© 2020. Published by The Company of Biologists Ltd | Journal of Experimental Biology (2020) 223, jeb214890. doi:10.1242/jeb.214890

RESEARCH ARTICLE Extra-gastric expression of the H+/K+-ATPase in the gills and kidney of the teleost Oreochromis niloticus Ebtesam Ali Barnawi1, Justine E. Doherty1,Patrıciá Gomes Ferreira1 and Jonathan M. Wilson1,2,*

ABSTRACT drive direct and indirect ion fluxes are the basolateral Na+/K+- Potassium regulation is essential for the proper functioning of excitable ATPase that provides a sodium motive force and an apical vacuolar- tissues in vertebrates. The H+/K+-ATPase (HKA), which is composed type proton-ATPase that provides a proton motive force (Evans of the HKα1 (: atp4a)andHKβ (gene: atp4b) subunits, has an et al., 2005; Hwang et al., 2011). However, Choe et al. (2004) have + + established role in potassium and acid–base regulation in mammals provided some evidence of a role of the H /K -ATPase (HKA) in and is well known for its role in gastric acidification. However, the role of ion regulation in elasmobranch fish gills. HKA in extra-gastric organs such as the gill and kidney is less clear, The main site of HKA expression is the stomach in vertebrates especially in fishes. In the present study in Nile tilapia, Oreochromis (Shin et al., 2009; Wilson and Castro, 2010). HKA is composed of α β niloticus, uptake of the K+ surrogate flux marker rubidium (Rb+)was HK 1 (gene: ATP4A)andHK (ATP4B) subunits (Pedersen and β demonstrated in vivo; however, this uptake was not inhibited with Carafoli, 1987). The -subunit is unique to the gastric HKA and is omeprazole, a potent inhibitor of the gastric HKA. This contrasts with involved in stabilizing cellular targeting of the transporter, α gill and kidney ex vivo preparations, where tissue Rb+ uptake was while the larger -subunit catalyses ATP hydrolysis and ion α significantly inhibited by omeprazole and SCH28080, another gastric translocation. The gastric HKA HK 1 subunit belongs to the large HKA inhibitor. The cellular localization of this pump in both the gill and subgroup of the P-type ATPase IIc family that also includes the α + + α kidney was demonstrated using immunohistochemical techniques with non-gastric/colonic HKA (HK 2) and Na /K - (NK ) custom-made antibodies specific for Atp4a and Atp4b. Antibodies (Pedersen and Carafoli, 1987). – against the two subunits showed the same apical ionocyte distribution Most studies of ion and/or acid base regulation in FW and + − + pattern in the gill and collecting tubules/ducts in the kidney. Atp4a saltwater (SW) teleost studies have focused on Na ,Cl and Ca + antibody specificity was confirmed by western blotting. RT-PCT was ions (Evans et al., 2005). However, K is also an ion of interest, as used to confirm the expression of both subunits in the gill and kidney. it is an essential and natural element that plays a critical role in nerve, Taken together, these results indicate for the first time K+ (Rb+)uptake muscle and many other vital cell functions, such as metabolism, + in O. niloticus and that HKA is implicated, as shown through the ex vivo growth and repair (Talling, 2010). In addition, K affects the ’ uptake inhibition by omeprazole and SCH28080, verifying a role for kidney s ability to reabsorb bicarbonate as the main extracellular HKA in K+ absorption in the gill’s ionocytes and collecting tubule/duct buffer to metabolic acids. During periods of positive potassium segments of the kidney. balance, potassium efflux pathways in SW tilapia (Furukawa et al., 2012) and FW zebrafish (Abbas et al., 2011) and medaka (Horng KEY WORDS: Potassium regulation, Fresh water, Acid–base et al., 2017) have been studied showing that orthologues to the regulation mammalian renal outer medullary potassium channel (ROMK) are present apically in branchial and skin ionocytes. In FW larval medaka, INTRODUCTION Horng et al. (2017) have proposed a paracellular uptake pathway In the aquatic environment, fishes are exposed to many challenges between skin keratinocytes that requires a high transepithelial that require regulation of ions and acid–base balance (Evans et al., potential (TEP) to operate using a SIET (scanning ion-selective 2005; Marshall and Grosell, 2005). In fresh water (FW), fishes electrode technique) approach. Studies by Eddy (1985) and Gardaire compensate for the diffusive loss of ions by active uptake of ions, and colleagues (Gardaire and Isaia, 1992; Gardaire et al., 1991) driven by pumps (Evans et al., 2005). They also maintain water suggest the possibility of branchial uptake of K+ from the water in homeostasis by getting rid of osmotically gained water by excreting rainbow trout, although the mechanism remains unknown. According copious amounts of dilute urine (Marshall and Grosell, 2005). In to Caplan (1998), H+/K+-ATPase is certainly also involved in extra- FW teleost fishes, acid–base transport takes place across the gill gastric roles such as renal acid–base regulation in tetrapods including epithelium via exchange mechanisms located in ionocytes mammals. Welling (2013) and Gumz et al. (2010) additionally [mitochondrion-rich ‘chloride’ cells (CCs)] and possibly in summarized that kidney HKA has an established role in potassium pavement or respiratory cells (PVCs) (Evans et al., 2005; Wilson, and acid–base regulation, which controls potassium absorption, and 2011). The two main pumps that have been identified in fish gill to could potentially contribute to efficient renal potassium excretion. Proton pump inhibitors (PPIs) and acid pump antagonists (APAs) are drugs that are the mainstay treatments for all acid-related 1Department of Biology, Wilfrid Laurier University, Waterloo, ON, Canada, N2L 3C5. diseases by inhibiting acid secretion with varying efficiency 2Molecular Physiology, Centro Interdisciplinar de Investigação Marinha e Ambiental, 4450-208 Matosinhos, Portugal. (Codina and DuBose, 2006; Shin et al., 2009). Omeprazole was the first drug of the PPIs class to be introduced into clinical use (in *Author for correspondence ( [email protected]) 1989) that inhibits the gastric HKA by covalent binding; hence, the P.G.F., 0000-0002-6132-1356; J.M.W., 0000-0003-3681-1166 duration of its effect is longer than expected from its levels in the blood. Unlike PPIs such as omeprazole, APAs do not require acid

Received 17 September 2019; Accepted 26 June 2020 activation. Journal of Experimental Biology

1 RESEARCH ARTICLE Journal of Experimental Biology (2020) 223, jeb214890. doi:10.1242/jeb.214890

Oreochromis niloticus is also a well-studied species for fish Rubdium uptake rate was determined by measuring rubidium biology and is widely used in aquaculture globally (Maclean et al., accumulation in muscle over time using terminal sampling. 2002). Therefore, it was chosen as a model species of significance as Oreochromis niloticus (n=16, 6–15 g) were captured randomly its genome has been sequenced and annotated (ensembl.org). The from the 180 l holding tanks using a hand net, then moved to one of O. niloticus genome only has the HKα1 gene atp4a (Castro et al., four 3 l tanks (n=4 per tank) on flow through with aeration. The water 2014) and lacks the non-gastric proton pump paralogue HKα2 gene was maintained at ∼pH 7.8. Fish were acclimated overnight, and the , which simplifies analysis of the results. The apparent HKα2 Rb+ flux was initiated by stopping flow to the tanks and adding a gene (atp12a) teleost orthologue (ENSHHUG00000034153) in specific final concentration of Rb+ (0.1, 0.5, 1 or 2.5 mmol l−1 RbCl) Hucho hucho has been incorrectly annotated in ensembl.org and to each tank. An initial water sample of 10 ml was taken to check the although at least two orthologues do exist in the ancient water [Rb+]. At 0, 3, 6 and 12 h, O. niloticus were killed with an Polypteriform ray-finned fish Erpetoichthys calabaricus overdose of MS222 (0.2 g l−1; Syndel Laboratories, Nanaimo, BC, (ENSECRG00000015423 and ENSECRG00000016690), none Canada) and epaxial muscle samples (usually 0.2–1 g) were excised are found in teleost genomes (J.M.W., unpublished observation). for measurement of [Rb+]. Sample processing is described in The potential significance of extra-gastric HKA expression in ‘Analytical techniques: Rb+ kinetics’,below. teleost fishes remains to be resolved. In this paper, we provide the first evidence for the expression of the gastric proton pump HKA (: In vivo pharmacological inhibition of the proton pump with atp4a and atp4b) in a teleost fish and demonstrate its potential omeprazole function in potassium regulation. The central objective was to Omeprazole was used as a proton pump inhibitor to evaluate the investigate the role of this pump in extra-gastric potassium regulation activity of HKA for this experiment by comparing sham-injected in the gill and kidney of the teleost fish O. niloticus.Rb+ wasusedasa controls and omeprazole-injected fish. Treatment fish were injected surrogate flux marker for K+ and two experimental approaches were with 5 mg kg−1 omeprazole dissolved in DMSO and 0.9% sodium taken: in vivo and ex vivo Rb+ uptake assays to analyse HKA activity chloride, 12 h before and at the start of the Rb uptake periods. The in the gill and kidney. To determine whether HKA was specifically control fish were injected with an equivalent volume of 0.9% NaCl involved in Rb+ uptake, we performed a pharmacological inhibition with 2% DMSO. For injection, fish were anaesthetized with 0.2 g l−1 assay of Rb+ uptake with the gastric HKA inhibitors omeprazole of MS222, weighed and the injection volume calculated. and SCH28080. In addition, a tissue viability assay confirmed the Oreochromis niloticus were moved to individual 500 ml tanks with stability of the ex vivo preparations. Specifically, the lactate aeration and flow to the tanks was stopped after the second injection. dehydrogenase (LDH) assay was used as an indicator of cell Rb+ was added to each tank at a final concentration of 0, 0.1, 0.5, 1 or damage by measuring the activity of this in the culture 2.5 mmol l−1 at time zero. After 3 or 6 h, fish (n=4) were killed with medium leaked from damaged cells. Finally, the expression of Atp4a an overdose of MS222, their length and mass were measured, and and Atp4b in the gill and kidney was established using muscle samples were collected as described above in ‘Rb+ kinetics’. immunohistochemistry (IHC) with custom-made antibodies to identify the presence and subcellular location of these subunit Analytical techniques: Rb+ kinetics within these tissues, followed by validation by western Muscle samples were weighed, dried at 60°C for 72 h, dissolved in blotting and confirmation of atp4a and atp4b expression by RT-PCR. 2.5 ml concentrated HNO3, and diluted 50× with Milli-Q water and spiked with CsCl at a final concentration of 0.2%. Rb+ concentration MATERIALS AND METHODS was determined using a PinAAcle 900T Atomic Absorption Animals Spectrophotometer (Perkin Elmer, Waltham, MA, USA) in flame Nile tilapia, Oreochromis niloticus (Linnaeus 1758), were supplied mode. Rubidium standards were prepared in 0.2% CsCl and 2% −1 by Sand Plains Aquaculture facility (London, ON, Canada), with HNO3 in Milli-Q water in a range from 0 to 9.6 μmol l . mass ranges of 5–25 g for in vivo and 50–200 g for ex vivo Rb+ kinetics were characterized from Rb+ uptake rate at the tested experiments. Upon arrival, the fish were placed in 180 l tanks concentrations of 0.1, 0.5 and 1 mmol l−1 using Michaelis–Menten supplied with dechlorinated city water and reverse osmosis water at kinetics with GraphPad Prism (version 6.01 for Windows, a room temperature of 25±1°C. Fish were fed twice daily with Ewos GraphPad Software, La Jolla, CA, USA). Vmax (maximum pellets ad libitum. All experiments were approved by Wilfrid velocity) and Km ( concentration at which the reaction rate is Laurier University’s Animal Care Committee (R14002 and half Vmax) were calculated by linear regression analysis of the R18003) and followed the guidelines from the Canadian Council Lineweaver–Burk plot equation plotted as 1/substrate (Rb on Animal Care. concentration) on the x-axis and 1/velocity (uptake rate) on the y-axis. The x-intercept represents −1/Km and the y-intercept is 1/Vmax. In vivo experiments Rb+ kinetics Ex vivo experiments Rubidium offers several advantages that makes it a suitable Measurement of omeprazole- and SCH28080-sensitive Rb+ uptake replacement for potassium for flux studies. Rubidium has a Tissue preparation similar physicochemical behaviour and characteristics to those of Oreochromis niloticus (n=3) were randomly sampled from the 180 l potassium. For instance, both are alkali metals and their hydration holding tanks using a hand net, and killed with an overdose of MS222 radii are similar. Also, Rb+ at high concentrations of exposure has as described above for sample collection. The gill arches and kidney limited toxicity compared with potassium. In the environment and were excised and rinsed in Ringer’ssolution[inmmoll−1:140 NaCl, + in the body, Rb is present at very low concentrations; therefore, 15 NaHCO3, 1.5 CaCl2, 1.0 NaH2PO4, 0.8 MgSO4,5.0D-glucose accumulation within the organism during flux studies is indicative and 5.0 N-(2-hydroxyethyl)-piperazinepropanesulfonic acid (Hepps); −1 of uptake from the water during exposure (Dietz and Byrne, 1990; 310 mosmol kg , pH 7.8] equilibrated with 99% O2/1% CO2.Each Sanders and Kirschner, 1983; Tipsmark and Madsen, 2001; Mähler gill arch was then cut transversely into blocks of 3–5pairsof and Persson, 2011; Wilcox and Dietz, 1995). filaments (5–10 mg), and kidney tissue was cut into blocks of Journal of Experimental Biology

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4–8 mg. Pieces were placed in 24-well culture plates and incubated In a 96-well plate, 10 μl of sample (tissue or medium) was added in 1 mmol l−1 Rb+ Ringer’s solution for up to 30 min in various RbCl to each well containing 200 µl of 0.32 mmol l−1 NADH dissolved −1 −1 concentrations (0, 0.1, 0.5 and 1 mmol l , and a higher concentration in phosphate buffer (PB; 50 mmol l KH2PO4, pH 7.4). Then, of 2.5 mmol l−1). Following incubation, tissues were washed free of 10 μl of 7.36 mmol l−1 of pyruvate in PB was added and a linear + −1 Rb , using Tris-sucrose buffer (in mmol l : 2.5 KCl, 1.5 CaCl2,1.0 (negative) slope obtained using a microplate reader (SpectroMax KH2PO4, 0.8 MgSO4, 10 Tris, 260 sucrose, pH 7.8), equilibrated with M2 plate reader; Molecular Devices, San Jose, CA, USA) at 340 nm 99% O2/1% CO2. The samples were then blotted gently on filter for 10 min, using SOFTmax Pro 4.0 software. LDH leakage was paper, weighed to the nearest 0.1 mg and placed in 1.5 ml tubes. expressed as a percentage of LDH activity in the extracellular Sample ion extractions were performed overnight in 0.5 ml of 5% medium versus the total activity. trichloroacetic acid (TCA) at 24°C. The next day, samples were homogenized with a bead homogenizer (Precellys 24, Bertin IHC Technologies SAS, Montigny-le-Bretonneux, France) and Tissue preparation centrifuged (14,000 g for 15 min at 4°C; Thermo Scientific Sorvall Gill, kidney and stomach tissues collected from the in vivo Legend Micro 21R Microcentrifuge, Thermo Scientific, Waltham, experiments were placed in embedding cassettes in 3% MA, USA). Finally, the supernatants were diluted 50× with Milli-Q paraformaldehyde in PBS (pH 7.4) fixative for 24 h. The tissues water and spiked with CsCl (0.2% final concentration). Rubidium were then transferred to decalcification solution (30% formic acid, standards prepared in 2% TCA, 0.2% CsCl ranged from 1.2 to 13% sodium citrate) for 48 h, and then stored in 70% alcohol 12.25 μmol l−1.Rb+ concentration was measured by PinAAcle 900T (histological grade) for at least 24 h. Tissues were then embedded in Atomic Absorption Spectrophotometer (Perkin Elmer) in flame mode paraffin using a Shandon Citadel 1000 processor (Thermo at 780.8 nm with a slit width of 2.0 nm. The rate of Rb+ uptake is Scientific, Pittsburgh, PA, USA). The paraffin blocks were expressed as μmol g−1 wet mass h−1. Additional gill, kidney, sectioned (5 μm) using a Leica RM2125 RTS microtome (Leica stomach, anterior intestine, rectum, liver, brain, testis, urinary bladder Biosystems, Wetzlar, Germany), collected on to aminopropylsilane- and spleen samples were collected at the time of killing for western coated slides (aminopropylsilane from Sigma-Aldrich, St Louis, blotting and PCR analyses. MO, USA), air dried and stored for future use or prepared for IHC.

Omeprazole Custom-made polyclonal antibodies Gill and kidney samples were transferred to 24-well microplates and Antigenic peptides specific for Atp4a and Atp4b were incubated with 1 mmol l−1 Rb+ Ringer solution equilibrated with determined from predicted O. niloticus amino acid sequences from 99% O2/1% CO2 for 30 min and omeprazole was added to the the Nile tilapia genome (Ensembl ENSONIG00000005974 and solution at a final concentration of 0.5 mmol l−1 from a stock ENSONIG00000003180, respectively). To avoid cross-reactivity solution of 19.2 mmol l−1 in methanol. An initial time course with NKAα and β subunits, the predicted peptide sequences were experiment over 0, 10, 20 and 30 min to determine an optimal screened against O. niloticus Atp1a1 (.org I3K3G3) and inhibition time showed a linear uptake of Rb+ over time in the Atp1b3a (ENSONIG00000027418.1), respectively. Peptide presence or absence of omeprazole. Omeprazole inhibition of Rb+ prediction, synthesis and conjugation to the carrier KLH uptake with different concentrations of Rb+ (0.5, 1, 1.5 and were performed by Genetel (Madison, WI, USA). The O. niloticus 3.5 mmol l−1 RbCl) was then carried out; the control was an Atp4a carboxyl-terminal peptide DEIRKLGVRRHPGSWWDQE equivalent volume of methanol. LYY (amino acids 999–1020) and Atp4b amino terminal peptide MATLKEKRTCGQRCEDFG (amino acids 1–18) were selected. SCH28080 Two chickens per antigenic peptide were used and IgYs were isolated Gill tissue Rb+ uptake was also assessed in the presence of from egg yolks as Atp4b-B1349 and Atp4b-B1350 antibodies and SCH28080 (Santa Cruz Biotech) added to a final concentration of Atp4a-B1351 and Atp4a-B1352 antibodies. Pre-immune isolated 0.2 mmol l−1 from a stock solution of 36.1 mmol l−1 dissolved in IgYs served as negative controls. Screening of staining in tilapia DMSO. An initial experiment was carried out for the time course of stomach gastric glands was used as a positive control of antibody Rb+ uptake at 0, 10, 20 and 30 min. We then evaluated Rb flux and specificity (Table S1). inhibition rates with different Rb+ concentrations (0.5, 1, 1.5 and 3.5 mmol l−1 RbCl); the control was an equivalent volume of IHC DMSO. Tissues were dewaxed at 60°C for 20 min, passed through three xylene baths (5 min) and an alcohol series of 2×100% (5 min) and Tissue viability assay (LDH activity) 70% (3 min), with or without antigen retrieval. This included 0.05% LDH assays were performed to evaluate LDH activity in the tissue citraconic anhydride for 30 min at pH 7.4 at 100°C (Namimatsu et al., (intracellular) and the leakage of enzyme into the medium 2005) followed by 1% SDS/PBS (pH 7.4) treatment (Brown et al., (extracellular), which can be used as a marker of dead or damaged 1996). The sections were then washed three times with deionized cells following treatment. Total LDH was calculated as the sum of water (5 min) and TPBS (5 min; 0.05% Tween-20 in PBS). Sections extracellular LDH plus the intracellular (tissue) LDH. To measure were blocked in a humidity chamber (20 min) with BLØK (Life extracellular LDH, 1 ml of culture medium (Rb+ Ringer’ssolution) Technologies) or 1% fish skin gelatin (Sigma-Aldrich) in TPBS, then was transferred from the 24-well microplates into 1.5 ml tubes and probed with the primary antibodies Atp4a-B1352 and Atp4b-B1349 snap frozen on dry ice. To measure intracellular LDH, tissues were at 1:5000 and 1:500 for the gill and kidney, respectively, and mouse rinsed with phosphate-buffered saline (PBS; in mmol l−1: 137 NaCl, monoclonal antibody for Na+/K+-ATPase (NKA) α5 (1:100; clone 2.7 KCl, 8.5 Na2HPO4,1.5KH2PO4, pH 7.4), blotted dry and a5, Developmental Studies Hybridoma Bank, Iowa, USA); chicken homogenized in 500 μl PBS to release all intracellular LDH and then pre-immune sera diluted 1:5000 or 1:500 for the gill and kidney, centrifuged. The LDH in the supernatant was assayed on the same respectively, were the negative controls (Burry, 2011). Slides were day or frozen at −80°C for later measurement. incubated for 1–2 h at 37°C in a humidity chamber, then washed three Journal of Experimental Biology

3 RESEARCH ARTICLE Journal of Experimental Biology (2020) 223, jeb214890. doi:10.1242/jeb.214890 times with TPBS (5, 10, 15 min) with intermittent agitation. The PCR was run using BESTaq master mix (Applied Biological secondary antibodies were goat anti-mouse IgG Alexa 555 (1:500) Materials Inc.) with either atp4a-oratp4b-specific primers at and goat anti-chicken IgY Alexa 488 (1:500) applied for 1–2 h at 37° 250 nmol l−1 each in 10 μl reaction volumes with 0.5 μl of cDNA C, followed by a final series of washes with TPBS, adding 5 μlDAPI (Table S2). The PCR consisted of an initial denaturation at 94°C for (4′,6-diamidino-2-phenylindole) to the second wash buffer. Double 3 min, followed by cycling steps of denaturation at 94°C for 10 s, sequential chicken labelling experiments were also done using the annealing at 60°C for 10 s and extension at 72°C for 5 s for a total of secondary antibodies goat anti-chicken IgY Fab Alexa 488 (1:500), 30 cycles. Subsequently, 5 μl of each sample were run on 2% and goat anti-chicken IgY CF640 (1:500) antibodies and a Fab agarose TBE (89 mmol l−1 Tris-borate and 2 mmol l−1 EDTA, pH blocking step (Negoescu et al., 1994). Coverslips were mounted with 8.3) gels containing GelRed (Biotium Inc., Fremont, CA, USA) at 1:1 glycerol PBS, pH 7.4, containing 0.1% NaN3. Sections were 50 V for 80 min using a Multipet-one electrophoresis apparatus. viewed on a Leica DM5500 B microscope with a Hamamatsu Gels were imaged using an Azure c300 imager. C11440 Orca-Flash 4.0 digital camera. Statistical analyses Western blotting Data were analysed as means±s.e.m. Two-way ANOVAs were used Frozen gill, kidney, stomach, anterior intestine, liver, testis, urinary to test for differences between treatment groups and time or Rb+ bladder and spleen collected from fish used in the ex vivo experiments concentrations for uptake/inhibition rate data. If a significant were thawed and homogenized in ice-cold imidazole buffer (IB; difference was found by ANOVA, a post hoc Holm–Šidák test 50 mmol l−1 imidazole, pH 7.5) with a Precellys24+ bead homogenizer was performed. In the case of non-normality and/or unequal (6500 rpm, 2×10 s; Bertin) and centrifuged at 5000 g for 10 min at 4°C. variance, an equivalent, non-parametric test was used (Mann– The supernatant (S1) was decanted and centrifuged at 21,100 g for Whitney rank sum test). A paired t-test was used to compare 20 min at 4°C, and the second supernatant (S2) was removed, and the differences in the ex vivo gill SCH28080 experiment. Significance pellet resuspended in IB by sonication. An equal volume of the was accepted when P<0.05. All statistical analyses were carried out homogenized pellet was mixed with 2× Laemmli’s buffer, heated at using SigmaPlot (v.11, Systat, San Jose, CA, USA). 70°Cfor10minandstoredat4°C.Totalproteinintheresuspended pellet was measured with the BCA assay, and samples in Laemmli’s RESULTS buffer were then adjusted to 1 μg μl−1. Western blotting was performed In vivo experiments as described in Wilson et al. (2007) using a Bio-Rad Tetra mini Rubidium uptake was successfully measured over time at a water Rb+ electrophoresis system (Hercules, CA, USA) with a 10% resolving gel concentration of 2.5 mmol l−1.Rb+ uptake, measured as and 4% stacking gel. Transfer to PVDF membranes (Immun-Blot®, accumulation in white muscle, showed an increase over time to a Bio-Rad) was performed using a Hoeffer TE22 wet cell (1 h at 100 V). maximum at 12 h (Fig. 1A). Using a range of Rb+ concentrations in Membranes were dried, marked, stained with 0.5% Ponceau S, and the millimolar range, the data showed a Michaelis–Menten blocked with 10% BLOTTO in TTBS (Tris-buffered saline with 0.05% relationship and linear regression of the Lineweaver–Burk plot + −1 −1 Tween-20) before probing with Atp4a and Atp4b antibodies. where the Rb Vmax was 20.4 nmol g h and the Km value was Preimmune IgY was used as a negative control. Goat anti-chicken 0.4 mmol l−1 (Fig. 1B). Rb+ uptake was not inhibited by omeprazole HRP-conjugated secondary antibody (1:50,000) was used to detect in vivo as there was no significant difference in Rb+ uptake rate immunoreactive bands by enhanced chemiluminescence (Clarity, Bio- between sham-injected (control) and omeprazole-injected O. Rad) using an imager (c300, Azure Biosystems, Dublin, CA, USA). niloticus (n=4, P=0.842) (Fig. 1C).

RT-PCR Ex vivo experiments Total RNA was extracted from a panel of tilapia organs (gill, kidney, As the in vivo methods performed to evaluate the activity of gastric stomach, anterior intestine, rectum, liver and brain) using the Bio-Rad HKA in Rb+ uptake showed no effect, another methodology using Aurum Total RNA extraction kit with on-column DNaseI digestion ex vivo gill filaments was examined. While ex vivo experiments according to the manufacturer’s instructions. The concentration and facilitate manipulation of the experimental conditions, tissue purity of the RNA were assessed by spectrophotometry using a viability can be an issue. Therefore, tissue viability was Biotek Take-3 plate and only samples with 260 nm/280 nm ratios monitored over the course of the experiments. between 1.8 and 2.2 were processed further. For each sample, 1 μgof RNA was converted to cDNA using the Applied Biosystems™ High- Tissue viability Capacity cDNA Reverse Transcription Kit according to the The appearance of the intracellular enzyme LDH in the culture manufacturer’s instructions. medium was used as an indicator of tissue viability. Higher levels of

ABCFig. 1. In vivo rubidium (Rb+) uptake in 15 10 2.0 Oreochromis niloticus white muscle. Control (A) Example Rb+ flux measured over 12 h at −1 8 1.5 Omeprazole 3 h intervals during exposure to 2.5 mmol l 10 RbCl (n=3). (B) Lineweaver–Burk plot used wet mass) 6 wet mass) −1 −1 –1 –1 1.0 to calculate Vmax (20.4 nmol g h ) and Km −1

1/V (0.4 mmol l ). (C) Effects of omeprazole 5 4 −1 + 0.5 injection (25 mg kg )onRb uptake over 2 time versus control (sham injection). No 0 significant difference in the uptake rate with [Rb] (nmol g 0 [Rb] (nmol g omeprazole was observed (P=0.842). Two- 036912 –5 0 5 10 036way ANOVA, Holm-Šidák post hoc test, n=3–4.

Time (h) 1/[Rb] Time (h) Journal of Experimental Biology

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Table 1. Lactate dehydrogenase (LDH) activity in Oreochromis niloticus gills and kidney and percentage leak into the medium in control and omeprazole treatment groups over time Control Omeprazole OrganTime (min) LDH activity (U g−1) % Leak LDH activity (U g−1) % Leak Gill 0 82.9±6.8 0.12±0.05 84.6±5.0 0.11±0.05 10 61.9±7.7 0.01±0.01 97.1±1.1 0.04±0.04 20 76.8±6.8 0.01±0.01 85.5±3.8 0.00±0.00 30 88.8±5.9 0.02±0.01 92.3±3.2 0.01±0.01 Kidney 0 43.1±11.9 0.04±0.02 60.2±4.8 0.02±0.02 10 61.9±8.0 0.00±0.00 45.1±6.7 0.00±0.00 20 43.8±6.0 0.06±0.06 42.4±6.4 0.01±0.01 30 44.5±12.6 0.00±0.00 48.1±6.3 0.05±0.05 n=3; two-way ANOVA, P=0.693.

LDH activity were found in gill compared with kidney tissue Optimized IHC staining conditions for the gill and kidney were (Table 1). Gill and kidney showed stable viability throughout the found: (1) at dilutions of 1:5000 and 1:500, respectively; (2) using examination phases, whether with control or treated O. niloticus fish skin gelatin in the blocking medium instead of BLØK for gills tissues, even at the longer times examined (30 min). In gill tissue – no difference in staining was found in the kidney; (3) with preparations, the highest leakage measured was only 0.12% in 1–2 h incubation at 37°C rather than overnight at 4°C; and (4) control and 0.11% in omeprazole treatments and this was in the without the need for staining with antigen retrieval steps [0.05% initial 0 min group. All other groups showed leakage of less than citraconic anhydride (Namimatsu et al., 2005) or 1% SDS/PBS 0.01% and there was no significant relationship with time or (Brown et al., 1996)]. treatment group. In the kidney, leakage rate was also very low. The Ionocytes, identified by NKA staining, were present mainly in the highest leakage for control and omeprazole treatment groups was filament epithelium with a round shape (Figs 3B and 4B). 0.06% and 0.04%, respectively. Consequently, no differences were Immunolocalization of both Atp4a and Atp4b was found in the found (P=0.693).

Omeprazole AB 10 350 Rubidium uptake by the gill was measured over 30 min in media Control −1 300 containing 1 mmol l RbCl (Fig. 2A). Omeprazole in ex vivo 8 Omeprazole c,* d,* + 250 treated gills significantly inhibited Rb uptake compared with the b,

wet mass) * c, + 6 200 * control and no significant difference in tissue Rb concentration was –1 a,* b,* observed between 10 and 30 min within this treatment group. In ex 4 150 vivo kidney preparations, a similar trend was observed, with A A 100 a 2 A A significant increases in tissue Rb+ concentration over 20 min in 50 A A A control tissue and a significant inhibition after 10 min in [Rb] (μmol g 0 0 omeprazole-treated kidney tissue for the duration of the 0 102030 0 5 10 15 20 experiment (Fig. 2B; n=3, P≤0.001). Time (min) Time (min) +

Flux measured over 15 min with Rb concentrations of 0.5, 1, 1.5 ) −

–1 C and 3.5 mmol l 1 indicated a significant inhibition by omeprazole D

h 250 1000 Control ≤ –1 (n=3) in the gill (Fig. 2C) and kidney (Fig. 2D) (P 0.001). In the B,* c,* gill, a significant inhibition was observed at 3.5 mmol l−1 Rb+ while 200 Omeprazole 800 mol g in the kidney, a significant inhibition was observed at 1.5 and μ 150 600 3.5 mmol l−1 Rb+. 100 400 b,* A SCH28080 50 A 200 a SCH28080-treated O. niloticus gill filaments showed a significant A a − − 0 0 + μ 1 1 0.5 11.53.5 0.5 1 1.5 3.5 inhibition of Rb uptake at 10 min (n=3, 2.88±0.11 mol g h ) Rb uptake rate ( compared with the control (5.75±1.22 μmol g−1 h−1; paired t-test, [Rb] (mmol l–1) [Rb] (mmol l–1) P≤0.001). Rb+ uptake in the kidney following treatment with Fig. 2. Ex vivo Rb+ uptake in isolated gill filaments and kidney of O. SCH28080 was not measured. niloticus. (A,B) Rb+ accumulation in gill filaments (A) and kidney (B) over 30 and 20 min, respectively, in the presence and absence of omeprazole. (C,D) IHC Rb+ uptake rate measured at different Rb+ concentrations (0.5–3.5 mmol l−1)in The stomach was used as a positive control for IHC as all antibodies the presence and absence of omeprazole in the gill (C) and kidney (D) over cross-reacted strongly with the gastric glands (Fig. S1). However, 15 min. Different letters indicate a significant difference over time within each ≤ the Atp4a and Atp4b antibodies B1350 and B1351, respectively, treatment group (A,B: control, lowercase; omeprazole, uppercase; P 0.001) or with omeprazole dose (D); in C, different letters indicate a concentration effect showed additional non-specific staining in the stomach lumenal independent of omeprazole treatment. Asterisks indicate a significant epithelium and were therefore not used for gill and kidney IHC difference between control and omeprazole treatment group at a given time staining experiments. The reported Atp4a and Atp4b localizations point or concentration. Two-way ANOVA, Holm-Šidák post hoc test; n=3–6inA were performed with the B1352 and B1349 antibodies, respectively. and B; n=4 in C and D. Journal of Experimental Biology

5 RESEARCH ARTICLE Journal of Experimental Biology (2020) 223, jeb214890. doi:10.1242/jeb.214890 apical region of these ionocytes (Figs 3A,C and 4A,C). The although weak expression of a slightly smaller protein was detected appearance of staining varied from diffuse apical to restricted along in urinary bladder and testis, while in the latter a larger diffuse band the apical membrane. (∼150 kDa) was also detected (Fig. S4b)]. We were unable to The different nephron regions of the Nile tilapia kidney can be produce consistent western blotting results with our Atp4b distinguished based on tubule cell morphology and NKA antibodies and thus no results are shown. immunolocalization. In the proximal tubule, NKA staining was weak and restricted to the basal membrane; the distal tubule had the Gene expression in organ panel strongest NKA staining with an extensive basolateral location, while RT-PCR confirmed that both atp4a and atp4b were expressed in the the collecting tubule/duct also had strong NKA staining in a gill and kidney although at apparently much lower levels than in the basolateral location. HKA staining with both Atp4a and Atp4b stomach (Fig. 8). In brain, atp4a was also detectable, while weak antibodies was found apically in the collecting tubule and was atp4b expression was detected in anterior intestine. No expression absent from proximal and distal tubule regions (Fig. 5A,D,E,H). was detected in rectum or liver samples for either HKA gene. Through the chicken–chicken double labelling technique, the Atp4a and Atp4b antibodies showed clear apical overlap in staining DISCUSSION ionocytes (positive NKA immunoreactivity) in gills (Fig. 6) and We show here, for the first time, the expression and potential role tubular cells in the kidney collecting tubule segment (Fig. S2). of HKA in the gill and kidney of a teleost, O. niloticus, Negative control pre-immune IgYs showed no staining comparable functioning as an extra-gastric proton pump based on in vivo to either Atp4a or Atp4b antibodies and this was not affected by and ex vivo experimental approaches and immunohistochemistry. changes in the IHC conditions (Fig. S3). Also, pre-incubation of The in vivo and ex vivo experiments used rubidium as a surrogate antibodies with excess peptides eliminated specific staining (data flux marker for potassium uptake: carrier-mediated uptake was not shown). demonstrated in vivo and pharmacological inhibition was demonstrated ex vivo by omeprazole and SCH28080 in the Western blotting kidney and gill. It is well recognized in tetrapods that the stomach The tilapia Atp4a and Atp4b peptides have a predicted molecular is the primary organ for acid secretion mediated by the proton mass of 113 and 33 kDa, respectively. In Atp4a western blots, a pump HKA. Consequently, in this study, the stomach was used as diffuse band around 110 kDa was detected in the gill and a more a positive control to validate custom-made HKA α and β subunit compact band in stomach although not in the kidney (Fig. 7). Band antibodies to confirm the molecular identity of the extra-gastric immunoreactivity increased with protein loading and was not proton pump in the gill and kidney. Specific gastric gland present in blots probed with preimmune IgY, as demonstrated in the immunoreactivity was observed in O. niloticus stomach, gill (Fig. S4a). Atp4a expression at the predicted molecular mass confirming antibody specificity, whereas staining was found was not found in other organs [anterior intestine, liver and spleen, apically in gill ionocytes and kidney collecting duct, consistent

AA′ Fig. 3. Immunohistochemistry staining of O. niloticus gill with Atp4a-B1352. (A,C) Atp4a (green) and (B,C) NKA (fuchsia) localization; sections were stained with DAPI (blue) and overlaid with the corresponding DIC image (C). Arrows indicate Atp4a-positive NKA immunoreactive ionocytes. Insets (A′–C′) are at 3× magnification. Scale bar: A–C50μm, A′–C′ 16.6 μm.

BB′

CC′ Journal of Experimental Biology

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AA′ Fig. 4. Immunohistochemistry staining of O. niloticus gill with Atp4b-B1349. (A,C) Atp4b (green) and (B,C) Na+/K+- ATPase (NKA; fuchsia) localization; sections were stained with DAPI (blue) and overlaid with the corresponding DIC image (C). Arrows indicate Atp4b-positive NKA immunoreactive ionocytes. Insets (A′–C′)areat3× magnification. Scale bar: A–C50μm, A′–C′ 16.6 μm.

BB′

CC′

with the role of these cells in ion and acid–base regulation. The We demonstrated that the uptake of potassium by the tilapia gill + kidney immunoreactivity in the collecting duct segment was was carrier mediated. The Vmax and the apparent K affinity Km in consistent with the contribution to K+ regulation through O. niloticus indicated a direct, substrate-independent mechanism reabsorbing K+ and acidifying the tubular fluid, as seen in and/or carrier-mediated potassium uptake. In rainbow trout, K+ mammals (Koeppen, 2009; Wingo and Cain, 1993). Taken uptake has also been measured, although the mechanism has not yet together with the findings for atp4a and atp4b RT-PCR, these been elucidated (Eddy, 1985; Gardaire and Isaia, 1992; Gardaire results strongly support the presence (IHC) and function (Rb+ et al., 1991). Eddy (1985) reported that in trout, K+ uptake rate was uptake) of HKA in gill and kidney potassium regulation. only 3–5% of Na+ and Cl− uptake (Eddy, 1975); however, in studies

ABCD

EFGH

Fig. 5. Immunohistochemistry staining of O. niloticus kidney with Atp4a-B1352 and Atp4b-B1349. (A,C,D) Atp4a (green) and (E,G,H) Atp4b (green) co- localized with (B–D,F–H) NKA (fuchsia); sections were stained with DAPI (blue) and overlaid with the corresponding DIC image (C,D,G,H). Insets (D,H) are at

3.3× magnification. Scale bar: A–C,E–G50μm, D,H 15 μm. pt, proximal tubule; dt, distal tubule; ct, collecting tubule; cd, collecting duct. Journal of Experimental Biology

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AA′ Fig. 6. Immunohistochemical localization of Atp4a, Atp4b and NKA in O. niloticus gill. (A,C) Atp4a (fuchsia), (B,C) Atp4b (green) and (C) NKA (aqua) localization; sections were stained with DAPI (blue) and overlaid with the corresponding DIC image (C). Insets (A′–C′) are at 2× magnification. Arrows indicate apical Atp4a and Atp4b localization to NKA-immunoreactive ionocytes using chicken–chicken double labelling. Scale bar: A–C50μm, A′–C′ 25 μm.

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with lungfish and lamprey, Rb+ flux rate was 20% and 10% of Na+ Omeprazole is available in both oral and injectable forms (Worden and Cl− flux, respectively (Doherty, 2016; Wilkie et al., 1998, and Hanna, 2017) and has few side effects (Schubert, 2017). 2007). In tilapia, Rb+ uptake rate was approximately Although less common, the injectable form is an effective means of 11 μmol kg−1 h−1, which was approximately 3% of Na+ uptake gastric ulcer treatment and gastric acidification inhibition (Worden rate (Oreochromis mossambicus 360 μmol kg−1 h−1; Flik et al., and Hanna, 2017). In the present study, 40 mg kg−1 omeprazole was 1989; Potts et al., 1967) and in agreement with the observation made injected intraperitoneally overnight 12 h before the experiment and by Eddy (1985). then at the beginning of the experiment. However, as no inhibition of Several classes of drugs have been investigated to evaluate the Rb+ uptake was observed, we assumed that either there was not activation of the pump by inhibiting the acid secretion by HKA sufficient time for the drug to reach the gills, considering the duration using, for example, omeprazole, ouabain, SCH28080 and vanadate of acid inhibition (48 h) allowing strong binding to HKA (Shin et al., (Hersey et al., 1988). However, in this study, only omeprazole and 2009) or the drug was not activated, given that binding and inhibition SCH28080 were tested for pharmacological inhibition as these PPIs of omeprazole are dependent on acidification, or maybe it was cleared and APAs have the highest binding affinity and covalently bind with and excreted by the fish. The second option is supported by a study of HKA (Singh et al., 2013). They are widely used for treating all acid Shin and Sachs (2010), which indicates that the activation rate of secretion-related disorders in the stomach, and omeprazole was the omeprazole correlates with the pH in the cells; therefore, it is possible first clinically useful acid-activated drug. However, it cannot be that under in vivo conditions, acidification was not sufficient to inhibit activated at neutral pH, and its activation is related to the pH level in the gill HKA as the pH was not adjusted during the experiments. In terms of other factors including dosage and duration of treatment future studies, in vitro measurements of HKA activity from the organs (Shin and Sachs, 2010). According to Shin et al. (2009), SCH28080 of treated fish would address this question of effective pump was a compound developed to control acid secretion by gastric inhibition. It is also possible that the omeprazole was cleared as in glands; however, unlike omeprazole, it does not require acid mammalian studies, omeprazole is rapidly cleared from the plasma activation to effectively reduce the acid secretion of HKA. The role with a half-life of less than 1 h because of rapid hepatic of the pumps in potassium uptake based on pharmacology was metabolization (Larsson et al., 1985). However, it is also possible demonstrated by pharmacological inhibition experiments using a that alternative mechanisms for K+ uptake might compensate for the proton pump inhibitor at a concentration predicted to be sufficient inhibition of branchial HKA such as the paracellular uptake for inhibition of HKA. However, only the ex vivo methods showed a mechanism described by Horng et al. (2017) in medaka. definitive result, whereas the in vivo results showed no clear As mentioned above, the kinetics of the accumulation of Rb+ over pharmacological support for the hypothesis that the gastric HKA time suggested the presence of carrier-mediated uptake of Rb+.This + plays a role in acid–base or K regulation by the gills of O. niloticus. promising observation led us to look for further pharmacological Journal of Experimental Biology

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proton pump inhibitors. The ex vivo findings along with the Rb+ GillKid Sto uptake results from the in vivo experiment support the hypothesis that kDa the gastric-type HKA plays a role in K+ and possibly acid–base 250 regulation in O. niloticus. This is the first evidence of the presence of 150 HKA in teleost gills and kidney. We then used omeprazole and SCH28080 as specific inhibitors for the gastric HKA, aimed at 100 reducing Rb+ uptake and demonstrating the presence of the gastric 75 HKA in the gills and kidney. Gumz et al. (2010), in their review, reported that the gastric HKA in tetrapod kidney is inhibited by 50 SCH28080 and it is sensitive to omeprazole, which is supported by our results showing an inhibition of Rb+ uptake with these inhibitors 37 in both the kidney and gill. This type of ex vivo approach was developed and validated by Tipsmark and Madsen (2001) in the gill and kidney of salmonid fish with the inhibitor ouabain to study NKA. As we were studying a 25 different pump (HKA) that is sensitive to different inhibitors (omeprazole and SCH28080), some modifications were made to the 20 approach to fit the experiments. As expected, gill and kidney inhibition rates were significantly noticeable within 10 min of the start of the experiment, demonstrating that the inhibitors were blocking the uptake of Rb+ and reducing the pump activity as time Fig. 7. Immunoblotting of gill, kidney and stomach crude membrane increased. Although inhibition with SCH28080 was only tested on preparations from O. niloticus. Blots were probed with Atp4a antibodies. A the gill at 10 min, it showed a high degree of inhibition (50%). band corresponding to the predicted molecular mass of Atp4a peptide A potential challenge with all ex vivo preparations is tissue (113 kDa) can be seen in the gill and stomach (Sto) but not the kidney (Kid). degradation. Although the tissues were capable of taking up Rb+ The dye front is indicated by the mark at the bottom of the blot. over 30 min, further compelling evidence for tissue integrity was provided by the LDH activity measurements. The leakage rate of evidence of HKA using a different methodology, an ex vivo this cytosolic enzyme into the medium was used as an indicator of preparation. With this preparation, we had more control over the cell damage and death. The resultant average leakage rate out of the tissue and the experimental surroundings, including the pH, which is tissue was extremely low (0.03%) during ex vivo experiments. known to be an essential factor for the activation and binding of the However, the only point when the gills showed a leakage rate higher than the average was at time 0 h (0.1%), when residual activity from sample preparation would be expected. These activity levels were atp4a L Gill Kid Sto Int Rec Liv Br B L still very low, indicating the maintenance of tissue integrity during the course of the experiment and significantly no correlation with inhibitor treatments was observed. In comparable studies that measured LDH activity levels, the same range of values was reported in Mugil auratus (81 U g−1 wet tissue mass; Krajnovic- Ozretic and Ozretic, 1987) and Fundulus grandis (51.8 U g−1 wet tissue mass; Diaz, 2014). The IHC results are the first to demonstrate that a gastric HKA in a atp4b L Gill Kid Sto Int Rec Liv Br B L FW teleost is expressed in extra-gastric sites where it may function in K+ absorption. Choe et al. (2004) were the first to detect expression of HKα1 (Atp4a) in the gills of a FW elasmobranch (Dasyatis sabina), and Hentschel et al. (1993) in the kidney of a marine elasmobranch (Scyliorhinus caniculus). These results suggested that the HKα1 isoform of elasmobranchs is similar to that in rat gastric parietal cells and renal intercalated cells, with 81.4% similarity to the rat HKα1 protein sequence. Smolka et al. 18s L Gill Kid Sto Int Rec Liv Br B L (1994) had already confirmed the cross-reactivity of the antibodies used in these studies with proteins found in the gastric glands. While all the type IIc P-ATPases share homology, alignment of D. sabina with O. niloticus Atp4a isoforms from GenBank shows that the O. niloticus protein shares similarities in many regions, including the invariant phosphorylation site and the predicted transmembrane regions of type HKα1. Furthermore, the O. niloticus sequence is 86% identical to that of D. sabina. Nile tilapia (O. niloticus) stomach was shown to be Fig. 8. Tilapia organ expression of atp4a (223 bp) and atp4b (148 bp). PCR immunoreactive to antibodies generated against Atp4a and Atp4b, products were run against a Biotium 100 bp ladder (L): gill, kidney (Kid), indicating that they recognize the O. niloticus gastric proton pump, stomach (Sto), anterior intestine (Int), rectum (Rec), liver (Liv), brain (Br) and which was confirmed by Atp4a western blotting and transcript blank (B). 18S rRNA was included as a RT-PCR control. expression of both subunits by RT-PCR. The results in gills indicate Journal of Experimental Biology

9 RESEARCH ARTICLE Journal of Experimental Biology (2020) 223, jeb214890. doi:10.1242/jeb.214890 that gastric HKA immunoreactivity occurred apically in a H+ subpopulation of mitochondrion-rich chloride cells or ionocytes. This cell type with its different subtypes in teleost fish gills is the site of ion uptake (Wilson and Laurent, 2002; Dymowska et al., 2012), although the subtypes that are responsible for K+ uptake had not ATP + been identified until this work. In the diadromous Atlantic stingray, K Choe et al. (2004) identified a similar apical staining pattern in gill ionocytes (intercalated cells) for Atp4a using a heterologous antibody. However, the HKA uptake mechanism of ionocytes may not be the exclusive means of K+ uptake given that, in medaka, paracellular K+ uptake has been measured in-between keratinocytes in skin (Horng et al., 2017) with ionocytes instead having been + + K identified as sites of K excretion (Abbas et al., 2011; Horng et al., ATP 2017). The HKA-expressing ionocytes may constitute a new ionocyte subtype in FW tilapia; however, additional work is required to determine its relationship with Type I (NKA only), II (apical NCC and basolateral NBC1 and NKA) and III (apical NHE3 K+ Na+ and basolateral NKCC and NKA) described in Mozambique tilapia (O. mossambicus) (Hwang et al., 2011; Dymowska et al., 2012). Fig. 9. Summary of gill/kidney H+/K+-ATPase (HKA) function in O. niloticus. Both HKA isoforms are localized to the colleting duct of HKA is found apically in both gill ionocytes and kidney collecting tubule/duct (green). ATP hydrolysis by HKA (green) drives an electroneutral mammalian kidney, and similarly, the gastric HKA hydrogen and potassium exchange. The accumulated intracellular K+ exits the immunoreactivity was also found in the collecting duct of cell across the basolateral membrane down its electrochemical gradient via an + O. niloticus, thus emphasizing its role in K reabsorption and inwardly rectifying potassium channel (Kir; blue). In the same cells, basolateral possibly acid–base balance. This segment has the ability to acidify the NKA (purple) uses ATP hydrolysis to pump 3 Na+ out of the cell in exchange for tubular fluid by secreting H+ and reabsorbing K+, thus playing a role in 2K+, which also cycles out through the Kir. acid–base and potassium regulation in tetrapods (Koeppen, 2009; Gumz et al., 2010). In contrast, in the kidney of a insufficiency or other causes, then excess K+ intake can produce marine elasmobranch (Scyliorhinus caniculus), gastric HKA hyperkalaemia. In contrast, K+ depletion (hypokalaemia) by the immunoreactivity was found in the late distal tubule as well as the kidney is commonly caused by polyuria (higher rates of urination) proximal tubule (Hentschel et al., 1993). and polydipsia (excessive thirst), which can lead to excessive The IHC results in O. niloticus gill are supported by both Atp4a drinking and dilution of K+ in the blood. Sustained K+ depletion can western blotting and atp4a and atp4b RT-PCR. The absence of severely affect systems and organs, leading to cardiovascular and supporting western blotting data for Atp4b is not ideal but is also not neurological disturbances and impaired muscle and kidney function. without precedence when using antibodies (e.g. NHE2, Ivanis et al., In aquatic animals, maintenance of potassium homeostasis is also 2008; NHE3b, Ito et al., 2014). Antibodies may not necessarily be vital. Although little work has been done in fishes, it would be cross-reactive with peptides in different formats (histological predicted that strenuous exercise and a diet and environment (e.g. sections versus SDS-PAGE immunoblots) where protein freshwater) poor in K+ would result in hypokalaemia whereas diets conformation differs (native versus denatured, respectively) and and environments (e.g. seawater) rich in K+ would be predicted to therefore antibody binding sites or epitopes may change (Harlow result in hyperkalaemia if regulatory mechanisms were impaired. and Lane, 1988). In the case of the kidney, while IHC and RT-PCR Based on our findings of an apical localization of HKA in gill presented positive findings for both HKA subunits, western blotting ionocytes and kidney collecting duct, we predict the following for Atp4a was negative in contrast to the gill and stomach. However, model to explain HKA’s role in K+ uptake (Fig. 9). HKA uses the the rationale for the lack of Atp4b antibody cross-reactivity cannot energy from ATP hydrolysis to power the electroneutral exchange of be applied to the case of Atp4a in the kidney. There is the possibility intracellular H+ for extracellular K+ (Caplan, 1998). The uptake of of insufficient sensitivity in the technique to detect the kidney K+ would be against its electrochemical gradient. Intracellular K+ Atp4a. Future work is needed to address this possibility. would exit the cell down its electrochemical gradient across the The main finding of this study is that tilapia can take up potassium basolateral membrane via an inwardly rectifying K+ channel (Evans and that HKA is involved. Potassium regulation is important to et al., 2005). The IHC results show that these cells are also rich in animals. Animals, including humans, generally obtain potassium basolateral NKA; however, its role in K+ uptake is likely to be through food at levels that do not pose health risks; however, minor indirect and/or related to driving other transport processes. It should disturbances (increase–decrease) in potassium concentration might be noted that K+ is but one of many ions that may be transported by cause serious health problems. In mammals, for example, gill ionocytes for ion and acid–base regulation (Evans et al., 2005). unfavourable effects might occur when they are subjected to higher than normal K+ plasma concentrations (>5.0 mmol l−1: Conclusions hyperkalaemia), or when K+ plasma concentrations are lower than In summary, this research is the first to demonstrate the expression the normal range (3.5–5.0 mmol l−1: hypokalaemia). Both and role of the gastric proton pump HKA as an extra-gastric pump hyperkalaemia and hypokalaemia result from disruptions in (in the gill and kidney) in a teleost species. Evidence for this transcellular homeostasis or in the renal regulation of K+ excretion includes omeprazole- and SCH28080-induced inhibition of the (Talling, 2010). However, as a normal kidney can excrete hundreds of gastric proton pump in O. niloticus gills and kidney, suggesting a milliequivalents of K+ daily, excessive K+ intake is an uncommon role in K+ reabsorption and possibly acid–base regulation. cause of hyperkalaemia without other contributing factors. Therefore, Immunohistochemistry demonstrated that the pump was localized + if renal K excretion is impaired, whether through drugs, renal apically in the gill ionocyte and the kidney collecting duct segment. Journal of Experimental Biology

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Future ex vivo and in vivo experiments in different teleost species are Evans, D. H., Piermarini, P. M. and Choe, K. P. (2005). The multifunctional fish gill: vital to better understand the role of HKA in teleost physiology and dominant site of gas exchange, osmoregulation, acid-base regulation, and excretion of nitrogenous waste. Physiol. Rev. 85, 97-177. doi:10.1152/physrev. to determine how widespread it is. Of particular interest will be 00050.2003 determining what role the pump plays in acid–base regulation. Flik, G., van der Velden, J. A., Seegers, H. C. M., Kolar, Z. and Bonga, S. E. W. (1989). Prolactin cell activity and sodium fluxes in tilapia (Oreochromis Acknowledgements mossambicus) after long-term acclimation to acid water. Gen. Comp. We would like to thank Mr R. Busby of Sand Plains Aquaculture for providing tilapia Endocrinol. 75, 39-45. doi:10.1016/0016-6480(89)90005-1 used in this study and Drs J. C. McGeer, M. P. Wilkie and P. Craig for comments on Furukawa, F., Watanabe, S., Kimura, S. and Kaneko, T. (2012). Potassium the thesis on which this paper is based (Barnawi, 2017). The α5 monoclonal excretion through ROMK potassium channel expressed in gill mitochondrion-rich antibody developed by Douglas M. Fambrough at Johns Hopkins University was cells of Mozambique tilapia. Am. J. Physiol. Regul. Integr. Comp. Physiol. 302, obtained from the Developmental Studies Hybridoma Bank, created by the NICHD R568-R576. doi:10.1152/ajpregu.00628.2011 of the NIH and maintained at The University of Iowa, Department of Biology. Gardaire, E. and Isaia, J. (1992). Potassium balance in freshwater-adapted trout Onchorhynchus mykiss. Comp. Biochem. Physiol. 103, 657-660. doi:10.1016/ 0300-9629(92)90162-J Competing interests Gardaire, E., Isaia, J. and Bornancin, M. (1991). Kinetics of potassium transport The authors declare no competing or financial interests. across trout gills. Comp. Biochem. Physiol. A Physiol. 99, 615-620. doi:10.1016/ 0300-9629(91)90139-4 Author contributions Gumz, M. L., Lynch, I. J., Greenlee, M. M., Cain, B. D. and Wingo, C. S. (2010). Conceptualization: E.A.B., J.E.D., J.M.W.; Methodology: E.A.B., J.E.D., J.M.W.; The renal H+-K+-ATPases: physiology, regulation, and structure. Am. J. Physiol. Formal analysis: E.A.B., J.E.D., P.F., J.M.W.; Investigation: E.A.B., J.E.D., P.F., 298, F12-F21. doi:10.1152/ajprenal.90723.2008 J.M.W.; Resources: J.M.W.; Writing - original draft: E.A.B.; Writing - review & editing: Harlow, E. and Lane, D. (1988). Antibodies: A Laboratory Manual, p. 726. P.F., J.M.W.; Visualization: P.F., J.M.W.; Project administration: J.M.W.; Funding New York: Cold Spring Harbor Laboratory Press. acquisition: J.M.W. Hentschel, H., Mähler, S., Herter, P. and Elger, M. (1993). Renal tubule of dogfish, Scyliorhinus caniculus: a comprehensive study of structure with emphasis on + + Funding intramembrane particles and immunoreactivity for H -K -adenosine This work was supported by Natural Sciences and Engineering Research Council of . Anat. Rec. 235, 511-532. doi:10.1002/ar.1092350404 Canada (NSERC) discovery and Canadian Foundation for Innovation grants to Hersey, S. J., Steiner, L., Mendlein, J., Rabon, E. and Sachs, G. (1988). + + J.M.W. E.A.B. was supported by a scholarship from the Saudi Arabia Cultural SCH28080 prevents omeprazole inhibition of the gastric H /K -ATPase. Biochim. Bureau and J.E.D. had partial support from an Ontario Graduate Scholarship. Biophys. Acta Protein Struct. Mol. Enzymol. 956, 49-57. doi:10.1016/0167- 4838(88)90296-8 Horng, J.-L., Yu, L.-L., Liu, S.-T., Chen, P.-Y. and Lin, L.-Y. (2017). 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