) also resulted in a single immunoreactive band (approximately
26
W.-K. Yang et al. / Comparative Biochemistry and Physiology, Part B 202 (2016) 23–30
100 kDa for all NKA α-subunits/isoform-like proteins; Fig. 3a) in the
immunoblotting, similar to those of tilapia. Higher protein content of
renal NKA pan α-subunit and α1-isoform-like protein was found in
FW milkfish than those in the SW group (approximately 3.5- and 2.4fold, respectively; Fig. 3b,c). For protein abundance of both NKA α2and α3-isoform-like proteins, however, there was no difference
between different salinity groups (Fig. 3d,e). Similar to the expression
patterns of NKA pan α-subunit and α1-isoform-like protein, renal
NKA activity of FW-acclimated fish was higher than that of SWacclimated individuals (Table 1).
for triggering ionoregulation (Hwang and Lee, 2007; Evans and
Claiborne, 2009). Although the kidney is an important organ responsible for ion-regulation in teleosts, only a few studies focused on its
osmoregulatory mechanisms when compared with those of the gills.
Hence, the present study investigated the protein expression patterns
of NKA α-isoforms in kidneys of the tilapia and milkfish to illustrate
the mechanisms underlying the regulation of NKA activity in euryhaline
teleosts.
4. Discussion
In this study, the kidneys of both tilapia and milkfish exhibited
typical structures of FW or euryhaline teleosts (Marshall and Grosell,
2006), including the glomerulus, proximal tubule, distal tubule,
collecting tubule, and collecting duct. Moreover, the results of NKA immunohistochemical staining were similar between these two teleostean
species and between FW- or SW-acclimated individuals, indicating that
the NKA α-subunit was distributed in the basolateral membrane of the
proximal tubule and distal tubule, as well as the collecting tubules and
ducts, but not in the glomeruli. These results of NKA localization were
similar compared to that for other teleosts (Lin et al., 2004; Katoh
et al., 2008; Teranishi and Kaneko, 2010; Yang et al., 2016). The main
functions of vertebrate kidneys contain excretion and osmoregulation.
The kidneys of FW teleosts have high glomerular filtration rates, and
the distal tubule is the principal site for active absorption of salts; for
marine fishes, the proximal tubule appears to be the site of reabsorption
of monovalent ions and organic compounds (Marshall and Grosell,
2006; Madsen et al., 2015). In these processes, renal NKA activity
plays a crucial role in providing the driving force for secondary ion
transporters to maintain homeostasis (Marshall and Grosell, 2006;
Whittamore, 2012). Taken together, the renal NKA distribution was
similar between different salinity groups in either tilapia or milkfish
and between the two fish species or among them and other teleosts,
showing that NKA plays a role in renal osmoregulatory mechanisms
like the other teleosts.
When euryhaline teleosts were acclimated to FW or SW, the osmoregulatory systems used differed in the direction of ion and water
movements as well as in their molecular components (Tang et al.,
2009a; Takei et al., 2014). The current model shows that epithelial
NKA of teleostean osmoregulatory organs is the primary active pump
4.1. Similar renal NKA distribution in tilapia and milkfish
4.2. Different expression profiles of renal NKA activity between the two fish
species
In the present study, the expression patterns of renal NKA activity
were different between tilapia and milkfish following salinity
challenges. The higher levels of renal NKA activity were found in FWacclimated milkfish. In FW, the major role of the teleostean kidney is
to excrete excess water and reabsorb filtered solutes (Beyenbach,
2004; Marshall and Grosell, 2006). Hence, these results showed that
renal NKA might provide a greater driving force for increasing ion
reabsorption when the SW-preference milkfish survived in FW (Tang
et al., 2010). In tilapia, on the other hand, renal NKA activity did
Table 1
Na+/K+-ATPase (NKA) activity and localization in the kidneys of tilapia and milkfish.
NKA expression
Fig. 1. Immunohistochemical localization of Na+/K+-ATPase (NKA) recognized by α5
antibody in tilapia kidney acclimated to fresh water (a, b) and seawater (d–g). (c) The
representative nephron architecture of teleostean kidney. The distribution pattern of
renal NKA was similar between both salinity groups. The positive immunostaining was
found in the basolateral membrane of epithelial cells of proximal, distal, and collecting
tubules, as well as collecting ducts, but not in the glomerulus. Scale bars: 20 μm.
Tilapia
Milkfish#
NKA activity (μmol Pi/mg protein/h)
Fresh water
19.99 ± 3.44
Seawater
20.05 ± 2.39
6.86 ± 1.54⁎
1.71 ± 0.32
NKA localization§ (fresh water/seawater)
Glomerulus
−/−
Proximal tubule
+/+
Distal tubule
+/+
Collecting tubule
+/+
Collecting duct
+/+
−/−
+/+
+/+
+/+
+/+
Values was mean ± SE (N = 4–5).
−, no immunosignal; +, positive immunosignal.
#
The data were modified from Tang et al. (2010).
§
Using the antibody α5.
⁎ A significant difference between the freshwater and seawater groups (P b 0.05).
W.-K. Yang et al. / Comparative Biochemistry and Physiology, Part B 202 (2016) 23–30
27
Fig. 2. Salinity effects on protein expression of renal Na+/K+-ATPase (NKA) α-subunits in tilapia. (a) Representative immunoreactive bands of NKA α-isoforms; (b–e) relative protein
abundance of immunoreactive bands of NKA pan α-subunit, α1-, α2-, and α3-isoforms in the kidney of tilapia acclimated to environments of different salinities. Actin was used as an
internal control for the immunoblots. Values were mean ± SE (N = 4–8). The asterisk indicated a significant difference (P b 0.05). M, marker (kDa); FW, fresh water; SW, seawater;
a.u., arbitrary units.
not change significantly with changes in environmental salinities.
Similar results were reported in kidneys of some euryhaline fish
(Fuentes et al., 1997; Arjona et al., 2007; Tomy et al., 2009), considering
that a lower energy demand occurs in fish for osmoregulation
(Varsamos et al., 2002; Tomy et al., 2009). Our results revealed that
the requirements of osmoregulation might be different between tilapia
and milkfish, although the mechanisms for the differences are not clear
yet. In gills, similar findings about the relationship between NKA
28
W.-K. Yang et al. / Comparative Biochemistry and Physiology, Part B 202 (2016) 23–30
Fig. 3. Salinity effects on protein expression of renal Na+/K+-ATPase (NKA) α-subunits in milkfish. (a) Representative immunoreactive bands of NKA α-isoforms; (b–e) relative protein
abundance of immunoreactive bands of NKA pan α-subunit, α1-, α2-, and α3-isoforms in the kidney of milkfish acclimated to environments of different salinities. GAPDH was used as an
internal control for the immunoblots. Values were mean ± SE (N = 7–8). The asterisk indicated a significant difference (P b 0.05). M, marker (kDa); FW, fresh water; SW, seawater; a.u.,
arbitrary units.
expression and environmental salinity were reported in many euryhaline teleosts. Moreover, the differences between osmoregulatory capabilities of teleosts were considered correlating with their evolutionary/
life histories and/or natural/intrinsic habitats (Inoue and Takei, 2003;
Hwang and Lee, 2007; Freire et al., 2008; Takei et al., 2014). In this
study, therefore, one of the possible reasons for different expression
profiles of renal NKA activity between tilapia and milkfish might be
caused by their FW and SW preference, respectively.
W.-K. Yang et al. / Comparative Biochemistry and Physiology, Part B 202 (2016) 23–30
4.3. Distinct isoform compositions of NKA α-subunit following salinity
challenge
The switching of NKA α- and/or β-subunits is one of the modulatory
mechanisms of NKA expression (Scheiner-Bobis, 2002; Blanco, 2005).
Alteration of NKA α-isoforms (the catalytic subunit) protein expression
was supposed to be a crucial mechanism in euryhaline teleosts for
acclimation to environments of various salinities (Tang et al., 2009a).
There was accumulating evidence indicating that teleosts expressed
multiple NKA α-isoforms and the levels changed with environmental
salinities, especially in the gills (Lee et al., 1998; Richards et al., 2003;
Tang et al., 2009a; Armesto et al., 2014). In protein level, the identities
of amino acid sequences of individual isoforms across species are higher
than those of different isoforms within one species (Blanco and Mercer,
1998; Guynn et al., 2002; Armesto et al., 2014), including tilapia (Feng
et al., 2002). Therefore, in many fish studies, the heterologous antibodies to NKA α-isoforms were used to determine expression of NKA αsubunit/isoform-like proteins (D'Cotta et al., 2000; Guynn et al., 2002;
Brauer et al., 2005; Chen et al., 2013; Armesto et al., 2014; Hiong et al.,
2014), including tilapia and milkfish (Lee et al., 1998; Tang et al.,
2009a). In this study, we thus detected the individual NKA α-isoforms
by the heterologous antibodies for specific isoforms. Among them, two
of these anti-NKA antibodies, i.e., α5 (pan α-subunit) and a6F (NKA
α1-isoform), have been used widely to determine NKA expression of
various teleosts (Brauer et al., 2005; Ip et al., 2012; Chen et al., 2013;
Armesto et al., 2014; Hiong et al., 2014), including tilapia and milkfish
(Lee et al., 1998; Tang et al., 2009a; Kang et al., 2015; Yang et al.,
2015). Moreover, the antibody of NKA α3-isoform (sc-16,052) has
been reported for estimating expression of branchial NKA α3-isoform
in milkfish (Tang et al., 2009a). In addition, in the present study, the
results of the preliminary immunoblotting showed that different expression patterns of NKA α-subunit/isoform-like proteins were found
via using these heterologous antibodies. Differences in intensities of
the bands between each tissue with each isoform-specific antibody suggest that they are relatively specific and that is reasonably consistent
with the result of pan α-subunit (α5) antibody. However, there is no
guarantee of the absolute specificity or lack of cross-reactivity of the
antibodies for/or between each NKA isoform even though they have
been applied on the other fish species. Taken together, these results revealed that the antibodies are the appropriate tools to detect renal NKA
α-subunit/isoform-like proteins in these two fish species.
In this study, the expression patterns of renal NKA α-isoform-like
proteins in milkfish and tilapia varied with the environmental salinities
to which the fish were acclimated. Following salinity challenge, protein
expression of NKA pan α-subunit and α1-isoform-like protein, as well
as NKA activity, was parallel in the milkfish kidneys. Similar results
were reported in the gills of milkfish (Tang et al., 2009a). On the other
hand, in the tilapia kidneys, the expression patterns of NKA α1- and
α3-isoform-like proteins were similar to the protein abundance of the
NKA pan α-subunit. In gills of tilapia acclimated to either FW or SW,
the protein expression of NKA pan α-subunit, α1-, and α3-isoforms,
as well as NKA activity have shown similar salinity-dependent patterns
(Lee et al., 1998, 2003). Unlike the gill, however, no difference was
found in renal NKA activities between SW- and FW-acclimated tilapia.
In mammals, NKA α-subunit isoforms possess different affinities for
ouabain, ATP, and substrate ions (Blanco, 2005). Similarly, in teleosts,
including tilapia and milkfish, branchial NKA activities between FWand SW-acclimated groups had different affinities for Na+ or K+ (Lin
and Lee, 2005). These results indicated that the major kinetic
differences occurred because of the composition of different NKA αisoforms. Thus, tilapia might maintain internal water and ion balance
via switching of the three NKA α-isoforms (resulted in different affinities) in the kidney rather than regulating renal NKA activity between
FW and SW. In addition, in the milkfish, expression patterns of renal
NKA α2- and α3-isoforms were not salinity‐dependent, indicating
that they might not be involved in osmoregulation (Tang et al.,
29
2009a). Furthermore, in the tilapia kidney, the expression pattern of
the NKA α2-isoform-like protein was opposite to the other NKA αsubunit/isoform-like proteins, implying that the role of the NKA α2isoform might differ from those of the NKA α1- and α3-isoforms.
Taken together, following salinity challenge, distinct isoform compositions of the NKA α-subunit, as well as NKA activity between the kidneys
of tilapia and milkfish revealed their divergent modulatory mechanisms, which might be due to their FW and SW preference, respectively.
Salinity-dependent expression of the NKA α-isoforms, especially
the α1-isoform, in kidneys indicated their involvement in the
mechanisms of ion regulation in FW- and SW-acclimated tilapia and
milkfish.
In mammals, the NKA α-isoforms have a different tissue distribution, and the NKA α1-isoform was the most widely expressed isoform,
as well as the major isoform expressed in the kidney (Blanco, 2005;
Suhail, 2010). Thus, NKA α1 is predicted to be the crucial isoform for
osmoregulation of teleosts (Liao et al., 2009; Dalziel et al., 2014). In
this study, the protein expression patterns of NKA pan α-subunit and
α1-isoform-like protein were parallel in kidneys of both teleost species,
yet their patterns were different between the FW and the SW groups.
These results implied that the renal NKA α1-isoform might play a
crucial role in the physiological regulation of euryhaline teleosts in
different environmental salinities. Of note, several paralogous genes of
the NKA α1-isoforms have been identified in certain teleosts and have
been investigated in gills following salinity challenge (Richards et al.,
2003; Ip et al., 2012; Armesto et al., 2014). In salmonid gills, nka α1a
and α1b were the dominant isoform in FW and SW, respectively
(Richards et al., 2003; Dalziel et al., 2014). However, unlike salmonid
gills, no salinity-dependent changes were reported for branchial nka
α1b expression in Senegalese sole (Solea senegalensis; Armesto et al.,
2014). On the other hand, the mRNA levels of nka α1a and α1b were
investigated in tilapia gills following salinity challenge; however, the
results were not consistent (Tipsmark et al., 2011; Moorman et al.,
2014, 2015). These results showed that the roles of these NKA α1
paralogous genes in euryhaline teleosts remain poorly understood.
Therefore, future studies should focus on detailed molecular mechanisms of the NKA α-subunit in kidneys of these two euryhaline teleosts.
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.cbpb.2016.07.008.
Author contributions
C.H.T. and T.H.L. designed the project and experiments. C.H.C.,
W.K.Y., and H.C.C. performed the experiments and analyzed the data.
W.K.Y., C.H.C., C.H.T., and T.H.L. wrote the paper. All authors have read
and approved the final manuscript.
Conflict of interest
The authors have no competing interests to declare.
Acknowledgments
The monoclonal antibodies against the Na+/K+-ATPase pan αsubunit (α5) and α1-isoform (a6F) were purchased from the Developmental Studies Hybridoma Bank maintained by the Department of
Pharmacology and Molecular Sciences, Johns Hopkins University School
of Medicine, Baltimore, MD 21205, and the Department of Biological
Sciences, University of Iowa, Iowa City, IA 52242, under Contract N01HD-6-2915, NICHD, USA. This study was supported by grants to T.H.L.
from the Ministry of Science and Technology (MOST) of Taiwan (962313-B-005-010-MY3) and the Taiwan Comprehensive University
System (103TCUS03).
30
W.-K. Yang et al. / Comparative Biochemistry and Physiology, Part B 202 (2016) 23–30
References
Arjona, F.J., Vargas-Chacoff, L., Ruiz-Jarabo, I., Martín del Río, M.P., Mancera, J.M., 2007.
Osmoregulatory response of Senegalese sole (Solea senegalensis) to changes in environmental salinity. Comp. Biochem. Physiol. A 148, 413–421.
Armesto, P., Campinho, M.A., Rodríguez-Rúa, A., Cousin, X., Power, D.M., Manchado, M.,
Infante, C., 2014. Molecular characterization and transcriptional regulation of the
Na+/K+ ATPase α subunit isoforms during development and salinity challenge in a
teleost fish, the Senegalese sole (Solea senegalensis). Comp. Biochem. Physiol. B 175,
23–38.
Beyenbach, K.W., 2004. Kidneys sans glomeruli. Am. J. Physiol. Ren. Physiol. 286,
F811–F827.
Blanco, G., 2005. Na,K-ATPase subunit heterogeneity as a mechanism for tissue-specific
ion regulation. Semin. Nephrol. 25, 292–303.
Blanco, G., Mercer, R.W., 1998. Isozymes of the Na-K-ATPase: heterogeneity in structure,
diversity in function. Am. J. Physiol. Ren. Physiol. 275, F633–F650.
Brauer, P.R., Sanmann, J.N., Petzel, D.H., 2005. Effects of warm acclimation on Na+,K+ATPase α-subunit expression in chloride cells of Antarctic fish. Anat. Rec. A 285,
600–609.
Chen, X.L., Wee, N.L.J.E., Hiong, K.C., Ong, J.L.Y., Chng, Y.R., Ching, B., Wong, W.P., Chew,
S.F., Ip, Y.K., 2013. Properties and expression of Na+/K+-ATPase α-subunit isoforms
in the brain of the swamp eel, Monopterus albus, which has unusually high brain
ammonia tolerance. PLoS One 8, e84298.
Ching, B., Woo, J.M., Hiong, K.C., Boo, M.V., Choo, C.Y.L., Wong, W.P., Chew, S.F., Ip, Y.K.,
2015. Na+/K+-ATPase α-subunit (nkaα) isoforms and their mRNA expression levels,
overall Nkaα protein abundance, and kinetic properties of Nka in the skeletal muscle
and three electric organs of the electric eel, Electrophorus electricus. PLoS One 10,
e0118352.
Dalziel, A.C., Bittman, J., Mandic, M., Ou, M., Schulte, P.M., 2014. Origins and functional
diversification of salinity-responsive Na+, K+ ATPase α1 paralogs in salmonids.
Mol. Ecol. 23, 3483–3503.
D'Cotta, H., Valotaire, C., le Gac, F., Prunet, P., 2000. Synthesis of gill Na+-K+-ATPase in
Atlantic salmon smolts: differences in α-mRNA and α-protein levels. Am. J. Phys.
Regul. Integr. Comp. Phys. 278, R101–R110.
Evans, D.H., Claiborne, J.B., 2009. Osmotic and ionic regulation in fishes. In: Evans, D.H.
(Ed.), Osmotic and Ionic Regulation. CRC Press, Boca Raton, FL, pp. 295–366.
Feng, S.H., Leu, J.H., Yang, C.H., Fang, M.J., Huang, C.J., Hwang, P.P., 2002. Gene expression
of Na+-K+-ATPase α1 and α3 subunits in gills of the teleost Oreochromis
mossambicus, adapted to different environmental salinities. Mar. Biotechnol. 4,
379–391.
Freire, C.A., Amado, E.M., Souza, L.R., Veiga, M.P.T., Vitule, J.R.S., Souza, M.M., Prodocimo,
V., 2008. Muscle water control in crustaceans and fishes as a function of habitat,
osmoregulatory capacity, and degree of euryhalinity. Comp. Biochem. Physiol. A
149, 435–446.
Fuentes, J., Soengas, J.L., Rey, P., Rebolledo, E., 1997. Progressive transfer to seawater enhances intestinal and branchial Na+-K+-ATPase activity in non-anadromous rainbow
trout. Aquac. Int. 5, 217–227.
Guynn, S.R., Scofield, M.A., Petzel, D.H., 2002. Identification of mRNA and protein expression of the Na/K-ATPase α1-, α2- and α3-subunit isoforms in Antarctic and New
Zealand nototheniid fishes. J. Exp. Mar. Biol. Ecol. 273, 15–32.
Hiong, K.C., Ip, Y.K., Wong, W.P., Chew, S.F., 2014. Brain Na+/K+-ATPase α-subunit isoforms and aestivation in the African lungfish, Protopterus annectens. J. Comp. Physiol.
B. 184, 571–587.
Hwang, P.P., Lee, T.H., 2007. New insights into fish ion regulation and mitochondrion-rich
cells. Comp. Biochem. Physiol. A 148, 479–497.
Hwang, P.P., Sun, C.M., Wu, S.M., 1988. Characterization of gill Na+-K+-activated adenosine triphosphatase from tilapia Oreochromis mossambicus. Bull. Inst. Zool. Acad. Sin.
27, 49–56.
Inokuchi, M., Hiroi, J., Watanabe, S., Lee, K.M., Kaneko, T., 2008. Gene expression and morphological localization of NHE3, NCC and NKCC1a in branchial mitochondria-rich
cells of Mozambique tilapia (Oreochromis mossambicus) acclimated to a wide range
of salinities. Comp. Biochem. Physiol. A 151, 151–158.
Inoue, K., Takei, Y., 2003. Asian medaka fishes offer new models for studying mechanisms
of seawater adaptation. Comp. Biochem. Physiol. B 136, 635–645.
Ip, Y.K., Loong, A.M., Kuah, J.S., Sim, E.W.L., Chen, X.L., Wong, W.P., Lam, S.H., Delgado, I.L.S.,
Wilson, J.M., Chew, S.F., 2012. Roles of three branchial Na+-K+-ATPase α-subunit
isoforms in freshwater adaptation, seawater acclimation, and active ammonia excretion in Anabas testudineus. Am. J. Phys. Regul. Integr. Comp. Phys. 303, R112–R125.
Kang, C.K., Tsai, H.J., Liu, C.C., Lee, T.H., Hwang, P.P., 2010. Salinity-dependent expression
of a Na+, K+, 2Cl− cotransporter in gills of the brackish medaka Oryzias dancena: a
molecular correlate for hyposmoregulatory endurance. Comp. Biochem. Physiol. A
157, 7–18.
Kang, C.K., Chen, Y.C., Chang, C.H., Tsai, S.C., Lee, T.H., 2015. Seawater-acclimation abates
cold effects on Na+, K+-ATPase activity in gills of the juvenile milkfish, Chanos
chanos. Aquaculture 446, 67–73.
Katoh, F., Cozzi, R.R.F., Marshall, W.S., Goss, G.G., 2008. Distinct Na+/K+/2Cl−
cotransporter localization in kidneys and gills of two euryhaline species, rainbow
trout and killifish. Cell Tissue Res. 334, 265–281.
Lee, T.H., Tsai, J.C., Fang, M.J., Yu, M.J., Hwang, P.P., 1998. Isoform expression of Na+-K+ATPase α-subunit in gills of the teleost Oreochromis mossambicus. Am. J. Phys.
Regul. Integr. Comp. Phys. 275, R926–R932.
Lee, T.H., Feng, S.H., Lin, C.H., Hwang, Y.H., Huang, C.L., Hwang, P.P., 2003. Ambient salinity
modulates the expression of sodium pumps in branchial mitochondria-rich cells of
Mozambique tilapia, Oreochromis mossambicus. Zool. Sci. 20, 29–36.
Liao, B.K., Chen, R.D., Hwang, P.P., 2009. Expression regulation of Na+-K+-ATPase α1subunit subtypes in zebrafish gill ionocytes. Am. J. Phys. Regul. Integr. Comp. Phys.
296, R1897–R1906.
Lin, C.H., Lee, T.H., 2005. Sodium or potassium ions activate different kinetics of gill Na, KATPase in three seawater- and freshwater-acclimated euryhaline teleosts. J. Exp. Zool.
A 303, 57–65.
Lin, Y.M., Chen, C.N., Lee, T.H., 2003. The expression of gill Na, K-ATPase in milkfish,
Chanos chanos, acclimated to seawater, brackish water and fresh water. Comp.
Biochem. Physiol. A 135, 489–497.
Lin, C.H., Tsai, R.S., Lee, T.H., 2004. Expression and distribution of Na, K-ATPase in gill and
kidney of the spotted green pufferfish, Tetraodon nigroviridis, in response to salinity
challenge. Comp. Biochem. Physiol. A 138, 287–295.
Madsen, S.S., Engelund, M.B., Cutler, C.P., 2015. Water transport and functional dynamics
of aquaporins in osmoregulatory organs of fishes. Biol. Bull. 229, 70–92.
Marshall, W.S., Grosell, M., 2006. Ion transport, osmoregulation, and acid–base balance.
In: Evans, D.H., Claiborne, J.B. (Eds.), The Physiology of Fishes. CRC Press, Boca
Raton, pp. 177–230.
Moorman, B.P., Inokuchi, M., Yamaguchi, Y., Lerner, D.T., Grau, E.G., Seale, A.P., 2014. The
osmoregulatory effects of rearing Mozambique tilapia in a tidally changing salinity.
Gen. Comp. Endocrinol. 207, 94–102.
Moorman, B.P., Lerner, D.T., Grau, E.G., Seale, A.P., 2015. The effects of acute salinity
challenges on osmoregulation in Mozambique tilapia reared in a tidally changing
salinity. J. Exp. Biol. 218, 731–739.
Morrison, J.F., Guynn, S.R., Scofield, M.A., Dowd, F.J., Petzel, D.H., 2006. Warm acclimation
changes the expression of the Na+,K+-ATPase α subunit isoforms in Antarctic fish
gills. J. Exp. Mar. Biol. Ecol. 333, 129–139.
Pagliarani, A., Ventrella, V., Ballestrazzi, R., Trombetti, F., Pirini, M., Trigari, G., 1991.
Salinity-dependence of the properties of gill (Na++K+)-ATPase in rainbow trout
(Oncorhynchus mykiss). Comp. Biochem. Physiol. B 100, 229–236.
Pressley, T.A., 1992. Phylogenetic conservation of isoform-specific regions within alphasubunit of Na+,K+-ATPase. Am. J. Phys. Cell Physiol. 262, C743–C751.
Richards, J.G., Semple, J.W., Bystriansky, J.S., Schulte, P.M., 2003. Na+/K+-ATPase αisoform switching in gills of rainbow trout (Oncorhynchus mykiss) during salinity
transfer. J. Exp. Biol. 206, 4475–4486.
Scheiner-Bobis, G., 2002. The sodium pump. Eur. J. Biochem. 269, 2424–2433.
Suhail, M., 2010. Na+, K+-ATPase: ubiquitous multifunctional transmembrane protein
and its relevance to various pathophysiological conditions. J. Clin. Med. Res. 2, 1–17.
Takei, Y., Hiroi, J., Takahashi, H., Sakamoto, T., 2014. Diverse mechanisms for body fluid
regulation in teleost fishes. Am. J. Phys. Regul. Integr. Comp. Phys. 307, R778–R792.
Takeyasu, K., Lemas, V., Fambrough, D.M., 1990. Stability of Na+-K+-ATPase alphasubunit isoforms in evolution. Am. J. Phys. Cell Physiol. 259, C619–C630.
Tang, C.H., Chiu, Y.H., Tsai, S.C., Lee, T.H., 2009a. Relative changes in the abundance of
branchial Na+/K+-ATPase α-isoform-like proteins in marine euryhaline milkfish
(Chanos chanos) acclimated to environments of different salinities. J. Exp. Zool. A
311, 521–529.
Tang, C.H., Tzeng, C.S., Hwang, L.Y., Lee, T.H., 2009b. Constant muscle water content and
renal HSP90 expression reflect osmotic homeostasis in euryhaline teleosts acclimated
to different environmental salinities. Zool. Stud. 48, 435–441.
Tang, C.H., Wu, W.Y., Tsai, S.C., Yoshinaga, T., Lee, T.H., 2010. Elevated Na+/K+-ATPase
responses and its potential role in triggering ion reabsorption in kidneys for homeostasis of marine euryhaline milkfish (Chanos chanos) when acclimated to hypotonic
fresh water. J. Comp. Physiol. B. 180, 813–824.
Teranishi, K., Kaneko, T., 2010. Spatial, cellular, and intracellular localization of Na+/K+ATPase in the sterically disposed renal tubules of Japanese eel. J. Histochem.
Cytochem. 58, 707–719.
Tipsmark, C.K., Breves, J.P., Seale, A.P., Lerner, D.T., Hirano, T., Grau, E.G., 2011. Switching of
Na+, K+-ATPase isoforms by salinity and prolactin in the gill of a cichlid fish.
J. Endocrinol. 209, 237–244.
Tomy, S., Chang, Y.M., Chen, Y.H., Cao, J.C., Wang, T.P., Chang, C.F., 2009. Salinity effects on
the expression of osmoregulatory genes in the euryhaline black porgy Acanthopagrus
schlegeli. Gen. Comp. Endocrinol. 161, 123–132.
Varsamos, S., Diaz, J.P., Charmantier, G.U.Y., Flik, G., Blasco, C., Connes, R., 2002. Branchial
chloride cells in sea bass (Dicentrarchus labrax) adapted to fresh water, seawater, and
doubly concentrated seawater. J. Exp. Zool. 293, 12–26.
Whittamore, J.M., 2012. Osmoregulation and epithelial water transport: lessons from the
intestine of marine teleost fish. J. Comp. Physiol. B. 182, 1–39.
Yang, S.H., Kang, C.K., Hu, Y.C., Yen, L.C., Tsai, S.C., Hsieh, Y.L., Lee, T.H., 2015. Comparisons
of two types of teleostean pseudobranchs, silver moony (Monodactylus argenteus)
and tilapia (Oreochromis mossambicus), with salinity-dependent morphology and
ion transporter expression. J. Comp. Physiol. B. 1–17.
Yang, W.K., Hseu, J.R., Tang, C.H., Chung, M.J., Wu, S.M., Lee, T.H., 2009. Na+/K+-ATPase
expression in gills of the euryhaline sailfin molly, Poecilia latipinna, is altered in
response to salinity challenge. J. Exp. Mar. Biol. Ecol. 375, 41–50.
Yang, W.K., Kang, C.K., Chen, T.Y., Chang, W.B., Lee, T.H., 2011. Salinity-dependent expression of the branchial Na+/K+/2Cl− cotransporter and Na+/K+-ATPase in the sailfin
molly correlates with hypoosmoregulatory endurance. J. Comp. Physiol. B. 181,
953–964.
Yang, W.K., Kang, C.K., Chang, C.H., Hsu, A.D., Lee, T.H., Hwang, P.P., 2013. Expression
profiles of branchial FXYD proteins in the brackish medaka Oryzias dancena: a potential saltwater fish model for studies of osmoregulation. PLoS One 8, e55470.
Yang, W.K., Kang, C.K., Hsu, A.D., Lin, C.H., Lee, T.H., 2016. Different modulatory mechanisms of renal FXYD12 for Na+-K+-ATPase between two closely related medakas
upon salinity challenge. Int. J. Biol. Sci. 12, 730–745.