Abstract
Estrogen-related receptors (ERRs) are known to function in mammalian kidney as key regulators of ion transport-related genes; however, a comprehensive understanding of the physiological functions of ERRs in vertebrate body fluid ionic homeostasis is still elusive. Here, we used medaka (Oryzias melastigma), a euryhaline teleost, to investigate how ERRs are involved in ion regulation. After transferring medaka from hypertonic seawater to hypotonic freshwater (FW), the mRNA expression levels of errγ2 were highly upregulated, suggesting that Errγ2 may play a crucial role in ion uptake. In situ hybridization showed that errγ2 was specifically expressed in ionocytes, the cells responsible for Na+/Cl− transport. In normal FW, ERRγ2 morpholino knockdown caused reductions in the mRNA expression of Na+/Cl− cotransporter (Ncc), the number of Ncc ionocytes, Na+/Cl− influxes of ionocytes, and whole-body Na+/Cl− contents. In FW with low Na+ and low Cl−, the expression levels of mRNA for Na+/H+ exchanger 3 (Nhe3) and Ncc were both decreased in Errγ2 morphants. Treating embryos with DY131, an agonist of Errγ, increased the whole-body Na+/Cl− contents and ncc mRNA expression in Errγ2 morphants. As such, medaka Errγ2 may control Na+/Cl− uptake by regulating ncc and/or nhe3 mRNA expression and ionocyte number, and these regulatory actions may be subtly adjusted depending on internal and external ion concentrations. These findings not only provide new insights into the underpinning mechanism of actions of ERRs, but also enhance our understanding of their roles in body fluid ionic homeostasis for adaptation to changing environments during vertebrate evolution.
Introduction
Vertebrates likely originated in marine (seawater (SW)) environments and may have evolved to become iono-/osmo-regulators upon their initial invasion of hypotonic freshwater (FW) habitats (Robertson 1957, Evans et al. 2005). To achieve body fluid ionic and osmotic homeostasis, teleosts, which are the most successful and diverse group of vertebrates (Venkatesh 2003), developed sophisticated mechanisms for uptake and secretion of ions in the gills (the major organ for fish osmoregulation) (Evans et al. 2005). These mechanisms were conserved in the kidney of mammals, in terms of the underlying transporters and pathways (Evans et al. 2005, Hwang & Lee 2007, Hwang et al. 2011). As such, studies into fish ion regulation mechanisms can provide valuable insights into the mechanisms in mammalian kidney, and at the same time, enhance our understanding of vertebrate evolutionary physiology.
Hormones are known to tightly control ion transport functions critical for body fluid ionic and osmotic homeostasis in fishes and mammals (Bradshaw & McCormick 2006, Takei et al. 2014, Yan & Hwang 2019). Upon the binding of hormones to specific receptors, downstream signaling pathways are triggered to increase salt secretion or uptake capacity by regulating the expression, trafficking and activity of ion transporters, or by regulating the proliferation and differentiation of ionocytes in the ion-transporting epithelia (Bradshaw & McCormick 2006, Guh & Hwang 2017, Yan & Hwang 2019). Interestingly, several receptors with undiscovered ligands, known as orphan receptors, may affect body fluid ionic and osmotic homeostasis. Estrogen-related receptors (ERRs), which principally include ERRα, ERRβ, and ERRγ, are considered to be orphan receptors belonging to the nuclear receptor superfamily (Giguère 2008). In their role as transcription factors, ERRs regulate the mRNA expression of specific downstream genes involved in various physiological processes, such as energy metabolism, blood pressure control, and ion balance (Giguère 2008, Alaynick et al. 2010, Tremblay et al. 2010, Wang et al. 2016).
Recent studies have suggested possible roles for mammalian ERRs in body fluid ionic homeostasis, although functional characterizations of related ion transport pathways are still lacking. For instance, increased serum Na+ and decreased urine Na+ were found in ERRα knockout (KO) mice, and decreased serum K+ was found in both ERRα and ERRγ KO mice. Furthermore, the mRNA expression of some ion transporters and renin–angiotensin system-related genes were changed in ERRα or ERRγ mutants (Alaynick et al. 2010, Tremblay et al. 2010). However, the mechanisms connecting altered gene expression to the observed defects in serum/urine ion contents are still unclear, largely due to the difficulty of conducting in vivo functional assays to measure the ion transport in the kidney. As a model system, fish do not suffer from this problem because the major organs for ion regulation (i.e. adult gills and embryonic skin) are rich in ionocytes and exposed to the environment. To exploit this advantage, a non-invasive scanning ion-selective electrode technique (SIET) was developed to directly detect real-time ion currents of ionocytes, which can provide convincing physiological evidence at a cellular level (Lin et al. 2006, Wu et al. 2010, Yan et al. 2020).
Medaka (Oryziasmelastigmus), a euryhaline teleost, is an emerging and powerful model to study fish ion regulation, and we have recently used this species to study ion transport pathways in gills and embryonic skin ionocytes (Hsu et al. 2014, Yan et al. 2020). Here, we aimed to test the hypothesis that ERRs are involved in the regulation of ionocyte ion transport functions to maintain fish body fluid ionic homeostasis. Overall, our findings demonstrated that Errγ2 affects Na+/Cl− uptake by regulating the expression of ion transporters in medaka, providing new insights into the underpinning mechanism of actions of ERRs in vertebrate body fluid ionic homeostasis.
Materials and methods
Experimental animals
Mature medaka (Oryziasmelastigma) were reared in local tap FW or in 35- parts per trillion (ppt) SW at 28°C under a 14-h light:10-h darkness photoperiod at the Institute of Cellular and Organismic Biology, Academia Sinica, Taipei, Taiwan. Fertilized eggs were collected for the experiments, and the culturing water was changed daily to maintain water quality. The experimental protocols were approved by the Academia Sinica Institutional Animal Care and Utilization Committee (approval no.: BSF0415-00003197), and all methods were performed in accordance with relevant guidelines and regulations.
Salinity acclimation experiments
For experiments on adults, SW (35-ppt)-reared medaka were transferred to FW for 7 days. The gills were sampled after the 7-day acclimation. The ratio of male and female medaka used in each experimental set was about 50%. For experiments on embryos, fertilized eggs spawned in 35-ppt SW were immediately transferred to FW for culturing, and then sampled at 6 days post-fertilization (dpf).
Embryo dechorination
For most experiments, embryos were dechorinated with hatch enzyme (National BioResource Project Medaka, Okazaki, Japan) before drug treatment, fixation, or functional analyses. Timing details are shown in the experimental schema of figures.
Complementary DNA (cDNA) preparation
Adult gills isolated from two individuals or ten embryos were pooled as one sample. Samples were homogenized in TRIzol Reagent (Invitrogen). Total RNA was purified following the manufacturer’s protocol and further treated with DNase I (Roche) at 37°C for 20 min to remove genomic DNA. NanoDrop 2000 (Thermo Scientific) was then used to determine the quality and quantity of the total RNA. Total RNA (1 μg) was used to synthesize cDNA with SuperScript IV reverse transcriptase (Thermo Scientific), following the manufacturer’s protocol.
Quantitative real-time polymerase chain reaction (qRT-PCR)
A Light Cycler real-time PCR system (Roche) was used to perform qRT-PCR in a final reaction volume of 10 μL, containing 5 μL 2× SYBR Green I Master Mix (Roche), 300 nM of the primer pairs, and 20–30 ng cDNA. Expression of the medaka gene for ribosomal protein L7 (rpl7) was used as an internal control. The primer sets used for qRT-PCR are provided in Supplementary Table 1 (see section on supplementary materials given at the end of this article). The amplification efficiency was confirmed by serial cDNA dilution for each primer set, and the specificity of primer sets was confirmed by Sanger sequencing of the amplicons.
In situ hybridization (ISH)
The cDNA fragments of medaka Errγ2, Na+/H+ exchanger 3 (Nhe3), and Na+/Cl− cotransporter (Ncc) were amplified with the specific primers (Supplementary Table 1) and inserted into pGEM-T easy vectors (Promega), respectively. These plasmids were then used to synthesize the digoxigenin-/fluorescein-labeled riboprobes. ten to twenty dechorinated embryos (6 dpf) were fixed, and ISH of the whole-mount embryos was performed by the protocol from a previous study (Wang et al. 2015). For cell counting, sample images were obtained with Axioplan 2 Upright Microscope (ZEISS). ImageJ was used to count the ionocyte number in the yolk-sac skin of embryos.
Morpholino oligonucleotide (MO) knockdown
MOs were purchased from Gene Tools (Philomath, OR, USA). The sequences of the MO against Errγ2 (ENSOMEG00000017888) and standard control (Ctrl) MO were 5’-ATCACTCATGGATTTAGTCGACCTG-3’ and 5’-CCTCTTACCTCAGTTACAATTTATA-3’, respectively. Medaka fertilized eggs were injected with 2 ng ERRγ2 or Ctrl MO. The dose of Errγ2 MO was selected based on Supplementary Fig. 1. Fertilized eggs were injected with 2 ng Errγ2 MO, and the survival rate was higher than 80% with no obvious defects at 6 dpf. Morphants were then reared in FW or in low-Na+/low-Cl− (LNLC)-FW until the desired stage. The LNLC-FW was prepared according to a previously published protocol (Wang et al. 2009).
Measurement of whole-body ion contents
Five dechorinated embryos were pooled as one sample. Samples were briefly rinsed in deionized water and then dried at 60°C for 2–3 h. To measure Na+ and Ca2+ contents, samples were digested with 70% HNO3 at 60°C overnight. Digested samples were then diluted with 14% HNO3 to a proper volume for measurement. Na+ and Ca2+ concentrations were measured with an atomic absorption spectrophotometer ZA3000 (Hitachi). For the measurement of Cl− content, each sample was homogenized in 1 mL deionized water and centrifuged at 14,600 g for 30 min. Supernatants were collected, and diluted with deionized water to proper volume for measurement. Thereafter, the ferricyanide method was conducted as described previously (Wang et al. 2009). Cl− concentration was measured with U-2000 double-beam spectrophotometer (Hitachi).
Scanning ion-selective electrode technique (SIET)
To record the ion fluxes of ionocytes in 6 dpf embryos, an ion-selective microelectrode was prepared and moved to a position about 2 μm above the apical surface of an ionocyte. Na+ and Cl− fluxes in the yolk-sac skin were measured by Na+-/Cl−-selective microelectrodes with ASET software, following the previous study (Shen et al. 2011).
Western blot
Ten dechorinated embryos were pooled as one sample and homogenized in 100 µL SEID buffer (150 mM sucrose, 10 mM EDTA, 50 mM imidazole, 0.1% sodium deoxycholate, and pH 7.4) with Complete Protease Inhibitor Cocktail (Roche) and centrifuged at 14,600 g for 30 min at 4°C. The supernatant was collected. A mixture of embryo homogenate (30 µg total protein) and 6× Protein Sample Dye (β-Me) (ACE Biolabs) was first denatured at 95°C for 5 min, and then separated by the electrophoresis on SDS-containing 8% polyacrylamide gel. Proteins were transferred to an Immobilon-P PVDF membrane (Millipore), pre-incubated with TOOLSpeed Blocking Reagent (BIOTOOLS) at room temperature for 5 min, and immunoblotted with the custom rabbit anti-Errγ2 antiserum (1:1000 dilution) or mouse anti-actin antibody, clone C4 (1:5000 dilution) (Sigma-Aldrich) overnight at 4°C, followed by reaction with goat anti-rabbit IgG (H+L) secondary antibody, HRP (1:5000 dilution) (Invitrogen) or mouse IgG HRP-linked whole antibody (1:5000 dilution) (Cytiva) for 1 h at room temperature. Signals were detected by UVP BioSpectrum 600 with WesternBright ECL (Advansta) and quantitated by ImageJ. The anti-Errγ2 antiserum was generated in a rabbit by the injection of the synthetic peptide (AAALAGNGGSTRRC) of medaka Errγ2.
Pharmacological treatment
The stock of 100 mM DY131 (Sigma-Aldrich) was prepared in DMSO. Dechorionated embryos were incubated in FW containing 50 μM DY131 for 12, 18, and 24 h. The final concentration of DMSO in working solution was 0.05%. DY131 concentration of 50 μM was selected based on Supplementary Fig. 4.
Statistical analysis
Values are presented as the mean ± s.d. The Shapiro–Wilk normality test was first applied to evaluate normality of the datasets before statistical analysis. Values from each group were analyzed using Mann–Whitney U test, Student’s t-test, or one-way ANOVA followed by Tukey’s pair-wise comparison when applicable. Statistical analysis was performed using GraphPad Prism 8. P values of one-way ANOVA are provided in Supplementary Table 2.
Results
Medaka errγ2 mRNA expression is upregulated after transfer from SW to FW
To examine if the ERRs are involved in fish ion regulation, adult medaka were reared in 35-ppt SW and then transferred to FW for 1 week, or fertilized eggs spawned in 35-ppt SW were immediately transferred to FW for 6 days. The mRNA expression levels of errα, errβ, errγ1, and errγ2 in adult gills and 6 dpf embryos were analyzed by qRT-PCR. The expression of errγ2 was significantly increased both in adult gills and embryos after transfer to FW (Fig. 1A and B). Therefore, we subsequently explored the localization and functions of Errγ2 in FW-acclimated medaka.
Effects of salinity change on the mRNA expression levels of estrogen-related receptors (ERRs). The mRNA expression levels of errα, errβ, errγ1, and errγ2 in adult gills (A) and 6 dpf embryos (B) were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR) after transfer from sea water (SW) to fresh water (FW). Gene expression data were normalized to rpl7. Bars represent the mean ± s.d. Sample size is shown in the parentheses. Student’s t-test, *P < 0.05, **P < 0.01, ***P < 0.001. ns, not significant.
Citation: Journal of Endocrinology 251, 2; 10.1530/JOE-21-0112
Effects of salinity change on the mRNA expression levels of estrogen-related receptors (ERRs). The mRNA expression levels of errα, errβ, errγ1, and errγ2 in adult gills (A) and 6 dpf embryos (B) were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR) after transfer from sea water (SW) to fresh water (FW). Gene expression data were normalized to rpl7. Bars represent the mean ± s.d. Sample size is shown in the parentheses. Student’s t-test, *P < 0.05, **P < 0.01, ***P < 0.001. ns, not significant.
Citation: Journal of Endocrinology 251, 2; 10.1530/JOE-21-0112
Effects of salinity change on the mRNA expression levels of estrogen-related receptors (ERRs). The mRNA expression levels of errα, errβ, errγ1, and errγ2 in adult gills (A) and 6 dpf embryos (B) were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR) after transfer from sea water (SW) to fresh water (FW). Gene expression data were normalized to rpl7. Bars represent the mean ± s.d. Sample size is shown in the parentheses. Student’s t-test, *P < 0.05, **P < 0.01, ***P < 0.001. ns, not significant.
Citation: Journal of Endocrinology 251, 2; 10.1530/JOE-21-0112
Medaka errγ2 is expressed in ionocytes
To test whether the Errγ2 may be expressed in ionocytes, where it might play a role in FW acclimation, the localization of errγ2, nhe3 (a NHE ionocyte maker), and ncc (a NCC ionocyte maker) was analyzed by ISH. Double ISH of errγ2 and ncc showed that some of the errγ2 mRNA signals were expressed in ncc-expressing ionocytes (Fig. 2A, B, C and D); besides, double ISH of errγ2 and nhe3 showed that some of the errγ2 mRNA signals were also expressed in nhe3-expressing ionocytes (Fig. 2E, F, G and H). A negative control for ISH, using a sense probe for errγ2, did not produce any clear signal (Fig. 2I). These data suggested that Errγ2 is expressed and may act specifically in NHE and NCC ionocytes, which are responsible for Na+/Cl− uptake (Yan & Hwang 2019).
Localization of errγ2 in 6 dpf FW-acclimated embryos. In situ hybridization (ISH) was used to analyze the expression of errγ2 (A, C, E and G), ncc (B and D), and nhe3 (F and H). (C), (D), (G) and (H) show enlargements of the indicated areas in (A), (B), (E) and (F), respectively. ISH with a sense probe of errγ2 was performed (I). Arrowheads indicate co-localization of errγ2 with nhe3 or ncc signals. Scale bar, 100 μm.
Citation: Journal of Endocrinology 251, 2; 10.1530/JOE-21-0112
Localization of errγ2 in 6 dpf FW-acclimated embryos. In situ hybridization (ISH) was used to analyze the expression of errγ2 (A, C, E and G), ncc (B and D), and nhe3 (F and H). (C), (D), (G) and (H) show enlargements of the indicated areas in (A), (B), (E) and (F), respectively. ISH with a sense probe of errγ2 was performed (I). Arrowheads indicate co-localization of errγ2 with nhe3 or ncc signals. Scale bar, 100 μm.
Citation: Journal of Endocrinology 251, 2; 10.1530/JOE-21-0112
Localization of errγ2 in 6 dpf FW-acclimated embryos. In situ hybridization (ISH) was used to analyze the expression of errγ2 (A, C, E and G), ncc (B and D), and nhe3 (F and H). (C), (D), (G) and (H) show enlargements of the indicated areas in (A), (B), (E) and (F), respectively. ISH with a sense probe of errγ2 was performed (I). Arrowheads indicate co-localization of errγ2 with nhe3 or ncc signals. Scale bar, 100 μm.
Citation: Journal of Endocrinology 251, 2; 10.1530/JOE-21-0112
Knockdown of Errγ2 impairs Na+ and Cl- uptake
To explore the function of Errγ2 in ionic homeostasis, a MO was used to knockdown Errγ2 expression. Fertilized eggs were injected with Errγ2 MO (2 ng/embryo), and 6 dpf morphants were analyzed (Fig. 3A). The whole-body Na+ and Cl− contents were significantly decreased in the ERRγ2 MO-injected group as compared with Ctrl MO and WT groups (Fig. 3B and C); however, knockdown of ERRγ2 did not affect Ca2+ contents (Supplementary Fig. 2). To determine the possible reasons for decreased Na+/Cl− contents, we examined Na+/Cl− absorption in individual ionocytes using SIET, and we quantified the number of ionocytes by ISH in the yolk-sac skin of 6 dpf morphants. As shown in Fig. 3D and E, ionocyte Na+ and Cl− influxes were significantly decreased in ERRγ2 morphants compared to controls. Moreover, the number of ncc-expressing cells (NCC ionocytes) was decreased in ERRγ2 morphants (Fig. 3F), but the number of nhe3-expressing cells (NHE ionocytes) was not altered (Fig. 3G).
Effects of Errγ2 knockdown on Na+/Cl− uptake in FW-acclimated embryos. Fertilized eggs were first injected with Errγ2 MO and dechorionated at 5 dpf. Errγ2 morphants were then used at 6 dpf for analyses of ion contents, scanning ion-selective electrode technique (SIET), and cell counting. The detailed timeline for the experiments is shown (A). The whole-body Na+ and Cl− contents were measured (B and C). Na+ and Cl− fluxes of ionocytes in the yolk-sac skin were detected by SIET (D and E). The ionocyte numbers of ncc- and nhe3-expressing cells in the yolk-sac skin were calculated after ISH in 6 dpf morphants (F and G). Bars represent the mean ± s.d. Sample size is shown in the parentheses. One-way ANOVA, Tukey’s pair-wise comparison. Different letters indicate a significant difference between groups, P < 0.05. Same letters indicate no significant difference between groups.
Citation: Journal of Endocrinology 251, 2; 10.1530/JOE-21-0112
Effects of Errγ2 knockdown on Na+/Cl− uptake in FW-acclimated embryos. Fertilized eggs were first injected with Errγ2 MO and dechorionated at 5 dpf. Errγ2 morphants were then used at 6 dpf for analyses of ion contents, scanning ion-selective electrode technique (SIET), and cell counting. The detailed timeline for the experiments is shown (A). The whole-body Na+ and Cl− contents were measured (B and C). Na+ and Cl− fluxes of ionocytes in the yolk-sac skin were detected by SIET (D and E). The ionocyte numbers of ncc- and nhe3-expressing cells in the yolk-sac skin were calculated after ISH in 6 dpf morphants (F and G). Bars represent the mean ± s.d. Sample size is shown in the parentheses. One-way ANOVA, Tukey’s pair-wise comparison. Different letters indicate a significant difference between groups, P < 0.05. Same letters indicate no significant difference between groups.
Citation: Journal of Endocrinology 251, 2; 10.1530/JOE-21-0112
Effects of Errγ2 knockdown on Na+/Cl− uptake in FW-acclimated embryos. Fertilized eggs were first injected with Errγ2 MO and dechorionated at 5 dpf. Errγ2 morphants were then used at 6 dpf for analyses of ion contents, scanning ion-selective electrode technique (SIET), and cell counting. The detailed timeline for the experiments is shown (A). The whole-body Na+ and Cl− contents were measured (B and C). Na+ and Cl− fluxes of ionocytes in the yolk-sac skin were detected by SIET (D and E). The ionocyte numbers of ncc- and nhe3-expressing cells in the yolk-sac skin were calculated after ISH in 6 dpf morphants (F and G). Bars represent the mean ± s.d. Sample size is shown in the parentheses. One-way ANOVA, Tukey’s pair-wise comparison. Different letters indicate a significant difference between groups, P < 0.05. Same letters indicate no significant difference between groups.
Citation: Journal of Endocrinology 251, 2; 10.1530/JOE-21-0112
As a transcription factor, Errγ2 may regulate the mRNA expression of ion transporters related to Na+/Cl− uptake. qRT-PCR analyses revealed that nhe3 expression levels were not significantly different in 2–6 dpf morphants than those in controls (Fig. 4A). The ncc expression levels were also not significantly altered in 4–6 dpf morphants but were decreased in 3 dpf morphants (Fig. 4B). In further experiments, we kept the ERRγ2 morphants in LNLC-FW before qRT-PCR analyses. Interestingly, both nhe3 and ncc mRNA expression levels were decreased in 2 and 4 dpf morphants (Fig. 4C and D), indicating that Errγ2 enhances both nhe3 and ncc mRNA expressions when the environmental Na+ levels are low. Furthermore, morphants at 6 dpf had increased nhe3 and ncc mRNA expression levels in LNLC-FW compared to controls (Fig. 4C and D). This effect might be the consequence of compensatory mechanisms, since the knockdown efficiency of a morpholino decreases following development of the embryos. The specificity and efficiency of the Errγ2 MO was confirmed by Western blotting for relative ERRγ2 protein abundance. ERRγ2 MO treatment effectively diminished ERRγ2 protein expression in 3 dpf Errγ2 morphants (Supplementary Fig. 3A and C), but not in 6 dpf Errγ2 morphants (Supplementary Fig. 3B and D).
Effects of Errγ2 knockdown on ncc and nhe3 mRNA expression in FW- and low-Na+/low-Cl− (LNLC)-FW-acclimated embryos. Fertilized eggs were first injected with Errγ2 MO and reared in normal FW or LNLC-FW. Errγ2 morphants were then sampled at 2–6 dpf for qRT-PCR analyses. The mRNA expression levels of ncc and nhe3 were analyzed after the acclimation to normal FW (A and B). The mRNA expression levels of ncc and nhe3 were analyzed after the acclimation to LNLC-FW (C and D). Gene expression data were normalized to rpl7. Bars represent the mean ± s.d. Sample size is shown in the parentheses. Mann-Whitney U test or Student’s t-test, *P < 0.05. ns, not significant.
Citation: Journal of Endocrinology 251, 2; 10.1530/JOE-21-0112
Effects of Errγ2 knockdown on ncc and nhe3 mRNA expression in FW- and low-Na+/low-Cl− (LNLC)-FW-acclimated embryos. Fertilized eggs were first injected with Errγ2 MO and reared in normal FW or LNLC-FW. Errγ2 morphants were then sampled at 2–6 dpf for qRT-PCR analyses. The mRNA expression levels of ncc and nhe3 were analyzed after the acclimation to normal FW (A and B). The mRNA expression levels of ncc and nhe3 were analyzed after the acclimation to LNLC-FW (C and D). Gene expression data were normalized to rpl7. Bars represent the mean ± s.d. Sample size is shown in the parentheses. Mann-Whitney U test or Student’s t-test, *P < 0.05. ns, not significant.
Citation: Journal of Endocrinology 251, 2; 10.1530/JOE-21-0112
Effects of Errγ2 knockdown on ncc and nhe3 mRNA expression in FW- and low-Na+/low-Cl− (LNLC)-FW-acclimated embryos. Fertilized eggs were first injected with Errγ2 MO and reared in normal FW or LNLC-FW. Errγ2 morphants were then sampled at 2–6 dpf for qRT-PCR analyses. The mRNA expression levels of ncc and nhe3 were analyzed after the acclimation to normal FW (A and B). The mRNA expression levels of ncc and nhe3 were analyzed after the acclimation to LNLC-FW (C and D). Gene expression data were normalized to rpl7. Bars represent the mean ± s.d. Sample size is shown in the parentheses. Mann-Whitney U test or Student’s t-test, *P < 0.05. ns, not significant.
Citation: Journal of Endocrinology 251, 2; 10.1530/JOE-21-0112
ERR agonist treatment enhances Na+ and Cl− uptake
To further confirm the functional effect of Errγ2 in ion regulation, we used DY131, which was reported to serve as a specific ligand of ERRγ in mammalian cells (Kang et al. 2018). Dechorionated embryos (5 dpf) were treated with FW containing 50 μM DY131 for 12, 18, and 24 h (Fig. 5A). After treating with DY131 for 18 or 24 h, whole-body Cl− content was increased (Fig. 5C). Na+/Cl− influxes were increased at 24 h of treatment (Fig. 5D and E), and ncc mRNA expression levels were elevated at 18 h (Fig. 5F); however, Na+ content was not affected by DY131 (Fig. 5B). Notably, treating 5 dpf dechorionated Errγ2 morphants with DY131 for 24 h (Fig. 6A) rescued the morphant phenotype that whole-body Na+ and Cl− contents were recovered after treatments (Fig. 6B and C). The rescue effect was further observed after 2.25 dpf dechorionated Errγ2 morphants treated with DY131 for 18 h (Fig. 7A). These treated embryos had increased ncc mRNA expression (Fig. 7B).
Effects of DY131 treatment on Na+/Cl− uptake in FW-acclimated embryos. Embryos were dechorionated at 4 dpf and treated with 50 μM DY131 at 5 dpf for 12, 18, and 24 h. Measurement of ion contents and qRT-PCR were performed at 12, 18, 24 h, while SIET was performed at 24 h. The detailed timeline of the experiments is shown (A). The whole-body Na+ and Cl− contents were measured (B and C). Na+ and Cl− fluxes of ionocytes in the yolk-sac skin were detected by SIET (D and E). The mRNA expression levels of ncc were analyzed by qRT-PCR (F). Gene expression data were normalized to rpl7. Bars represent the mean ± s.d. Sample size is shown in the parentheses. Student’s t-test, *P < 0.05, **P < 0.01. ns, not significant.
Citation: Journal of Endocrinology 251, 2; 10.1530/JOE-21-0112
Effects of DY131 treatment on Na+/Cl− uptake in FW-acclimated embryos. Embryos were dechorionated at 4 dpf and treated with 50 μM DY131 at 5 dpf for 12, 18, and 24 h. Measurement of ion contents and qRT-PCR were performed at 12, 18, 24 h, while SIET was performed at 24 h. The detailed timeline of the experiments is shown (A). The whole-body Na+ and Cl− contents were measured (B and C). Na+ and Cl− fluxes of ionocytes in the yolk-sac skin were detected by SIET (D and E). The mRNA expression levels of ncc were analyzed by qRT-PCR (F). Gene expression data were normalized to rpl7. Bars represent the mean ± s.d. Sample size is shown in the parentheses. Student’s t-test, *P < 0.05, **P < 0.01. ns, not significant.
Citation: Journal of Endocrinology 251, 2; 10.1530/JOE-21-0112
Effects of DY131 treatment on Na+/Cl− uptake in FW-acclimated embryos. Embryos were dechorionated at 4 dpf and treated with 50 μM DY131 at 5 dpf for 12, 18, and 24 h. Measurement of ion contents and qRT-PCR were performed at 12, 18, 24 h, while SIET was performed at 24 h. The detailed timeline of the experiments is shown (A). The whole-body Na+ and Cl− contents were measured (B and C). Na+ and Cl− fluxes of ionocytes in the yolk-sac skin were detected by SIET (D and E). The mRNA expression levels of ncc were analyzed by qRT-PCR (F). Gene expression data were normalized to rpl7. Bars represent the mean ± s.d. Sample size is shown in the parentheses. Student’s t-test, *P < 0.05, **P < 0.01. ns, not significant.
Citation: Journal of Endocrinology 251, 2; 10.1530/JOE-21-0112
Effects of DY131 treatment on the whole-body Na+/Cl− contents in FW-acclimated Errγ2 morphants. Fertilized eggs were first injected with Errγ2 MO, dechorionated at 4 dpf stage. Then, embryos were treated with 50 μM DY131 at 5 dpf for 24 h, followed by measurement of ion contents. The detailed timeline of the experiments is shown (A). The whole-body Na+ and Cl− contents were measured (B and C). Bars represent the mean ± s.d. Sample size is shown in the parentheses. One-way ANOVA, Tukey’s pair-wise comparison. Different letters indicate a significant difference between groups, P < 0.05. Same letters indicate no significant difference between groups.
Citation: Journal of Endocrinology 251, 2; 10.1530/JOE-21-0112
Effects of DY131 treatment on the whole-body Na+/Cl− contents in FW-acclimated Errγ2 morphants. Fertilized eggs were first injected with Errγ2 MO, dechorionated at 4 dpf stage. Then, embryos were treated with 50 μM DY131 at 5 dpf for 24 h, followed by measurement of ion contents. The detailed timeline of the experiments is shown (A). The whole-body Na+ and Cl− contents were measured (B and C). Bars represent the mean ± s.d. Sample size is shown in the parentheses. One-way ANOVA, Tukey’s pair-wise comparison. Different letters indicate a significant difference between groups, P < 0.05. Same letters indicate no significant difference between groups.
Citation: Journal of Endocrinology 251, 2; 10.1530/JOE-21-0112
Effects of DY131 treatment on the whole-body Na+/Cl− contents in FW-acclimated Errγ2 morphants. Fertilized eggs were first injected with Errγ2 MO, dechorionated at 4 dpf stage. Then, embryos were treated with 50 μM DY131 at 5 dpf for 24 h, followed by measurement of ion contents. The detailed timeline of the experiments is shown (A). The whole-body Na+ and Cl− contents were measured (B and C). Bars represent the mean ± s.d. Sample size is shown in the parentheses. One-way ANOVA, Tukey’s pair-wise comparison. Different letters indicate a significant difference between groups, P < 0.05. Same letters indicate no significant difference between groups.
Citation: Journal of Endocrinology 251, 2; 10.1530/JOE-21-0112
Effects of DY131 treatment on ncc mRNA expression in FW-acclimated Errγ2 morphants. Fertilized eggs were first injected with Errγ2 MO and dechorionated at 2 dpf stage. Errγ2 morphants at 2.25 dpf were then treated with 50 μM DY131 for 18 h and sampled at 3 dpf for qRT-PCR analyses. The detailed timeline of the experiments is shown (A). The mRNA expression levels of ncc were analyzed (B). Gene expression data were normalized to rpl7. Bars represent the mean ± s.d. Sample size is shown in the parentheses. One-way ANOVA, Tukey’s pair-wise comparison. Different letters indicate a significant difference between groups, P < 0.05. Same letters indicate no significant difference between groups.
Citation: Journal of Endocrinology 251, 2; 10.1530/JOE-21-0112
Effects of DY131 treatment on ncc mRNA expression in FW-acclimated Errγ2 morphants. Fertilized eggs were first injected with Errγ2 MO and dechorionated at 2 dpf stage. Errγ2 morphants at 2.25 dpf were then treated with 50 μM DY131 for 18 h and sampled at 3 dpf for qRT-PCR analyses. The detailed timeline of the experiments is shown (A). The mRNA expression levels of ncc were analyzed (B). Gene expression data were normalized to rpl7. Bars represent the mean ± s.d. Sample size is shown in the parentheses. One-way ANOVA, Tukey’s pair-wise comparison. Different letters indicate a significant difference between groups, P < 0.05. Same letters indicate no significant difference between groups.
Citation: Journal of Endocrinology 251, 2; 10.1530/JOE-21-0112
Effects of DY131 treatment on ncc mRNA expression in FW-acclimated Errγ2 morphants. Fertilized eggs were first injected with Errγ2 MO and dechorionated at 2 dpf stage. Errγ2 morphants at 2.25 dpf were then treated with 50 μM DY131 for 18 h and sampled at 3 dpf for qRT-PCR analyses. The detailed timeline of the experiments is shown (A). The mRNA expression levels of ncc were analyzed (B). Gene expression data were normalized to rpl7. Bars represent the mean ± s.d. Sample size is shown in the parentheses. One-way ANOVA, Tukey’s pair-wise comparison. Different letters indicate a significant difference between groups, P < 0.05. Same letters indicate no significant difference between groups.
Citation: Journal of Endocrinology 251, 2; 10.1530/JOE-21-0112
Discussion
The findings of the present study suggest that medaka Errγ2 affects Na+/Cl− uptake functions by positively regulating ncc mRNA expression and the number of Ncc-expressing ionocytes, and that its actions are further extended to regulation of nhe3 mRNA expression under a Na+-deficient conditions. These functions of Errγ2 provide new insights into the role of ERRs in vertebrate body fluid ionic homeostasis.
Three ERR isoforms exist in mammals, including α, β, and γ (Giguère 2008). In line with their known physiological functions, mammalian ERRs are expressed in several organs and tissues with high energy demand, including the kidney (Giguère 2008). By comparison, teleosts have some additional ERR isoforms, including α, β, γ1, and γ2 in medaka and α, β, γ1, γ2, and δ in zebrafish. Therefore, we may consider how different actions and targets of ERRs may affect body fluid ionic homeostasis by the kidney and other osmoregulatory organs (i.e. the gills and skin of fish); similar to fish epithelia, the mammalian kidney has diverse ion transport functions that correspond to specific cell types in the renal tubules. Studies in mammals have shown that ERRα and ERRγ are expressed in the renal tubules and collecting ducts (CD) without further information regarding co-localization with transporters (Alaynick et al. 2010, Wang et al. 2016). Meanwhile, studies in fish can provide additional clues to reveal the diverse functions of ERR isoforms in ion homeostasis. In zebrafish, Errα is specifically expressed in H+-ATPase rich (HR) ionocytes, and it regulates acid secretion function (Guh et al. 2014). In medaka, we show that Errγ2 is expressed in nhe3- and ncc-expressing ionocytes (Fig. 2) (Hsu et al. 2014) responsible for the function of Na+ and/or Cl− uptake (Fig. 3) (Yan & Hwang 2019). Notably, Nhe3- and Ncc-expressing ionocytes are analogous to proximal tubular and distal convoluted tubular cells, respectively, in terms of the expression and functions of major ion transporters (Hwang & Lee 2007, Hwang & Chou 2013). As such, the findings in fish warrant further studies on mammalian kidney to explore the actions of ERRs on the ion transporters, other than the known effects on epithelial Na+ channels (ENaC) and K+ channels (Kcn) (Alaynick et al. 2010, Wang et al. 2016).
On the basis of knockout rats and cell biological experiments, ERRα was proposed to control renal Na+ reabsorption, probably through regulation of ENaC expression by binding to its promoter (Wang et al. 2016). In addition, ERRγ was suggested to affect K+ excretion by regulating the expression of Kcnj1, Kcne1, and Kcne2 (Alaynick et al. 2010). However, there have been no reports of functional studies to clarify the underlying mechanisms of the proposed actions of ERRs in mammalian kidney. This lack of direct evidence is probably due to the difficulty of conducting in vivo functional assays of ion transport in the kidney. By contrast, our loss-of-function (via morpholino) and gain-of-function (via agonist) experiments were combined with the SIET functional assay in order to directly show that Errγ2 is involved in body fluid ionic homeostasis by enhancing Na+/Cl− uptake functions (Fig. 3B, C, D and E). This action is coincident with and probably mediated by the stimulation of ncc mRNA expression (Fig. 4B) and increases in ncc-expressing ionocyte number (Fig. 3F). Similarly in zebrafish, Errα controls acid secretion and body fluid acid–base homeostasis through the regulation of HR ionocyte numbers (cells specific for acid secretion) and atp6v1a (H+-ATPase) expression (Guh et al. 2014). Together with the results in this study, these fish works demonstrate that the ionoregulatory effects of ERRs are mediated by processes that control both the numbers and the functions of ionocytes in fish. The effects of ERRs on ionocyte number in fish models imply their possible roles in ionocyte differentiation. Indeed, mammalian ERRs have been reported to be involved in cell proliferation and differentiation, and cell cycle progression (Giguère 2008, Eichner & Giguère 2011, Murray et al. 2013, Poidatz et al. 2015). Thus, a further question is raised about whether ERRs might regulate the differentiation of renal cells. As such, fish models, such as medaka and zebrafish, provide informative and competent platforms with which to study the detailed physiological mechanisms of ERR participation in vertebrate body fluid ionic homeostasis.
Different segments of mammalian nephrons display a sophisticated division of labor for transport of Na+, Cl− and other ions to achieve body fluid ionic homeostasis. About 90–95% of total Na+ reabsorption takes place in the proximal segments via NHE3 (in the proximal convoluted tubules; PCT) and in the thick limbs via Na+-K+-2Cl− cotransporter; meanwhile, the distal segments only contribute 5–10% of total Na+ reabsorption, with NCC and ENaC as the major transporters in the distal convoluted tubules (DCT) and the CD, respectively (Palmer & Schnermann 2015). In contrast to the PCT, which displays a rather constant rate of Na+ transport, the DCT and CD have Na+ reabsorption activities that are highly regulated by hormones and play a major role in the fine-tuning of urine Na+ ion concentrations (Greger 2000, Eladari & Hübner 2011, Palmer & Schnermann 2015). This difference in functional regulation between NHE3- and NCC (and/or ENaC)-mediated Na+ transport pathways in mammalian kidney is also found in fish (Hwang & Chou 2013), although teleost fish lack ENaC (Wichmann & Althaus 2020). In the skin and gills of zebrafish, Nhe3 is the major transporter, with Ncc playing a minor or backup role for Na+ uptake (Wang et al. 2009, Chang et al. 2013). The action of ERRs on body fluid Na+ homeostasis via differential regulations of these two Na+ transport pathways appears to result from convergent evolution in teleost gills (and embryonic skin) and mammalian kidney. As we found in medaka, Errγ2 affects Ncc but not Nhe3, in terms of mRNA expression and numbers of transporter-expressing ionocytes (Figs 3 and 4). Similarly in mammals, ERRα- and ERRγ-null kidneys respectively exhibit altered expression levels of Scnn1a (α subunit of ENaC) and Slc12a3 (NCC); however, no studies have shown altered expression of Slc9a3 (NHE3) upon loss- or gain-of-function for ERRs (Tremblay et al. 2010, Wang et al. 2016, Zhao et al. 2018). Our further loss-of-function experiments in a Na+-deficient environment elaborated the actions of ERRs on body fluid homeostasis in fish under external or internal disturbances. Loss of Errγ2 function affected both nhe3 and ncc expression in medaka embryos in LNLC-FW (0.04 mM of Na+) (Fig. 4C and D). This Na+-deficient condition is known to stimulate compensatory Na+ uptake function to counteract disturbed body fluid Na+ homeostasis (Yan et al. 2007, Shih et al. 2012), suggesting that the actions of Errγ2 are subtly extended to Nhe3, in addition to the major effects on Ncc (Fig. 4). This observation provides a reason to investigate ERR regulation of the expression and function of NHEs in mammalian kidney under conditions of low Na+, potentially revealing novel actions of ERRs on body fluid Na+ homeostasis in vertebrates.
ERRα null mice was reported to reveal phenotypes similar to Bartter syndrome with defects in blood Na+ and K+ concentrations and blood pressure, and ERRα was proposed to act as a transcriptional regulator for blood pressure probably through control the expression of renal Na+ and K+ transporters (Scnn1a, Atp1a1, Atp1b, Bsnd, and Kcnq1) and the genes involved in the renin–angiotensin pathway (Ren1, Agt, and Ace2) (Tremblay et al. 2010). As such, ERRα and other isoforms were suggested to be possible pharmacological targets for blood pressure disorders (Tremblay et al. 2010). The present findings provide additional information and reference to explore this issue. The direct actions of Errγ2 on Ncc- and Nhe3-mediated Na+ and/or Cl− uptake functions may be another pathway to control blood pressure because Na+ and Cl− are the key components in blood pressure control (McCallum et al. 2015, Nakajima et al. 2016).
Mammalian ERRγ has been reported to promote energy-generating mitochondrial functions and cellular metabolism, including the tricarboxylic acid cycle, fatty acid β-oxidation, and oxidative phosphorylation (Giguère 2008, Eichner et al. 2010, Rangwala et al. 2010, Eichner & Giguère 2011, Zhao et al. 2018). In renal epithelial cells of mice, ERRγ transcriptionally controls energy-generating mitochondrial metabolism and energy-consuming renal reabsorption (Zhao et al. 2018). In the present study, we considered that the decreased ncc mRNA expression in 3 dpf morphants was a direct consequence of the Errγ2 MO (Fig. 4B and Supplementary Fig. 3A, C), and the impaired Na+/Cl− uptake ability observed in 6 dpf Errγ2 morphants (Fig. 3B,C, D and E) may have resulted from decreased Ncc protein expression/activity; however, we still cannot exclude the possibility that energy deficiency and mitochondrial dysfunction in ionocytes (mitochondrial-rich cells) contribute to the observed effects, because the functions of fish gill/skin ionocytes have been well documented to be closely associated with energy metabolism pathways (Hwang et al. 2011, Takei & Hwang 2016, Tseng et al. 2020).
For summary, we show that medaka, an emerging model for fish ion regulation, activates Na+/Cl− uptake functions through transcriptional regulation of Errγ2 on Ncc and Nhe3 transporters. Moreover, these regulatory actions on the transporters may be subtly adjusted depending on internal and external ion concentrations. These findings explored further details about the roles of ERRs in vertebrate body fluid ionic homeostasis, providing additional reference to future studies on human renal physiology and related diseases.
Supplementary materials
This is linked to the online version of the paper at https://doi.org/10.1530/JOE-21-0112.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Funding
This work was financially supported by grants to P-P Hwang from Academia Sinica (AS-IA-108-L03) and the Ministry of Science and Technology (MOST107-2321-B-001-030), Taiwan.
Author contribution statement
S-W Shih, J-J Yan, and P-P Hwang conceived and designed the research. S-W Shih carried out most of the experiments. Y-H Wang and L Chiu conducted part of molecular analyses. Y-L Tsou assisted in SIET analyses. Y-C Tseng and M-Y Chou provided suggestions to the whole project. S-W Shih wrote the manuscript draft. P-P Hwang supervised the project and finalized the manuscript. All authors approved the manuscript for publication.
Acknowledgements
We thank the ICOB Core Facility for technical supports. We also thank Drs. Kiyoshi Naruse and Satoshi Ansai (National BioResource Project, National Institute for Basic Biology) for providing the medaka strains and the technical assistance in medaka rearing and manipulation.
References
Alaynick WA, Way JM, Wilson SA, Benson WG, Pei L, Downes M, Yu R, Jonker JW, Holt JA & Rajpal DK et al. 2010 ERRγ regulates cardiac, gastric, and renal potassium homeostasis. Molecular Endocrinology 24 299–309. (https://doi.org/10.1210/me.2009-0114)
Bradshaw D & & McCormick S 2006 Hormonal control of salt and water balance in vertebrates – a symposium. General and Comparative Endocrinology 147 1–2. (https://doi.org/10.1016/j.ygcen.2005.09.024)
Chang WJ, Wang YF, Hu HJ, Wang JH, Lee TH & & Hwang PP 2013 Compensatory regulation of Na+ absorption by Na+/H+ exchanger and Na+-Cl- cotransporter in zebrafish ( Danio rerio). Frontiers in Zoology 10 46. (https://doi.org/10.1186/1742-9994-10-46)
Eichner LJ & & Giguère V 2011 Estrogen related receptors (ERRs): a new dawn in transcriptional control of mitochondrial gene networks. Mitochondrion 11 544–552. (https://doi.org/10.1016/j.mito.2011.03.121)
Eichner LJ, Perry MC, Dufour CR, Bertos N, Park M, St-Pierre J & & Giguère V 2010 miR-378∗ mediates metabolic shift in breast cancer cells via the PGC-1β/ERRγ transcriptional pathway. Cell Metabolism 12 352–361. (https://doi.org/10.1016/j.cmet.2010.09.002)
Eladari D & & Hübner CA 2011 Novel mechanisms for NaCl reabsorption in the collecting duct. Current Opinion in Nephrology and Hypertension 20 506–511. (https://doi.org/10.1097/MNH.0b013e3283486c4a)
Evans DH, Piermarini PM & & Choe KP 2005 The multifunctional fish gill: dominant site of gas exchange, osmoregulation, acid-base regulation, and excretion of nitrogenous waste. Physiological Reviews 85 97–177. (https://doi.org/10.1152/physrev.00050.2003)
Giguère V 2008 Transcriptional control of energy homeostasis by the estrogen-related receptors. Endocrine Reviews 29 677–696. (https://doi.org/10.1210/er.2008-0017)
Greger R 2000 Physiology of renal sodium transport. American Journal of the Medical Sciences 319 51–62. (https://doi.org/10.1097/00000441-200001000-00005)
Guh YJ & & Hwang PP 2017 Insights into molecular and cellular mechanisms of hormonal actions on fish ion regulation derived from the zebrafish model. General and Comparative Endocrinology 251 12–20. (https://doi.org/10.1016/j.ygcen.2016.08.009)
Guh YJ, Tseng YC, Yang CY & & Hwang PP 2014 Endothelin-1 regulates H+-ATPase-dependent transepithelial H+ secretion in zebrafish. Endocrinology 155 1728–1737. (https://doi.org/10.1210/en.2013-1775)
Hsu HH, Lin LY, Tseng YC, Horng JL & & Hwang PP 2014 A new model for fish ion regulation: identification of ionocytes in freshwater-and seawater-acclimated medaka ( Oryzias latipes). Cell and Tissue Research 357 225–243. (https://doi.org/10.1007/s00441-014-1883-z)
Hwang PP & & Chou MY 2013 Zebrafish as an animal model to study ion homeostasis. Pflugers Archiv 465 1233–1247. (https://doi.org/10.1007/s00424-013-1269-1)
Hwang PP & & Lee TH 2007 New insights into fish ion regulation and mitochondrion-rich cells. Comparative Biochemistry and Physiology: Part A, Molecular and Integrative Physiology 148 479–497. (https://doi.org/10.1016/j.cbpa.2007.06.416)
Hwang PP, Lee TH & & Lin LY 2011 Ion regulation in fish gills: recent progress in the cellular and molecular mechanisms. American Journal of Physiology: Regulatory, Integrative and Comparative Physiology 301 R28–R47. (https://doi.org/10.1152/ajpregu.00047.2011)
Kang MH, Choi H, Oshima M, Cheong JH, Kim S, Lee JH, Park YS, Choi HS, Kweon MN & Pack CG et al. 2018 Estrogen-related receptor gamma functions as a tumor suppressor in gastric cancer. Nature Communications 9 1920. (https://doi.org/10.1038/s41467-018-04244-2)
Lin LY, Horng JL, Kunkel JG & & Hwang PP 2006 Proton pump-rich cell secretes acid in skin of zebrafish larvae. American Journal of Physiology: Cell Physiology 290 C371–C378. (https://doi.org/10.1152/ajpcell.00281.2005)
McCallum L, Lip S & & Padmanabhan S 2015 The hidden hand of chloride in hypertension. Pflugers Archiv 467 595–603. (https://doi.org/10.1007/s00424-015-1690-8)
Murray J, Auwerx J & & Huss JM 2013 Impaired myogenesis in estrogen‐related receptor γ (ERRγ)‐deficient skeletal myocytes due to oxidative stress. FASEB Journal 27 135–150. (https://doi.org/10.1096/fj.12-212290)
Nakajima K, Oda E & & Kanda E 2016 The association of serum sodium and chloride levels with blood pressure and estimated glomerular filtration rate. Blood Pressure 25 51–57. (https://doi.org/10.3109/08037051.2015.1090711)
Palmer LG & & Schnermann J 2015 Integrated control of Na transport along the nephron. Clinical Journal of the American Society of Nephrology 10 676–687. (https://doi.org/10.2215/CJN.12391213)
Poidatz D, Dos Santos E, Gronier H, Vialard F, Maury B, De Mazancourt P & & Dieudonné MN 2015 Trophoblast syncytialisation necessitates mitochondrial function through estrogen-related receptor-γ activation. Molecular Human Reproduction 21 206–216. (https://doi.org/10.1093/molehr/gau102)
Rangwala SM, Wang X, Calvo JA, Lindsley L, Zhang Y, Deyneko G, Beaulieu V, Gao J, Turner G & & Markovits J 2010 Estrogen-related receptor γ is a key regulator of muscle mitochondrial activity and oxidative capacity. Journal of Biological Chemistry 285 22619–22629. (https://doi.org/10.1074/jbc.M110.125401)
Robertson JD 1957 The habitat of the early vertebrates. Biological Reviews 32 156–187. (https://doi.org/10.1111/j.1469-185X.1957.tb01561.x)
Shen WP, Horng JL & & Lin LY 2011 Functional plasticity of mitochondrion-rich cells in the skin of euryhaline medaka larvae ( Oryzias latipes) subjected to salinity changes. American Journal of Physiology: Regulatory, Integrative and Comparative Physiology 300 R858–R868. (https://doi.org/10.1152/ajpregu.00705.2010)
Shih TH, Horng JL, Liu ST, Hwang PP & & Lin LY 2012 Rhcg1 and NHE3b are involved in ammonium-dependent sodium uptake by zebrafish larvae acclimated to low-sodium water. American Journal of Physiology: Regulatory, Integrative and Comparative Physiology 302 R84–R93. (https://doi.org/10.1152/ajpregu.00318.2011)
Takei Y & & Hwang PP 2016 Homeostatic responses to osmotic stress. In Fish Physiology – Biology of Stress in Fish, pp. 207–249, 1st ed. Eds Schreck B, Tort CL, Farrell AP, & Brauber CJ. Cambridge, MA, USA: Academic Press.
Takei Y, Hiroi J, Takahashi H & & Sakamoto T 2014 Diverse mechanisms for body fluid regulation in teleost fishes. American Journal of Physiology: Regulatory, Integrative and Comparative Physiology 307 R778–R792. (https://doi.org/10.1152/ajpregu.00104.2014)
Tremblay AM, Dufour CR, Ghahremani M, Reudelhuber TL & & Giguère V 2010 Physiological genomics identifies estrogen-related receptor α as a regulator of renal sodium and potassium homeostasis and the renin-angiotensin pathway. Molecular Endocrinology 24 22–32. (https://doi.org/10.1210/me.2009-0254)
Tseng YC, Yan JJ, Furukawa F & & Hwang PP 2020 Did acidic stress resistance in vertebrates evolve as Na+/H+ exchanger‐mediated ammonia excretion in fish? BioEssays 42 e1900161. (https://doi.org/10.1002/bies.201900161)
Venkatesh B 2003 Evolution and diversity of fish genomes. Current Opinion in Genetics and Development 13 588–592. (https://doi.org/10.1016/j.gde.2003.09.001)
Wang YF, Tseng YC, Yan JJ, Hiroi J & & Hwang PP 2009 Role of SLC12A10.2, a Na-Cl cotransporter-like protein, in a Cl uptake mechanism in zebrafish (Danio rerio). American Journal of Physiology: Regulatory, Integrative and Comparative Physiology 296 R1650–R1660. (https://doi.org/10.1152/ajpregu.00119.2009)
Wang YF, Yan JJ, Tseng YC, Chen RD & & Hwang PP 2015 Molecular physiology of an extra-renal Cl- uptake mechanism for body fluid Cl- homeostasis. International Journal of Biological Sciences 11 1190–1203. (https://doi.org/10.7150/ijbs.11737)
Wang D, Wang Y, Liu FQ, Yuan ZY & & Mu JJ 2016 High salt diet affects renal sodium excretion and ERRα expression. International Journal of Molecular Sciences 17 480. (https://doi.org/10.3390/ijms17040480)
Wichmann L & & Althaus M 2020 Evolution of epithelial sodium channels: current concepts and hypotheses. American Journal of Physiology: Regulatory, Integrative and Comparative Physiology 319 R387–R400. (https://doi.org/10.1152/ajpregu.00144.2020)
Wu SC, Horng JL, Liu ST, Hwang PP, Wen ZH, Lin CS & & Lin LY 2010 Ammonium-dependent sodium uptake in mitochondrion-rich cells of medaka ( Oryzias latipes) larvae. American Journal of Physiology: Cell Physiology 298 C237–C250. (https://doi.org/10.1152/ajpcell.00373.2009)
Yan JJ & & Hwang PP 2019 Novel discoveries in acid-base regulation and osmoregulation: a review of selected hormonal actions in zebrafish and medaka. General and Comparative Endocrinology 277 20–29. (https://doi.org/10.1016/j.ygcen.2019.03.007)
Yan JJ, Chou MY, Kaneko T & & Hwang PP 2007 Gene expression of Na+/H+ exchanger in zebrafish H+-ATPase-rich cells during acclimation to low-Na+ and acidic environments. American Journal of Physiology: Cell Physiology 293 C1814–C1823. (https://doi.org/10.1152/ajpcell.00358.2007)
Yan JJ, Lee YC, Tsou YL, Tseng YC & & Hwang PP 2020 Insulin-like growth factor 1 triggers salt secretion machinery in fish under acute salinity stress. Journal of Endocrinology 246 277–288. (https://doi.org/10.1530/JOE-20-0053)
Zhao J, Lupino K, Wilkins BJ, Qiu C, Liu J, Omura Y, Allred AL, McDonald C, Susztak K & Barish GD et al. 2018 Genomic integration of ERRγ-HNF1β regulates renal bioenergetics and prevents chronic kidney disease. PNAS 115 E4910–E4919. (https://doi.org/10.1073/pnas.1804965115)
