Comprehensive Analysis of Genes Contributing to Euryhalinity in The

Home , Banded houndshark, Bull shark, Potamotrygonidae, Triakis

RESEARCH ARTICLE Comprehensive analysis of genes contributing to euryhalinity in the bull shark, Carcharhinus leucas;Na+-Cl− co-transporter is one of the key renal factors upregulated in acclimation to low-salinity environment Itaru Imaseki1,‡, Midori Wakabayashi1, Yuichiro Hara2,*, Taro Watanabe1, Souichirou Takabe1, Keigo Kakumura1, Yuki Honda1, Keiichi Ueda3, Kiyomi Murakumo3, Rui Matsumoto3, Yosuke Matsumoto3, Masaru Nakamura4, Wataru Takagi1, Shigehiro Kuraku2 and Susumu Hyodo1

ABSTRACT leucas, have the capacity to inhabit both seawater (SW) and Most cartilaginous fishes live principally in seawater (SW) freshwater (FW) environments (see Compagno, 1984; Hazon et al., environments, but a limited number of species including the bull 2003; Pillans and Franklin, 2004). Bull sharks spend most of their shark, Carcharhinus leucas, inhabit both SW and freshwater (FW) lifetime in SW, but they utilize a wide range of salinities throughout environments during their life cycle. Euryhaline elasmobranchs their life cycle (Thorson, 1971; Montoya and Thorson, 1982; maintain high internal urea and ion levels even in FW environments, Simpfendorfer et al., 2005; Carlson et al., 2010). Female bull sharks but little is known about the osmoregulatory mechanisms that enable are thought to give birth in estuaries or river mouths, and neonates and them to maintain internal homeostasis in hypoosmotic environments. juveniles are thought to migrate upstream to occupy inshore rivers as In the present study, we focused on the kidney because this is the only nursery habitats (Ortega et al., 2009; Werry et al., 2012). The use of organ that can excrete excess water from the body in a hypoosmotic FW habitats by juvenile bull sharks has been reported in the southern environment. We conducted a transfer experiment of bull sharks from USA (Simpfendorfer et al., 2005; Ortega et al., 2009; Matich and SW to FW and performed differential gene expression analysis Heithaus, 2015), central America (Thorson, 1971; Montoya and between the two conditions using RNA-sequencing. A search for Thorson, 1982), Australia (Pillans and Franklin, 2004; Werry et al., genes upregulated in the FW-acclimated bull shark kidney indicated 2012) and South Africa (Cliff and Dudley, 1991; McCord and that the expression of the Na+-Cl− cotransporter (NCC; Slc12a3) was Lamberth, 2009). Once bull sharks reach sexual maturity – 10 times higher in the FW-acclimated sharks compared with that in SW [approximately 160 180 cm in total length (TL) in Florida], they sharks. In the kidney, apically located NCC was observed in the late move from riverine and estuarine environments into full-strength SW distal tubule and in the anterior half of the collecting tubule, where for further growth and breeding (Curtis et al., 2011). basolateral Na+/K+-ATPase was also expressed, implying that these It is well recognized that marine cartilaginous fishes, including segments contribute to NaCl reabsorption from the filtrate for diluting SW-adapted bull sharks, conduct urea-based osmoregulation. Plasma the urine. This expression pattern was not observed in the houndshark, ion levels are maintained at approximately half that of SW, whereas Triakis scyllium, which had been transferred to 30% SW; this species total plasma osmolality is slightly hyperosmotic to the surrounding cannot survive in FW environments. The salinity transfer experiment SW environment through the retention of urea. As a result, combined with a comprehensive gene screening approach cartilaginous fish do not suffer dehydration in a SW environment demonstrates that NCC is a key renal protein that contributes to the (Smith, 1936; Robertson, 1975; Hazon et al., 2003; Hyodo et al., remarkable euryhaline ability of the bull shark. 2004b). In FW environments, bull sharks and other euryhaline elasmobranch species maintain high internal NaCl and urea levels, KEY WORDS: Kidney, Ion transporters, Euryhalinity, resulting in a plasma osmolality of approximately 600 mOsm (Janech Osmoregulation, Freshwater adaptation, Cartilaginous fish and Piermarini, 2002; Pillans and Franklin, 2004) that is almost twice that of FW teleosts (Evans, 1993). This strategy is in contrast with FW INTRODUCTION stingrays (Potamotrygonidae), which, like FW teleosts, do not Most cartilaginous fishes are principally marine species, and only a accumulate urea (Wood et al., 2002). Therefore, it is likely that bull limited number of species, including the bull shark, Carcharhinus sharks face incipient water gain and solute loss in a FW environment by branchial diffusion and urinary loss. Recently, the contribution of + + + + 1Laboratory of Physiology, Atmosphere and Ocean Research Institute, The Na /K -ATPase (NKA) and Na /H exchanger 3 was reported for University of Tokyo, Kashiwa, Chiba 277-8564, Japan. 2RIKEN Center for branchial Na+ uptake in bull sharks in a FW environment (Reilly Biosystems Dynamics Research, Kobe, Hyogo 650-0047, Japan. 3Okinawa Churaumi Aquarium, Motobu, Okinawa 905-0206, Japan. 4Okinawa Churashima et al., 2011). However, mechanisms enabling the euryhalinity in Foundation, Motobu, Okinawa 905-0206, Japan. cartilaginous fish are still largely unknown. *Present address: Research Institute of Environmental Medicine, Nagoya It is generally accepted that multiple organs are involved in University, Nagoya 464-8601, Japan. maintaining body–fluid homeostasis in vertebrates. Among them, ‡Author for correspondence ([email protected]) the kidney is one of the most important osmoregulatory organs in vertebrates including cartilaginous fishes. In a SW environment, I.I., 0000-0002-3003-926X; M.W., 0000-0003-0661-6052; K.K., 0000-0002- 0556-5110; W.T., 0000-0002-7882-0223 more than 90% of filtered urea is reabsorbed from the primary urine and returned to the blood system, thereby reducing the urinary loss

Received 14 February 2019; Accepted 18 May 2019 of urea (Smith, 1936; Kempton, 1953; Boylan, 1967). Reflecting Journal of Experimental Biology

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Japanese banded houndsharks, Triakis scyllium Müller & Henle List of abbreviations 1839 (n=9, TL=68.1±1.0 cm, BM=1.0±0.05 kg), were collected in BM body mass Koajiro Bay, near the Misaki Marine Biological Station of the CT collecting tubule University of Tokyo. They were transported to the Atmosphere and EDT early distal tubule Ocean Research Institute at the University of Tokyo and kept in FDR false discovery rate 2×103 liter holding tanks (20–22°C) under a constant photoperiod FPKM fragments per kilobase of exon per million reads mapped FW freshwater (12 h:12 h light:dark). The houndsharks were fed on squid for at GFR glomerular filtration rate least 2 weeks before the experiments and were kept without feeding LDT late distal tubule during the experimental period (see Yamaguchi et al., 2009). logFC log2 fold change All animal experiments were conducted according to the Guidelines − NCC Na+-Cl co-transporter for Care and Use of Animals approved by the committees of the α + + α NKA 1Na/K ATPase subunit 1 University of Tokyo and the Okinawa Churaumi Aquarium. NKCC2 Na+-K+-Cl− cotransporter 2 ORF open reading frame RNA-seq RNA-sequencing Transfer experiments Slc solute carrier In March of 2012, three bull sharks kept in holding tanks were SW seawater moved to an experimental tank (15×103 liters, filled with full- UFR urine flow rate strength SW, 35 ppt) 3 days before the onset of salinity change. It UT urea transporter was ascertained that they could swim comfortably in the new tank. On day 1 of the experiment, the salinity was adjusted by adding FW to achieve a salinity of 80% SW. On day 2, the same degree of their crucial function, the renal tubules of marine cartilaginous fish salinity change was performed to produce 60% SW. On days 3–8, are highly elaborate and show unique features compared with those the salinity of the tank was reduced by 10% per day, and the salinity of other vertebrates (Lacy and Reale, 1985; Hentschel et al., 1998; was deemed to have reached nearly FW (less than 2 ppt) on day 8 Hyodo et al., 2014). We have conducted mapping of various pumps, (Fig. S1). Bull sharks were killed on days 8–10. Two SW control channels and transporters in the nephron segments, and proposed a sharks were maintained in the holding tank with running SW, and model for urea reabsorption in the kidney of SW-adapted were killed on days 8 and 9, and two further SW sharks were killed cartilaginous fishes (Hyodo et al., 2004a, 2014; Yamaguchi et al., in November 2011 and February 2013, respectively. For sampling, 2009; Kakumura et al., 2015; Hasegawa et al., 2016). In FW bull sharks were initially anesthetized with an intramuscular environments, in contrast, it is assumed that the kidney of the bull injection of midazolam (2.5 mg kg−1 BM) and then euthanized shark must excrete excess water caused by the osmotic influx of with an intravenous injection of 2,6-diisoprophylphenol (0.6– water, while concomitantly retaining ions and urea. Therefore, it is 1.6 mg kg−1 BM). Blood samples were collected from the dorsal highly probable that the expression of key proteins involved in vasculature by a syringe and were centrifuged at 10,000 g at 4°C for osmoregulation is altered in the kidney when bull sharks move from 10 min to obtain the plasma. Urine samples were collected using a SW to FW habitats. Pillans et al. (2005) reported that the expression urethral catheter that was inserted under anesthesia (All Silicone Foley level of NKA decreased in the kidney of bull sharks following Balloon Catheter, Create Medic Co. Ltd, Kanagawa, Japan). Plasma transfer from FW to SW, supporting our notion that mechanisms for and urine samples were stored at −80°C until analysis. Ion and urea retaining ions and urea are upregulated in a FW environment. concentrations and the osmolality of plasma and urine were measured In the present study, we performed a transfer experiment of captive by Rapid Labo 1265 (Siemens Healthineers, Erlangen, Germany). bull sharks from SW to FW and conducted a comprehensive search Kidneys were dissected out and separated into the left and right halves. for transcripts exhibiting significant increases in expression levels in One half was frozen quickly in liquid nitrogen, while the other half FW-acclimated bull sharks by RNA-sequencing (RNA-seq). The was trimmed transversely to 1 cm slices and fixed in Bouin’s solution differential gene expression profiling with RNA-seq, and subsequent without acetic acid (saturated picric acid:formalin=3:1) or 4% quantitative PCR and in situ hybridization analyses revealed that the paraformaldehyde in 0.1 mol l−1 phosphate buffer (pH 7.4) expression of Na+-Cl− cotransporter (NCC) was substantially containing 150 mmol l−1 NaCl and 350 mmol l−1 urea at 4°C for upregulated in the late distal tubule (the fourth loop) and the 2 days. Other tissues (hypothalamus, pituitary, gill, ventricle, atrium, collecting tubule (the final segment) by transfer of bull sharks from liver, muscle, stomach, intestine, rectum and rectal gland) were also SW to FW. In contrast, similar changes in distribution and expression collected and were frozen or fixed as described above. level of NCC were not observed in the houndshark, Triakis scyllium, With regard to the houndshark, frozen and fixed tissues collected when it was acclimated to 30% SW; this species cannot survive in the in the previously conducted transfer experiment in 2009 were used. FW environment. Our findings indicate that the activation of NaCl In brief, houndsharks were kept in two tanks (1–2×103 liters, filled reabsorption associated with NCC is one of the crucial mechanisms with full-strength SW, 34 ppt). On day 1, one tank was adjusted to a that contributes to the euryhalinity of the bull shark. salinity of 80% SW by adding FW. On days 2 and 3, the same degree of salinity change was performed to produce 40% SW. FW was MATERIALS AND METHODS added on day 4 to produce a final salinity of 30% SW (10 ppt, n=4). Animals Control houndsharks (n=5) were kept in full-strength SW during the The bull sharks, Carcharhinus leucas (Müller & Henle 1839), used dilution protocol. Houndsharks were maintained for 1 week in each in this study [n=7, TL=199.9±16.0 cm, body mass (BM)=94.1± salinity and then euthanized and sampled. Sampling procedures 20.4 kg] were caught by set nets or reared in the Okinawa were as described in detail previously (Yamaguchi et al., 2009). Churaumi Aquarium. They were kept in holding tanks filled with SW (20–23°C) in the aquarium under a constant photoperiod RNA-seq analysis (12 h:12 h light:dark) and fed chopped fish. During the Total RNA was extracted from the frozen kidneys of SW (sharks no. 2 experimental period, they were kept without feeding. and 3) and FW-acclimated (sharks no. 5 and 6) bull sharks using Journal of Experimental Biology

2 RESEARCH ARTICLE Journal of Experimental Biology (2019) 222, jeb201780. doi:10.1242/jeb.201780 the guanidium thiocyanate–phenol–chloroform mixture (ISOGEN, Quantitative real-time PCR assay Nippon Gene, Toyama, Japan). The concentration and quality of The expression levels of mRNAs were examined by real-time the extracted RNA were assessed using a Qubit 2.0 Fluorometer quantitative PCR (qPCR) using a 7900HT Sequence Detection (Thermo Fisher Scientific, Waltham, MA, USA) and an Agilent 2100 System (Applied Biosystems) with a KAPA SYBR Fast qPCR kit Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA), (Kapa Biosystems), as previously described in detail (Hasegawa respectively. Non-stranded libraries were then constructed using a et al., 2016). The plasmids containing cloned cDNAs were serially TruSeq total RNA sample preparation kit (Illumina, San Diego, CA, diluted and were used as the known amounts of standard cDNAs for USA). Sequencing was performed on a HiSeq 1500 (Illumina) to absolute quantification in qPCR analyses. The copy numbers of the obtain 127 bp pair-end reads, as documented previously (Tatsumi standard cDNAs were calculated using BioMath Calculators et al., 2015). The adaptor sequences and low-quality reads were (https://www.promega.co.uk/resources/tools/biomath-calculators/). discarded using Trim Galore! v0.3.1 (https://www.bioinformatics. Primer sets for qPCR were designed using Primer Express software babraham.ac.uk/projects/trim_galore/) and a fastq quality filter v0.10.0 or PrimerQuest (https://sg.idtdna.com/pages) (Table S2). As an (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/). internal control, EF1α1 mRNA was used. First-strand cDNAs were The trimmed reads were subjected to de novo assembly to obtain prepared from 11 tissues (hypothalamus, pituitary, gill, liver, transcript contigs for each shark using Trinity version r20140717 muscle, ventricle, atrium, stomach, intestine, rectum and rectal (Grabherr et al., 2011). The contigs from each individual shark were gland) as described above, and were used for a tissue distribution then clustered using CD-HIT EST v4.6.1 (Fu et al., 2012) and analysis. TGICL v0.0.1 (Pertea et al., 2003) to produce the consensus contigs. The read trimming, transcriptome assembly and merging of In situ hybridization and morphological observation contigs were performed according to a previous study (Hara et al., Kidneys fixed in 4% paraformaldehyde solution were embedded in 2015). From these consensus contigs, open reading frames (ORFs) Paraplast (McCormick Scientific, Richmond, IL, USA). Serial and the deduced amino acid sequences were predicted using sections were cut at 7 µm thickness and mounted onto MAS-coated Transdecorder version r20140704 (Haas et al., 2013). The contigs slides (Matsunami Glass, Osaka, Japan). The cDNA fragments of were annotated using Trinotate version r20150708 (Bryant et al., bull shark NKAα1 (765 bp), NKCC2 (845 bp), NCC (1079 bp) and 2017), which provides the homology information of known proteins UT (664 bp), and houndshark NKAα1 (859 bp), NKCC2 (936 bp), and functional domains, as well as ab initio prediction of NCC (1080 bp) and UT (972 bp) were amplified using gene- transmembrane and signal peptides. The trimmed sequence reads specific primers (Table S2), and then used to synthesize digoxigenin were then mapped to the contigs using bowtie2 v2.1.0 (Langmead and (DIG)-labeled antisense and sense cRNA probes (DIG RNA Salzberg, 2012) and the expressions were quantified using eXpress labeling kit; Roche Applied Science, Mannheim, Germany), v1.5.1 (Cappé and Moulines, 2009). The differential expression following the manufacturer’s protocols. Hybridization and analysis between SW and FW individuals was performed using edgeR washing were conducted using a previously described protocol v3.0.0 (Robinson et al., 2010; McCarthy et al., 2012). The sequencing (Takabe et al., 2012). Stained sections were counterstained with information and assembly statistics are shown in Table S1. The Nuclear Fast Red (Vector Laboratories, Burlingame, CA, USA). accession numbers for the short-read data of RNA-seq analysis are: Micrographs were obtained using a virtual slide system (Toco; DRX162067, DRX162068, DRX162069 and DRX162070. Claro, Aomori, Japan). For morphological observations, kidney sections fixed in Bouin’s cDNA cloning solution were stained with periodic acid–Schiff (McManus, 1946). Complementary DNA cloning was performed as previously Briefly, deparaffinized sections were oxidized in 0.5% periodic acid described in detail (Hasegawa et al., 2016). Total RNA was solution (Wako, Osaka, Japan) for 5 min. After washing in tap water extracted from the kidney with ISOGEN. Two micrograms of total and distilled water, sections were placed in Schiff’s reagent (Wako) RNA was treated with DNase using a TURBO DNA-free kit (Life for 15 min, and then rinsed in sulfurous acid three times. Sections Technologies, Carlsbad, CA, USA) and reverse-transcribed to first- were then counterstained with Mayer’s hematoxylin (Wako). strand cDNA using a high-capacity cDNA reverse transcription kit (Life Technologies), following the manufacturer’s instructions. Immunohistochemistry Primers for cDNA cloning were designed to amplify entire ORFs A peptide C+GFEDEAIVKELRKD from bull shark NCC was based on the contig sequence data from the RNA-seq (Table S2). synthesized and coupled via cysteine to keyhole limpet hemocyanin. The target cDNAs were amplified with Kapa Taq Extra DNA The conjugated peptide was emulsified with complete Freund’s polymerase (Kapa Biosystems, Boston, MA, USA), electrophoresed adjuvant and injected into a rat for immunization (Eurofins on a 1.2% agarose gel, excised and purified using the UltraClean 15 Genomics, Tokyo, Japan). DNA Purification Kit (MO BIO Laboratories, Carlsbad, CA, USA). Kidneys fixed with Bouin’s solution without acetic acid were The purified fragments were ligated into a pGEM T-easy plasmid embedded in Paraplast. Serial sections were cut at 7 µm thickness (Promega, Madison, WI, USA), and the nucleotide sequence was and mounted onto MAS-coated slides (Matsunami Glass). determined using an automated DNA sequencer (3130xl Genetic Rehydrated tissue sections were treated with antigen activator Analyzer; Life Technologies). The accession numbers for the partial (Histo VT One; Nacalai Tesque, Kyoto, Japan) for 20 min at 90°C. sequences of bull shark Na+/K+-ATPase alpha subunit 1 (NKAα1), Immunohistochemical staining for NCC was performed with the Na+-K+-Cl− cotransporter-2 (NKCC2), Na+-Cl− cotransporter avidin-biotin-peroxidase complex kit (Vector Laboratories), as (NCC), urea transporter (UT) and elongation factor 1 alpha described previously (Hasegawa et al., 2016). NCC antiserum was subunit 1 (EF1α1) are LC462277, LC462278, AB769491, diluted with 2% normal goat serum in PBS (pH 7.4) (1:90,000). The LC462279 and LC462280, respectively. The accession numbers specificity of immunoreactive signals for NCC was confirmed by for the partial sequences of houndshark NKAα1, NKCC2, NCC, preabsorption of antibody with the synthetic antigen for 24 h at 4°C UT and EF1α1 mRNA are AB669491, AB769486, AB769487, prior to incubation. Stained sections were counterstained with

AB094993 and LC462281, respectively. Mayer’s hematoxylin (Wako). Micrographs were obtained using a Journal of Experimental Biology

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Table 1. Plasma and urine composition of bull sharks in seawater (SW) and freshwater (FW) Group N Osmolality (mOsm kg−1)Na+ (mmol l−1)Cl− (mmol l−1) Urea (mmol l−1) Plasma SW 4 1031.0±24.9 290.3±20.0 239.3±17.0 355.1±2.7 FW 3 617.7±13.0*** 213.7±9.0 177.0±3.5* 144.4±13.0** Urine SW 3 849.3±100.5 282.7±53.5 236.0±14.5 105.9±30.5 FW 3 480.7±43.0 162.7±13.9 143.7±10.8* 122.0±4.7 SW 1067.0 550.0 510.0 ND Values are means±s.e.m. ND, not detectable. Statistical analysis was performed using t-test (all parameters except for plasma Na+) or Mann–Whitney U-test (plasma Na+). Statistically significant differences between SW and FW are shown with asterisks (*P<0.05; **P<0.01; ***P<0.001; plasma osmolality, P=0.00005; plasma Cl−, P=0.03276; plasma urea, P=0.00272; urine Cl−, P=0.02882). digital camera (DSRi1; Nikon, Tokyo, Japan). For double-labeling In the present study, we could not measure glomerular filtration fluorescence immunohistochemistry, the NCC antiserum (1:30,000) rate (GFR) and urine flow rate (UFR) of SW- and FW-acclimated and anti-NKAα subunit antibody (1:2000; immunized in a rabbit; a bull sharks. In the Atlantic stingray, the transfer from ambient SW to gift from Prof. Kaneko, University of Tokyo) were mixed and 50% SW for 2 h increased GFR and UFR 3-fold and 9-fold, incubated with deparaffinized sections for 48 h at 4°C. Sections respectively (Janech et al., 2006). Therefore, in Table 2, the values were washed in PBS, and then incubated with fluorescein-labeled for GFR and UFR reported in the Atlantic stingray were used to anti-rat IgG antibody (Alexa Fluor 555) and anti-rabbit IgG antibody calculate the hypothetical absolute amount of reabsorbed NaCl in (Alexa Fluor 488) (Thermo Fisher Scientific) for 2 h at room the bull shark kidney. The estimated values of NaCl and urea temperature. Sections were mounted with ProLong Gold Antifade reabsorption in SW- and FW-acclimated bull shark kidneys were Reagent (Thermo Fisher Scientific). Micrographs were obtained calculated from the plasma and urine concentrations of NaCl and using a fluorescence microscope (BX53; Olympus, Tokyo, Japan). urea in bull sharks and from the stingray GFR and UFR values in ambient SW (used for SW-acclimated bull sharks) and 50% SW Statistical analysis (used for FW-acclimated bull sharks). The estimated values in Values are presented as means±s.e.m. and were compared using Table 2 indicate that the transfer of bull sharks from SW to FW Student’s t-test or the Mann–Whitney U-test, with the assumption of caused an approximately 50% increase in reabsorption of NaCl from normality checked by the Shapiro–Wilk test. P-values <0.05 were the glomerular filtrate. considered statistically significant. All analyses were performed The plasma parameters of houndsharks after transfer to 30% SW using KyPlot 5.0 software (Kyenslab, Tokyo, Japan). (10 ppt) were reported previously (Yamaguchi et al., 2009). Plasma osmolality and the concentration of urea and ions were decreased in RESULTS the 30% SW group. Our preliminary experiment showed that Changes in plasma and urine parameters after transfer Japanese banded houndshark cannot survive when environmental to low-salinity environments salinity was lowered to less than 25% SW (8 ppt) (see Yamaguchi The plasma and urine measurement of bull sharks kept in SW and et al., 2009). acclimated to FW are summarized in Table 1. Plasma osmolality, ion and urea concentrations were decreased after the transfer to FW, Expression profiles of Slc protein-family mRNAs in the bull and the changes in osmolality, Cl− and urea were statistically shark kidney significant. However, as previously reported (Pillans and Franklin, Renal mRNA expression profiles were examined in two SW and 2004), bull sharks maintained plasma levels of ion and urea twoFWbullsharksbyRNA-seqtosearchforcandidategenes concentrations at approximately 600 mOsm kg−1 in the FW exhibiting a significant difference in expression levels following environment. Although we could not investigate the time course the FW acclimation (see Materials and Methods). The clustering of changes in the plasma parameters during the transfer experiment, the overall expression profiles showed a clear distinction between osmolality and Na+ concentration of environmental water reached the two SW individuals and the two FW-acclimated individuals levels below those of FW-acclimated bull shark plasma on days 2 (Fig. S2), indicating that acclimation to the FW environment and 4, respectively (Fig. S1). Therefore, bull sharks were exposed to triggered changes in renal function at the molecular level. The hypo-osmotic environmental conditions for a period of 1 week application of dual cut-offs [false discovery rate (FDR)<0.05 and before sampling. Urine parameters, except for urea, were also log2 fold change (logFC)>1.5 of the fragment per kilobase of exon decreased in the FW environment (Table 1). per million (FPKM)] identified 138 genes upregulated in the FW-acclimated group. Among them, six genes encoding the solute carrier (Slc) superfamily proteins were found to be upregulated: Na+/K+/Ca2+ exchanger 5 (Slc24a5), carnitine transporter 2 Table 2. Hypothetical absolute amount of solutes reabsorbed from the (Slc22a5, OCNT2), Na+-coupled neutral amino acid transporter glomerular filtrate 8(Slc38a8),Ca2+-binding mitochondrial carrier protein 2B Amount of reabsorbed solutes (µmol h−1 kg−1) (Slc25a25), Na+-Cl− cotransporter (NCC, Slc12a3) and glucose Group N Na+ Cl− Urea transporter 12 (Slc2a12) (Table 3). The Slc superfamily comprises proteins that are responsible for osmolyte transport (Hediger et al., Bull shark (SW) 3 813.4±124.0 665.4±85.8 1256.4±30.7 Bull shark (FW) 3 1331.9±47.5* 1031.1±68.8* 802.6±190.4 2004). In our analysis, NCC stably showed the largest FPKM values in FW individuals (over 120 in both individuals) that Values are means±s.e.m. For the calculation, the values of glomerular filtration were approximately 8-fold higher than that of SW individuals. rate and urine flow rate of Atlantic stingray were used from Janech et al. (2006). Statistical analysis was performed using t-test (Na+ and Cl−) or Mann–Whitney Therefore, in the following investigations, we focused on U test (urea). Statistically significant differences between SW and FW are NCC as a candidate gene important for FW acclimation in the − shown with asterisks (*P<0.05; Na+, P=0.01748; Cl , P=0.02922). bull shark. Journal of Experimental Biology

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ABC D Bull shark NKAα1 Bull shark NKCC2 Bull shark NCC Bull shark UT 0.5 * 0.08 0.4 0.05 **

0.4 0.04 0.06 0.3

0.3 0.03 0.04 0.2

0.2 0.02

0.02 0.1 0.1 0.01 1 α 0 0 0 0 SW FWSW FW SWFW SW FW EFGH Houndshark NKAα1 Houndshark NKCC2 Houndshark NCC Houndshark UT

Target gene/EF1 Target 0.08 0.18 0.0009 0.012

0.06 0.12 0.0006 0.008

0.04

0.06 0.0003 0.004 0.02

0 0 0 0 SW 30% SWSW 30% SW SW30% SW SW 30% SW

Fig. 1. Transcript quantification with real-time RT-PCR. mRNA expression levels are shown for (A,E) Na+/K+ ATPase subunit α1 (NKAα1), (B,F) Na+-K+-Cl− cotransporter 2 (NKCC2), (C,G) Na+-Cl− co-transporter (NCC) and (D,H) urea transporter (UT) in bull shark (A–D) and houndshark (E–H) kidneys. Open bars represent seawater (SW; control) individuals, whereas filled bars show freshwater (FW)- or 30% SW-acclimated fish. Data are expressed as means±s.e.m. of n=4 SW bull sharks, n=3 FW bull sharks, n=5 (SW houndsharks) and n=4 (30% SW houndsharks). Statistically significant differences (t-test) are shown with asterisks (*P<0.05; **P<0.001; bull shark NKAα1, P=0.0192; bull shark NCC, P=0.000004).

Tissue distribution of NCC mRNA in the bull shark NCC, we examined the mRNA levels of Na+/K+-ATPase alpha Tissue distribution of NCC mRNA was examined with quantitative subunit 1 (NKAα1; Atp1a1; a driving force of NaCl reabsorption), RT-PCR in four bull sharks kept in SW and three bull sharks Na+-K+-2Cl− cotransporter 2 (NKCC2; Slc12a1; a protein important acclimated in FW (Fig. S3). NCC mRNA was predominantly for NaCl reabsorption in the mammalian thick ascending limb) and expressed in the kidney. Expression of NCC mRNA was urea transporter (UT; Slc14a2) in the kidneys of bull sharks, because significantly higher in the kidneys of FW-acclimated bull sharks of their involvement in osmoregulation. The expression of NKAα1 than SW individuals (Fig. S3 and Fig. 1). Minute amounts of mRNA was significantly higher in the kidneys of FW-acclimated expression were detected in the hypothalamus, pituitary and gills, individuals compared with that of bull sharks kept in SW. In contrast, but no significant change was observed between bull sharks in SW no significant change was observed in the expression levels of and FW (Fig. S3). NKCC2 and UT mRNA following the transfer of bull sharks from SW to FW, although the mean value of UT mRNA levels in FW was Changes in expression levels of mRNA encoding NKAα1, 1.5 times greater than that in SW (Fig. 1). NKCC2, NCC and UT in the kidneys of bull sharks and In contrast, in the kidneys of houndsharks, no significant change houndsharks was observed in the NKAα1, NKCC2, NCC and UT mRNA levels To validate the results of the RNA-seq and examine the possible after transfer to diluted SW (30% SW) (Fig. 1). Furthermore, NCC involvement of other genes, we performed quantitative RT-PCR for mRNA levels (NCC mRNA/EF1α1 mRNA) in houndsharks in full- some selected genes. As mentioned above, the expression of NCC strength SW were approximately 40 times lower compared with mRNA was 10-fold higher in the kidneys of bull sharks acclimated to those of bull sharks in SW. This resulted in approximately 500

FW compared with that in SW individuals (Fig. 1). In addition to times higher expression levels of the NCC mRNA/EF1α1 mRNA Journal of Experimental Biology

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Table 3. Differentially expressed Slc family genes identified in the transcriptome analysis FPKM value Transcript contig ID Gene symbol Putative gene product SW no. 2 SW no. 3 FW no. 5 FW no. 6 FW/SW logFC FDR c13993_g0 Slc24a5 Na+/K+/Ca2+ exchanger 5 0 0.05 1.19 5.09 125.60 6.616 1.21×10−5 c37168_g0 Slc22a5 Carnitine transporter 2 0.05 0.14 2.73 1.05 19.89 3.948 1.54×10−2 c218567_g0 Slc38a8 Na+-neutral amino acid transporter 8 0.04 0.17 1.29 3.05 20.67 3.806 1.33×10−2 c8778_g0 Slc25a25 Ca2+-binding mitochondrial carrier protein 2B 8.35 12.56 216.6 35.3 12.05 3.161 3.00×10−3 c24229_g0 Slc12a3 Na+-Cl− co-transporter (NCC) 12.73 19.55 125.48 124.87 7.76 2.596 3.52×10−5 c1052_g0 Slc2a12 Glucose transporter 12 16.91 25.75 156.77 94.23 5.88 2.269 1.44×10−3 Other functionally associated genes specifically analyzed for NCC c791_g0 Slc12a1 Na+-K+-Cl− cotransporter 2 (NKCC2) 201.37 159.48 185.2 256.42 1.22 c41982_g0 Slc14a2 Urea transporter (UT) 52.19 43.28 50.28 74.45 1.31 c88436_g0 Atp1a1 Na+/K+ ATPase subunit α1 (NKAα1) 646.42 780.25 1000.47 867.94 1.31 FPKM, fragments per kilobase of exon per million reads mapped. This list includes only the contigs annotated as solute carrier (Slc) family members that satisfy the condition applied [FDR (false discovery rate)<0.05; logFC (log2 fold change)>1.5]. in FW-acclimated bull sharks compared with values in 30% (Fig. 3A). In the magnified images, NKAα1 mRNA signals were SW-acclimated houndsharks. localized in the smaller diameter LDT in the sinus zone (filled arrows in Fig. 3B) and in tubules with the largest diameter in the Distribution of mRNA encoding NKAα1, NKCC2, NCC and UT bundle zone (early distal tubule, EDT) (open arrowheads in in the kidney of bull sharks Fig. 3C). Some tubules neighboring renal corpuscles also showed Similar to the kidneys of other marine cartilaginous fishes (Lacy and NKAα1 mRNA signals; these tubules correspond to the transitional Reale, 1985; Hentschel et al., 1998; Hyodo et al., 2004a; Kakumura segments from the EDT to the LDT (open arrow in Fig. 3B) and et al., 2015), the bull shark kidney consists of multiple lobules. Each from the LDT to the collecting tubule (CT) (filled arrowhead in lobule is separated into two morphologically distinguishable Fig. 3B). Weak signals for NKAα1 mRNA were observed in the CT regions, a sinus zone and a bundle zone (Fig. 2A). The sinus zone in the bundle zone (filled arrowhead in Fig. 3C). Therefore, positive occupied a large area in lobules and was filled with blood sinuses. signals for NKAα1 mRNA were continuously observed from the The sinus zone was composed of two types of tubules: the larger EDT to the CT (Fig. 3i). A similar distribution of NKAα1 mRNA diameter tubules with apical brush border correspond to the was observed in the kidneys of FW-acclimated bull sharks. proximal segment II of the second loop (open arrows in Fig. 2B), However, the signals were stronger compared with those of SW while the late distal tubules (LDT) of the fourth loop (filled arrows individuals, especially in the LDT, and were also evident in the in Fig. 2B) were characterized by a relatively smaller diameter and descending limb of the third loop (Fig. 3D–F,ii). Signals for lower epithelial cell height, and were without an apical brush-border NKCC2 mRNA were observed almost exclusively in tubules in membrane. In contrast, in the bundle zone, renal tubules were the bundle zone of both SW- and FW-acclimated bull sharks densely packed, and five tubular segments of a single nephron (the (Fig. 3G–L). In the bundle zone, intense signals of NKCC2 mRNA descending and ascending limbs of the first and the third loops, and were detected in the EDT (open arrowheads in Fig. 3I,L). In the final segment) were enclosed in a peritubular sheath (Fig. 2C,D). contrast, in the sinus zone, only a small number of tubules located in Fig. 3 shows mRNA signals of NKAα1, NKCC2, NCC and UT the vicinity of renal corpuscles were positive for NKCC2 mRNA in the bull shark kidney. In SW individuals, signals for NKAα1 (open arrows in Fig. 3H,K). Detailed observation using serial mRNA were observed in both the bundle and the sinus zones sections revealed that the NKCC2 mRNA-expressing tubules in the

A B SZ D Late distal tubule (LDT) BZ

Renal corpuscle SZ Central vessel (CV)

C BZ BZ

Collecting tubule (CT)

BZ Early distal tubule (EDT) Peritubular sheath

Fig. 2. Representative kidney sections showing the sinus zone (SZ) and the bundle zone (BZ) of the bull shark kidney. Sections were stained with hematoxylin and periodic acid–Schiff (A–C). B and C are magnified views of the SZ and BZ, respectively. Open and filled arrows represent the second loop and the late distal tubule (LDT; fourth loop), respectively. Scale bars=500 µm (A) and 50 µm (B,C). (D) Schematic representation of the bull shark nephron. Journal of Experimental Biology

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Fig. 3. In situ hybridization analyses of mRNA A NKAα1 (SW) B Sinus C Bundle i encoding NKAα1, NKCC2, NCC and UT in the kidneys of bull sharks acclimated to SW and FW. α – – – BZ SZ NKA 1(A F), NKCC2 (G L), NCC (M R) and UT (S–X) mRNAs in SW- (A–C,G–I,M–O,S–U) and FW-acclimated (D–F,J–L,P–R,V–X) bull sharks. B,E,H,K,N,Q,T,W and C,F,I,L,O,R,U,X are magnified views of the SZ and BZ, respectively. The localization of each mRNA in the nephron is D NKAα1 (FW) E F ii schematically illustrated (i–viii). Open arrows, filled arrows and open arrowheads represent the SZ transitional segment from the early distal tubule (EDT) to the LDT, the LDT and the EDT, respectively. Filled arrowheads represent the transition segment from the LDT to the collecting BZ tubule (CT) in the SZ, and the CT in the bundle zone. Note that NCC mRNA was intensely expressed in the LDT and the anterior part of the CT G NKCC2 (SW) H I iii in FW-acclimated bull shark kidney. Sections were counterstained with Nuclear Fast Red. Scale BZ SZ bars=400 µm (A,D,G,J,M,P,S,V) and 50 µm (B,C,E,F,H,I,K,L,N,O,Q,R,T,U,W,X).

J NKCC2 (FW) K L iv

M NCC (SW) N O v

BZ SZ

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S UT(SW) T U vii

BZ SZ

V UT(FW) W X viii SZ

BZ Journal of Experimental Biology

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sinus zone are the transitional segment from the EDT to the LDT A NKAα1 B (Fig. 3iii,iv). No difference was observed in the distribution and signal intensity of NKCC2 mRNA between SW and FW sharks. Signals for NCC mRNA were very weak in SW individuals (Fig. 3M–O). The signals were detected in the sinus zone, but not in the bundle zone. The morphological characteristics of the tubules showing positive signals revealed that these tubules are the LDT (filled arrows in Fig. 3N). Meanwhile, in FW-acclimated individuals, strong signals for NCC mRNA were observed in the LDT (Fig. 3P,Q). In addition, intense signals were also detected in the anterior half of the CT in the bundle zone (filled arrowheads in Fig. 3R, and schematically shown in Fig. 3vi). Signals for UT CDNKCC2 mRNA were detected almost exclusively in the CT in the bundle zone in both SW- and FW-acclimated bull sharks (Fig. 3S,V), and the signal intensity was higher in FW-acclimated bull sharks than in SW individuals (filled arrowheads in Fig. 3U,X). A small number of tubules located in the vicinity of renal corpuscles showed UT mRNA signals in the sinus zone, and these segments correspond to the transitional segment from the LDT to the CT (filled arrowheads in Fig. 3T,W). The intensity of UT mRNA signals in this segment was also higher in FW-acclimated bull sharks (Fig. 3vii,viii). α Co-expression of NKA 1, NKCC2, NCC and UT mRNA was EFNCC examined using the serial sections of FW individuals (Fig. 4). In the sinus zone, NCC mRNA was co-expressed with NKAα1mRNAin the LDT (filled arrows in Fig. 4A,E). The LDT did not express NKCC2 or UT mRNA (Fig. 4C,G). NKCC2 mRNA was moderately expressed in the transitional segment from the EDT to the LDT, where NKAα1 mRNA was also expressed (open arrows in Fig. 4A,C). In the bundle zone, NKCC2 mRNA was co-expressed with NKAα1 mRNA in the EDT (open arrowheads in Fig. 4B,D), while co-expression of NKAα1, NCC and UT mRNA was found in the CT (filled arrowheads in Fig. 4B,F,H). GHUT Distribution of NKAα1, NKCC2, NCC and UT mRNA in houndshark kidneys In the kidneys of houndsharks kept in full-strength SW, intense positive signals for NKAα1 mRNA were observed in the EDT (open arrowheads in Fig. 5C). The LDT (fourth loop of nephron) also showed positive signals for NKAα1 mRNA (filled arrows in Fig. 5B). In the LDT, the transitional segment from the EDT to the LDT showed higher expression (open arrows in Fig. 5A,B), while signals in the remaining part of LDT were weak (Fig. 5i). No difference was observed in the localization and signal intensity of NKAα1 mRNA between SW individuals and 30% SW-acclimated – Fig. 4. Co-localization of mRNA signals in the kidney of bull shark in FW. individuals (Fig 5A F,i,ii). The expression of NKCC2 mRNA was α α – NKA 1 (A,B), NKCC2 (C,D), NCC (E,F) and UT (G,H) mRNA in the SZ similar to that of NKA 1 mRNA (Fig. 5G L). Intense signals for (A,C,E,G) and BZ (B,D,F,H). Filled arrows and open arrows in the sinus zone NKCC2 mRNA were detected in the EDT in the bundle zone (open represent the LDT and the transitional segment from the EDT to the LDT, arrowheads in Fig. 5I,L), while weak or moderate levels of NKCC2 respectively. Filled arrowheads and open arrowheads in the bundle zone mRNA expression were seen in the LDT (open and filled arrows in represent the CT and the EDT, respectively. Sections were counterstained with Fig. 5H,K). No difference was observed in localization and signal Nuclear Fast Red. Scale bar=50 µm. intensity of NKCC2 mRNA between 100% SW and 30% SW groups (Fig 5G–L,iii,iv). transitional segment was decreased by the transfer of houndsharks NCC mRNA was expressed only in a limited region of the fourth from full-strength SW to 30% SW (filled arrowheads in Fig. 5W), loop, which was the transitional segment from the LDT to the CT similar to NCC mRNA signals. (Fig. 5M and filled arrowheads in N, schematically shown in v). In Co-expression of the four mRNAs was examined using the serial 30% SW-acclimated houndsharks, signals for NCC mRNA were sections of SW individuals (Fig. S4). NKCC2 mRNA was much weaker than that in SW individuals (filled arrowheads in consistently expressed with NKAα1 mRNA in the EDT (open Fig. 5Q). UT mRNA was expressed in the CT (Fig. 5S and filled arrowheads in Fig. S4B,D) and the LDT (open arrows in Fig. S4A,C). arrowheads in U). In the sinus zone, intense signals for UT mRNA In the sinus zone, NCC mRNA was co-expressed with UT mRNA in were detected in the transitional segment from the LDT to the CT the transitional segment from the LDT to the CT, where NKAα1

(filled arrowheads in Fig. 5T), and the signal intensity of the mRNA was also expressed (filled arrows in Fig. S4A,E,G). In Journal of Experimental Biology

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In situ A NKAα1 (SW) B Sinus C Bundle i Fig. 5. hybridization analyses of mRNA BZ encoding NKAα1, NKCC2, NCC and UT in the kidneys of houndsharks acclimated to SW and 30% SW. NKAα1(A–F), NKCC2 (G–L), NCC (M–R) and UT (S–X) mRNAs in SW- (A–C, G–I,M–O,S–U) and 30% SW-acclimated (D–F,J– SZ L,P–R,V–X) houndsharks. B,E,H,K,N,Q,T,W and C,F,I,L,O,R,U,X are magnified views of the SZ and BZ, respectively. The localization of each D NKAα1 (30% SW) E F ii mRNA in the nephron is schematically illustrated (i–viii). Open arrows, filled arrows and open arrowheads represent the transitional segment from the EDT to the LDT, the LDT and the EDT, respectively. Filled arrowheads represent the SZ BZ transitional segment from the LDT to the CT in the SZ, and the CT in the BZ. Sections were counterstained with Nuclear Fast Red. Scale bars=400 µm (A,D,G,J,M,P,S,V) and 50 µm (B,C, G NKCC2 (SW) H I iii E,F,H,I,K,L,N,O,Q,R,T,U,W,X). BZ

J NKCC2 (30% SW) K L iv

SZ BZ

MNCC (SW) N O v

P NCC (30% SW) Q R vi

S UT (SW) T U vii BZ

V UT (30% SW) W X viii

BZ Journal of Experimental Biology

9 RESEARCH ARTICLE Journal of Experimental Biology (2019) 222, jeb201780. doi:10.1242/jeb.201780 contrast to the results in bull sharks, only UT mRNA expression was A B found in the CT (filled arrowheads in Fig. S4B,D,F,H).

Localization of NCC protein in the LDT cells of bull sharks The intracellular localization of bull shark NCC was examined using the antiserum raised against a synthetic polypeptide of bull shark NCC. Consistent with the results of in situ hybridization, immunoreactive signals for NCC were observed in the LDT of the nephron in FW individuals (open arrows in Fig. 6A), but not in SW FW Preimmune individuals (Fig. 6D). Immunoreaction for NCC was localized to the C D apical membrane. The use of preimmune serum (Fig. 6B) and preabsorption of the anti-NCC serum with the synthetic NCC polypeptide (Fig. 6C) resulted in the disappearance of the NCC immunoreactive signals in the LDT. Double-labeling fluorescence immunohistochemistry showed co-localization of apically located NCC and basolaterally located NKA in the tubular cells of the LDT (Fig. 6F). Preabsorption SW DISCUSSION E F It is well recognized that most cartilaginous fishes are principally marine species, and that the bull shark is a rare euryhaline species with the capacity to inhabit both SW and FW environments. To further our understanding of the underlying mechanisms of euryhalinity in bull sharks, we focused on kidney function in the present study, as the kidney is the only organ by which excess water in the body can be excreted in a hypo-osmotic environment. By In situ means of RNA-seq analysis and the subsequent molecular and hybridization NKA and NCC in FW histochemical analyses, we found that NCC expressed in the LDT Fig. 6. Immunohistochemistry of NCC in the kidney of FW bull shark. and CT is one of the key molecules contributing to the successful Signals were localized on the apical membrane of the LDT (A, open arrows). The FW acclimation of the bull shark. use of preimmune serum (B) and pre-absorbed antiserum (C) resulted in the disappearance of the immunoreactive signals. Signals were not observed Reabsorption of NaCl from the glomerular filtrate in the kidney of SW individual (D). (E) In situ hybridization of NCC mRNA in the kidney of FW bull shark showing the co-localization of immunoreactive signals In the SW environment, bull sharks maintain their plasma slightly (A) and mRNA signals (E) in adjacent sections. Sections were counterstained hyperosmotic to surrounding SW by accumulating urea in the body, with hematoxylin (A–D) or Nuclear Fast Red (E). (F) Double-labeling which is similar to other marine cartilaginous fishes (see also Pillans fluorescence immunohistochemistry with anti-NCC antibody (magenta; and Franklin, 2004). Transfer to a low-salinity environment resulted indicated by arrows) and anti-NKA antibody (green). Scale bars=20 µm. in a decrease in plasma ion and urea levels. However, the FW- acclimated bull sharks still maintained high plasma Na+ (213.7± compared with Atlantic stingrays transferred to 50% SW. In other 9.0 mmol l−1) and Cl− (177.0±3.5 mmol l−1) concentrations, and words, bull sharks in the FW environment likely filter more plasma the resulting plasma osmolality was 617.7±13.0 mOsm, as has been and excrete more urine compared with stingrays. The calculated previously reported for this species (Pillans and Franklin, 2004; values in Table 2 indicate a 1.5 times increase in NaCl reabsorption, Pillans et al., 2006) and for other euryhaline cartilaginous fishes which is lower than the value in stingrays acutely transferred to 50% including the Atlantic stingray, Hypanus sabina (Piermarini and SW (2.5 times increase) (Janech et al., 2006), but a greater increase in Evans, 1998). These osmolality data indicate that bull sharks NaCl reabsorption is assumed in FW-acclimated bull sharks. exposed to severe hypo-osmotic conditions must be excreting large Therefore, we searched for upregulated mRNAs encoding solute amounts of diluting urine to compensate for the osmotic influx of carrier superfamily proteins involved in NaCl reabsorption. water from the environment. In accordance with this idea, a concomitant decrease in urine osmolality and NaCl concentrations NCC in the LDT: a key molecule contributing to euryhalinity was observed. In the present study, however, due to technical of the bull shark limitations, we could not measure the GFR and UFR of SW- and We previously identified the expression of NKAα1 and NKCC2 in FW-acclimated bull sharks, and thus could not calculate exact the kidney of another cartilaginous fish, Callorhinchus milli values for water excretion and NaCl reabsorption in the kidney after (Kakumura et al., 2015). NKCC2 (Slc12a1) is well known to be FW transfer. We therefore used the values for GFR and UFR that localized in the thick ascending limb of mammalian Henle’sloop were reported in Atlantic stingrays transferred from ambient SW to together with NKA, where NaCl is actively reabsorbed for dilution of 50%-diluted SW (Janech et al., 2006) to calculate the hypothetical primary urine (Fenton and Knepper, 2007). In the elephant fish absolute amount of reabsorbed NaCl in the bull shark kidney kidney, NKAα1 and NKCC2 were colocalized in the EDT and the (Table 2). The estimated values in Table 2 indicate that the transfer posterior half of the LDT (Kakumura et al., 2015), and the NaCl of bull sharks from SW to FW caused an approximately 50% uptake by NKCC2 is considered to be important for the urea increase in reabsorption of NaCl from the glomerular filtrate. reabsorption process (Hyodo et al., 2014). We thus initially focused It should be noted that, because of the larger osmotic difference on NKCC2 as a candidate molecule important for NaCl reabsorption between internal and environmental osmolalities, bull sharks in FW-acclimated bull sharks. However, no significant change was acclimated in FW are considered to receive a greater influx of water observed in the distribution and expression levels of NKCC2 mRNA Journal of Experimental Biology

10 RESEARCH ARTICLE Journal of Experimental Biology (2019) 222, jeb201780. doi:10.1242/jeb.201780 following the transfer of bull sharks to a FW environment. Therefore, NaCl reabsorption in the LDT is critical for the euryhalinity of this we performed RNA-seq analysis to search for other candidate genes species. contributing to NaCl reabsorption, and upregulated by FW acclimation. NCC in the collecting tubule: NaCl reabsorption and/or urea In the RNA-seq analysis, a remarkable increase was detected in the reabsorption? expression of NCC mRNA. Consistent with the results of qPCR, only In addition to the LDT, upregulation of NCC mRNA expression was a modest level of NCC mRNA signal was found in the LDT of bull also found in the CT. Expression of NKAα1 mRNA was also shark kidney in SW, whereas the intensity of hybridization signals for observed, implying that NaCl is reabsorbed in the CT of FW- NCC mRNA considerably increased in the LDT of FW-acclimated acclimated bull sharks. In contrast, neither NKAα1 nor NCC bull shark. NCC is known to be expressed in the apical membrane of mRNA signals were detected in the CT of houndshark kidney. the mammalian distal convoluted tubule (Fenton and Knepper, 2007) Therefore, NaCl reabsorption in the CT is another feature of the and in the distal segment of the teleost fish kidney (Kato et al., 2011; euryhaline bull shark kidney. The expansion of tubular segments Teranishi et al., 2013). Localization on the apical membrane was contributing to NaCl reabsorption, in comparison to the kidneys of confirmed also in the LDT of bull sharks. In the LDT, NCC is co- SW-acclimated bull sharks and houndsharks in SW and 30% SW, is expressed with NKAα1, which is well recognized as the generator for most likely important for highly effective NaCl retention in FW the Na+ electrochemical gradient that is the driving force for NaCl environments (Fig. 7). reabsorption via NCC (Fenton and Knepper, 2007). The upregulation Alternatively, NaCl reabsorption in the CT may contribute to in the NKAα1 mRNA signal was also observed in the LDT of FW- another important function, that is, urea retention. Based on the acclimated bull sharks. These results strongly suggest that the LDT exclusive localization of UT, the CT has been implicated as the urea- contributes significantly to NaCl retention in the FW environment, reabsorbing segment in elephant fish and houndsharks (Hyodo and that NCC is a key molecule for NaCl reabsorption in the LDT et al., 2004a, 2014; Yamaguchi et al., 2009; Kakumura et al., 2015). (Fig. 7). The limited expression of UT in the CT has also been demonstrated In cartilaginous fish, we initially identified NCC mRNA in the bull shark kidney, implying that urea reabsorption is a expression in the gills and kidney of the Japanese houndshark, common function of the CT in both marine and euryhaline T. scyllium (Takabe et al., 2016). The present qPCR analysis cartilaginous fishes. We have proposed a model for urea revealed that, in the kidney of houndsharks, expression levels of reabsorption in the tubular bundle, in which the first and the third NCC mRNA were considerably lower compared with those in the loops and the CT are wrapped with an impermeable peritubular kidney of FW-acclimated bull sharks, and no significant change was sheath (Hyodo et al., 2014). The first step of this model is massive observed in NCC mRNA levels between SW- and 30% SW- reabsorption of NaCl. The resulting increase in osmolality causes acclimated houndsharks. In situ hybridization further demonstrated reabsorption of water, which produces a low urea environment in the that NCC mRNA was expressed only in the limited region of the tubular bundle. Urea is then reabsorbed from the CT via UT using LDT in the houndshark kidney. In contrast to the kidney, NCC the concentration gradient of urea as a driving force (Hyodo et al., mRNA levels and the number of NCC-expressing cells in the gill 2014). In this model, NKA and NKCC2 expressed in the third loop increased in houndsharks after the transfer from SW to 30% SW (the EDT) contribute to the NaCl reabsorption. Expression of (Takabe et al., 2016). In houndsharks, NCC is more likely to be NKAα1 and NCC mRNA in the CT of FW-acclimated bull shark important in the gills for absorption of NaCl from the environment. most probably enhances NaCl uptake in the tubular bundle, which in Although the timelines of transfer experiments were not exactly the turn enhances water uptake, and finally urea reabsorption. When we same between bull sharks and houndsharks, our results revealed calculated the amount of reabsorbed urea in bull sharks using the that the significant upregulation of NCC mRNA expression in GFR and UFR data of the Atlantic stingray, the amount in the FW- the LDT was unique to the bull shark, further implying that the acclimated bull shark kidney was approximately two-thirds that of bull sharks in SW (Table 2). However, again, it would be reasonable ABα to consider that bull sharks in FW have greater GFR and UFR values SW FW NKA 1 + NCC compared with Atlantic stingrays in 50% SW, in order to excrete excess water in the body. If this is the case, bull sharks in FW NaCl environments reabsorb more urea than in the SW environment in order to maintain plasma urea levels (Fig. 7). Currently, the function of the LDT in SW environment remains to be clarified. In elephant fish and houndsharks, NKCC2 mRNA is NaCl expressed in the LDT (present study and Kakumura et al., 2015), Urea and the LDT is considered to be important for concentrating urea for Urea subsequent urea reabsorption in the CT (Hyodo et al., 2014). NKAα1 NKAα1 + UT + UT However, in the bull shark kidney in a SW environment, neither NKCC2 nor NCC was observed in the LDT (Fig. 7). Further studies NaCl NaCl are needed to understand the function of LDT in the kidney of bull shark in the SW environment by identifying transporters that are NKAα1 + NKCC2 NKAα1 + NKCC2 expressed in the LDT. In summary, we show for the first time that NCC expressed in the Fig. 7. Schematic diagram showing changes in kidney function between LDT and CT of the kidney is a key molecule for NaCl retention SW- and FW-acclimated bull sharks. (A) SW; (B) FW. Note that reabsorption required for euryhalinity of bull sharks. Our comprehensive of NaCl is enhanced in FW-acclimated bull shark kidney by the upregulated expression of NCC and NKAα1 in the LDT and the anterior part of the CT approach with RNA-seq effectively pinpointed several candidate (shown in black). The enhanced reabsorption of NaCl in the CT may contribute genes, of which we focused on NCC. The remaining genes with to urea retention. significant changes in expression profiles will be characterized in Journal of Experimental Biology

11 RESEARCH ARTICLE Journal of Experimental Biology (2019) 222, jeb201780. doi:10.1242/jeb.201780 further studies to ascertain their role in renal function in the Evans, D. H. (1993). Osmotic and ionic regulation. In The Physiology of Fishes (ed. euryhaline bull shark. In addition, other organs including the gill D. H. Evans), pp. 315-341. Boca Raton: CRC Press. Fenton, R. A. and Knepper, M. A. (2007). Mouse models and the urinary (Reilly et al., 2011), rectal gland (Pillans et al., 2005) and liver concentrating mechanism in the new millennium. Physiol. Rev. 87, 1083-1112. (Anderson et al., 2005) are thought to be involved in euryhaline doi:10.1152/physrev.00053.2006 mechanisms of bull sharks; RNA-seq analysis of bull shark gills is Fu, L., Niu, B., Zhu, Z., Wu, S. and Li, W. (2012). CD-HIT: accelerated for clustering the next-generation sequencing data. Bioinformatics. 28, 3150-3152. doi:10. now underway. Comprehensive studies on these osmoregulatory 1093/bioinformatics/bts565 organs will uncover a more complete picture of the mechanisms for Grabherr, M. G., Haas, B. J., Yassour, M., Levin, J. Z., Thompson, D. A., Amit, I., euryhalinity in this unique elasmobranch, the bull shark. Adiconis, X., Fan, L., Raychowdhury, R., Zeng, Q. et al. (2011). Full-length transcriptome assembly from RNA-seq data without a reference genome. Nat. Acknowledgements Biotechnol. 29, 644-652. doi:10.1038/nbt.1883 Haas, B. J., Papanicolaou, A., Yassour, M., Grabherr, M., Blood, P. D., Bowden, We thank Drs Y. Yamaguchi, K. Hasegawa, M. Inokuchi and K. Sato for their J., Couger, M. B., Eccles, D., Li, B. and Lieber, M. et al. (2013). De novo invaluable support and discussion, the rest of the Laboratory for Phyloinformatics, transcript sequence reconstruction from RNA-seq using the Trinity platform for RIKEN BDR, for their support in RNA-seq data production, Mr. Nomura and reference generation and analysis. Nat. Protoc. 8, 1494-1512. doi:10.1038/nprot. Ms. Y. Nakamatsu for their support on the transfer experiment of bull shark, and Dr 2013.084 A. Kato of the Tokyo Institute of Technology for the use of a virtual slide system. We Hara, Y., Tatsumi, K., Yoshida, M., Kajikawa, E., Kiyonari, H. and Kuraku, S. also thank Prof C. A. Loretz of State University of New York at Buffalo and Prof. J. A. (2015). Optimizing and benchmarking de novo transcriptome sequencing: from Donald of Deakin University for critical comments on the manuscript. The anti-NKA library preparation to assembly evaluation. BMC Genomics. 16, 977. doi:10.1186/ antiserum was provided courtesy of Prof T. Kaneko of the University of Tokyo. s12864-015-2007-1 Hasegawa, K., Kato, A., Watanabe, T., Takagi, W., Romero, M. F., Bell, J. D., Competing interests Toop, T., Donald, J. A. and Hyodo, S. (2016). Sulfate transporters involved in The authors declare no competing or financial interests. sulfate secretion in the kidney are localized in the renal proximal tubule II of the elephant fish (Callorhinchus milii). Am. J. Physiol. Regul. Integr. Comp. Physiol. Author contributions 311, 66-78. doi:10.1152/ajpregu.00477.2015 Conceptualization: S.H.; Methodology: I.I., S.H.; Validation: I.I., S.H.; Formal Hazon, N., Wells, A., Pillans, R. D., Good, J. P., Anderson, W. G. and Franklin, analysis: Y. Hara, Y. Honda, S.K.; Investigation: I.I., M.W., Y. Hara, T.W., S.T., K.K., C. E. (2003). Urea based osmoregulation and endocrine control in elasmobranch fish with special reference to euryhalinity. Comp. Biochem. Physiol. B 136, Y. Honda, K.U., K.M., R.M., Y.M., M.N., W.T., S.K., S.H.; Resources: K.U., K.M., 685-700. doi:10.1016/S1096-4959(03)00280-X R.M., Y.M., M.N.; Writing - original draft: I.I., S.H.; Writing - review & editing: I.I., M.W., Hediger, M. A., Romero, M. F., Peng, J.-B., Rolfs, A., Takanaga, H. and Bruford, Y. Hara, W.T., S.K., S.H.; Funding acquisition: I.I., S.H. E. A. (2004). The ABCs of solute carriers: physiological, pathological and therapeutic implications of human membrane transport proteins. Pflugers Arch. Funding Eur. J. Physiol. 447, 465-468. doi:10.1007/s00424-003-1192-y This study was supported by a Grant-in-Aid for Scientific Research from the Japan Hentschel, H., Storb, U., Teckhaus, L. and Elger, M. (1998). The central vessel of Society for the Promotion of Science (JSPS KAKENHI 26650110, 26291065, the renal countercurrent bundles of two marine elasmobranchs – dogfish 17H03868) and a Research Grant from the Okinawa Churashima Foundation to (Scyliorhinus caniculus) and skate (Raja erinacea) – as revealed by light and S.H., and a Grant-in-Aid for JSPS Fellows (16J07895) to I.I. I.I. is supported by JSPS electron microscopy with computer-assisted reconstruction. Anat. Embryol. 198, Research Fellowships for Young Scientists. 73-89. doi:10.1007/s004290050166 Hyodo, S., Kato, F., Kaneko, T. and Takei, Y. (2004a). A facilitative urea transporter Data availability is localized in the renal collecting tubule of the dogfish Triakis scyllia. J. Exp. Biol. The short-read data of RNA-seq analysis are available from the DNA Data Bank of 207, 347-356. doi:10.1242/jeb.00773 Japan (DDBJ): DRX162067, DRX162068, DRX162069 and DRX162070. The Hyodo, S., Tsukada, T. and Takei, Y. (2004b). Neurohypophysial hormones of partial sequences of bull shark NKAα1, NKCC2, UT and EF1α1 and houndshark dogfish, Triakis scyllium: structures and salinity-dependent secretion. Gen. Comp. Endocrinol. 138, 97-104. doi:10.1016/j.ygcen.2004.05.009 EF1α1 are also available from the DDBJ: LC462277, LC462278, LC462279, Hyodo, S., Kakumura, K., Takagi, W., Hasegawa, K. and Yamaguchi, Y. (2014). LC462280 and LC462281, respectively. 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