ABSTRACT Regulation of water permeability in the collecting duct is important for osmoregulatory acclimation in teleost fish. In hyperosmotic environments such as seawater (SW), the teleost kidney functions as a site of divalent ion excretion. The collecting ducts reabsorb Na + , Cl − , and water, thereby reducing urine volume and producing small amounts of isotonic urine with high concentrations of divalent ions. In hypoosmotic environments such as freshwater (FW) or low-salinity brackish water (BW), the kidney produces large volumes of hypotonic urine and serves as a site of water excretion; under these conditions, the collecting ducts reabsorb Na + and Cl − but not water. To identify aquaporins (Aqps) involved in regulating water permeability in the collecting ducts of teleosts, we analyzed renal Aqp expression in a euryhaline marine fish, the Japanese pufferfish ( Takifugu rubripes ), which possesses 16 Aqp genes in its genome, seven of which (Aqp1aa, 1ab, 3a, 4a, 7, 8bb, and 11a) are expressed in the kidney. Quantitative RT-PCR analysis showed that Aqp1aa and Aqp4a were highly expressed in collecting duct tissues, and that Aqp1aa expression was markedly reduced in fish acclimated to BW. Immunohistochemistry revealed apical localization of Aqp1aa and basolateral localization of Aqp4 in collecting duct cells, with apical Aqp1aa downregulated in BW. These results suggest that Aqp1aa and Aqp4 mediate water reabsorption in SW and that downregulation of Aqp1aa contributes to hypotonic urine production in BW. NEW & NOTEWORTHY Regulation of water permeability in the collecting duct is important for osmoregulation in teleost fish. Expression analyses of aquaporins (Aqps) in the marine pufferfish Takifugu rubripes showed that Aqp1aa and Aqp4a are highly expressed in the collecting duct and localized to the apical and basolateral membranes, respectively. Renal Aqp1aa expression was markedly reduced in fish acclimated to hypoosmotic brackish water. These results indicate that collecting duct water permeability is regulated by Aqp1aa expression.

Slc12a10 is a homolog of the Na+-Cl- cotransporter Ncc (Slc12a3) and is also referred to as Nccβ or Ncc2. The Slc12a10 gene is widely distributed among bony vertebrates; however, it has been lost or pseudogenized in birds, most mammals including humans, and some teleost fishes. Slc12a10 is expressed in the intestinal epithelium of anguillid eels and in ionocytes of the zebrafish gill, where it is thought to contribute to epithelial NaCl transport. Despite these observations, only a limited number of studies have directly evaluated the transport activity of Slc12a10, and its ion-coupling properties and electrical characteristics remain incompletely defined. To address this gap, we examined the transport activity of Slc12a10 from European and Japanese eels (Anguilla anguilla and Anguilla japonica) using a Xenopus laevis oocyte expression system combined with ion-selective microelectrode measurements. When oocytes expressing eel Slc12a10 were exposed to Na+-free solution, intracellular Na+ and Cl- activities decreased significantly. Similar decreases in intracellular Na+ and Cl- activities were observed upon exposure to Cl--free solution. These changes were not observed in control oocytes and were not accompanied by significant changes in membrane potential. Furthermore, under voltage-clamp conditions at -20 mV, exposure of Slc12a10-expressing oocytes to Na+-free solution induced a significant decrease in intracellular Na+ activity without generating detectable membrane currents. These activities were comparable to those previously reported for zebrafish Slc12a10.1. Together, these results provide new evidence that eel Slc12a10 functions as an electroneutral Na+-Cl- cotransporter and suggest that this transport mechanism is conserved across fish species.

The aquaporin genes (aqps) are widely distributed among vertebrates. In cartilaginous fish, aqp3 and aqp10 are duplicated, whereas many species have lost aqp8. While the solute permeability of aquaglyceroporins and Aqp8 exhibits significant diversity, systematic evaluations of their activity within species are limited. Consequently, we conducted a comprehensive analysis of the aqps in various cartilaginous fish genome databases. Our analysis revealed that the sharpnose sevengill shark (Heptranchias perlo) and North Pacific spiny dogfish (Squalus suckleyi) possess all aqp genes found in cartilaginous fish, rendering them suitable for comparative analysis of Aqp activity. The activity of all aquaglyceroporins and Aqp8 in the sharpnose sevengill shark was analyzed using Xenopus oocyte swelling assays. The water permeability of Aqp3c2, Aqp8, Aqp9, Aqp10c1, and Aqp10c2; the glycerol permeability of Aqp3c2, Aqp9, and Aqp10c2; the urea permeability of Aqp3c2, Aqp8, Aqp9, and Aqp10c2; and the boric acid permeability of Aqp8 and Aqp9 were determined. The urea and boric acid permeabilities of Aqp9 were higher than those of other Aqps. Our results provide genomic evidence for the loss of aqp7 and further elucidate the diversification of aqp8 in cartilaginous fishes. Moreover, the urea permeability of Aqp9 suggests a functional role in their unique urea-based osmoregulation strategy.

Aquaporin (Aqp)-10 is an aquaglyceroporin permeable to both water and uncharged small-molecule compounds. In ray-finned fish Aqp10 paralogs, urea and boric acid permeabilities of Aqp10.2-but not its glycerol permeability-are much weaker than those of Aqp10.1 and plesiomorphic Aqp10; however, the molecular mechanisms responsible for urea and boric acid permeabilities remain unclear. In this study, we constructed structural models of these sequences and found that two aromatic amino acid residues at positions 1 and 3 of the four amino acid sites in the aromatic/arginine (ar/R) selectivity filter were important in reducing urea and boric acid permeabilities, but not glycerol permeability. Moreover, the characteristics of these amino acid residues could be quantified by calculating the sum of molecular weights of the two amino acid residues. Site-directed mutagenesis revealed that replacement of one of the two aromatic amino acid residues at positions 1 and 3 in the ar/R region with a small amino acid residue enhanced the urea and boric acid permeabilities of Aqp10. In the examined Aqp10s, sum of the molecular weights of amino acid residues at positions 1 and 3 in the ar/R selectivity filter was inversely correlated with the pore diameter and urea and boric acid permeabilities. Overall, our results indicate that the two bulky amino acid residues in the ar/R selectivity filter contribute to the formation of a filter that influences the urea and boric acid permeabilities of aquaglyceroporins.NEW & NOTEWORTHY Urea and boric acid permeabilities of aquaporin (Aqp)-10.2 are lower than those of Aqp10.1 and plesiomorphic Aqp10, and the molecular weight sum of the two amino acid residues in the aromatic/arginine (ar/R) selectivity filter plays a filtering role that affects permeability. Therefore, urea and boric acid permeabilities of Aqp10s can be assessed using the sum of the molecular weights of the two amino acids in the ar/R region, which represents a significant advancement in this field.

Aquaporin (Aqp) 10 is a member of the aquaglyceriporin family, which transports small, uncharged solutes in addition to water. Although the solute selectivity of aquaglyceroporins varies, the mechanism of solute selectivity has not yet been fully elucidated. The common ancestor of ray-finned fish possessed two paralogous genes for aquaporin 10, aqp10.1 and aqp10.2, which produce Aqps with different solute selectivities. Most teleosts possess one or more ohnologs derived from aqp10.1 and aqp10.2; however, the common ancestor of Anguilliformes species lost all aqp10.1-derived ohnologs. Anguilliformes species, except Anguilla species, have one aqp10.2b, but recent tandem duplications in the European eel have generated three aqp10.2b paralogs (aqp10.2b1-aqp10.2b3), whose activities remain ambiguous. In this study, we found that the four sites forming the aromatic/arginine (ar/R) selectivity filter in European eel Aqp10.2b1 were identical to those in Aqp10.2b of other species. However, the Y residue at position 3 was replaced with G in the ar/R selectivity filter of Aqp10.2b2 and b3. When expressed in Xenopus oocytes, Aqp10.2b2 and b3 showed higher permeability to urea and boric acid than Aqp10.2b1, indicating that Aqp10.2b2 and b3 acquired broad solute selectivity similar to that of Aqp10.1, which was lost in the ancestral Anguilliformes species. Urea and boric acid permeabilities of Aqp10.2b1 increased when the Y residue at position 3 of the ar/R selectivity filter was replaced with G. Overall, our results outline the history of the loss and gain of Aqp10 paralogs with broad solute selectivity in anguillid eels.

Intron gain and loss are rare events in vertebrates; however, comparative genome analysis of elephant sharks, tetrapods, and teleosts revealed a higher level of intron turnover in teleosts. slc26a1 and slc26a2 are members of the anion-exchanger gene family. Human, zebrafish, and Japanese pufferfish slc26a1 consist of two, two, and seven exons, respectively, and slc26a2, two, three, and four exons, respectively. To better understand intron turnover in teleosts, we analyzed the exon-intron organization of slc26a1 and slc26a2 in 81 vertebrates, including 62 ray-finned fish. In most Eurypterygii, which comprise the majority of the Neoteleostei and include Acanthomorpha, Aulopiformes, and Myctophiformes, slc26a1 and slc26a2 have seven and four exons, respectively, whereas those of most other ray-finned fishes consist of two and three exons, respectively, suggesting that intron gain occurred in both slc26a1 and slc26a2 of the Eurypterygii ancestor. In addition, notothenioid slc26a2 has six exons, suggesting that two introns were inserted into the notothenioid ancestor. The two newly acquired introns in the notothenioid consist of transposon-like sequences, suggesting that they were generated via transposon insertion. The positions of some of the newly acquired introns of slc26a1 and slc26a2 in Eurypterygii are identical or very close to those of other slc26 members. These results demonstrate the lineage-specific intron gains of slc26a1 and slc26a2 in ray-finned fish and convergence at the insertion sites of some of the newly acquired introns.

Vertebrates exhibit diversity in the presence and number of aquaporin (Aqp)-10 genes. In Rodentia, mice possess an Aqp10 pseudogene, whereas guinea pigs possess an intact Aqp10. However, Aqp10 retention and pseudogenization history in various rodent lineages remains unclear. Therefore, in this study, we aimed to investigate the molecular evolution of Aqp10 using the recent increasingly decoded rodent genome sequences. We analyzed Aqp10 in the genomes of 43 rodent species belonging to 14 families and found that Aqp10 was pseudogenized in 13 species of three families in the Myomorpha suborder. In contrast, a single intact Aqp10 was retained in the other 30 rodent species, with no Aqp10 pseudogene found in the Castorimorpha, Hystricomorpha, and Sciuromorpha suborders. Additionally, we investigated the tissue expression levels of aquaglyceroporin genes in guinea pigs and rats via reverse transcription-polymerase chain reaction and detected Aqp10 expression in the guinea pig intestines. Notably, none of the examined rat organs expressed Aqp10; however, Aqp7 was expressed in the rat intestines. In situ hybridization showed that guinea pig Aqp10 was expressed in the intestinal epithelial cells. Moreover, AQP10 was permeable to water, glycerol, urea, and boric acid in Xenopus oocytes. Overall, these results clarify the Aqp10 pseudogenization history in Rodentia and suggest guinea pigs as excellent small animal models to analyze the intestinal AQP10 functions.

Aquaglyceroporins are water channels that are permeable to uncharged, low-molecular-weight compounds such as glycerol. Aquaglyceroporins are conserved across many species. However, our knowledge of aquaglyceroporins in cartilaginous fish is limited, particularly regarding the functional differences in cartilaginous fish-specific paralogs. We analyzed the evolutionary relationship between Aqp10 paralogs in two elasmobranchs and one holocephalan and compared their solute permeabilities. Molecular phylogenetic and synteny analyses confirmed that the elasmobranch Aqp10 paralogs, Aqp10C1 and Aqp10C2, arose from a cartilaginous-fish-specific tandem gene duplication. The holocephalan lacks Aqp10C1 and possesses two paralogs (Aqp10C2a and Aqp10C2b) probably derived from Aqp10C2. Swelling assays showed that Aqp10C2 maintained glycerol permeability when expressed in Xenopus oocytes. However, the glycerol permeability of Aqp10C1s were lower than those of other paralogs and the activity was similar to those of water-specific aquaporins. Aqp10 function is highly differentiated in elasmobranchs, and Aqp10C1 may have lost its glycerol permeability during a unique evolution through tandem gene duplication and sub/neofunctionalization.

Cellular and organism survival depends upon the regulation of pH, which is regulated by highly specialized cell membrane transporters, the solute carriers (SLC) (For a comprehensive list of the solute carrier family members, see: https://www.bioparadigms.org/slc/ ). The SLC4 family of bicarbonate (HCO3−) transporters consists of ten members, sorted by their coupling to either sodium (NBCe1, NBCe2, NBCn1, NBCn2, NDCBE), chloride (AE1, AE2, AE3), or borate (BTR1). The ionic coupling of SLC4A9 (AE4) remains controversial. These SLC4 bicarbonate transporters may be controlled by cellular ionic gradients, cellular membrane voltage, and signaling molecules to maintain critical cellular and systemic pH (acid–base) balance. There are profound consequences when blood pH deviates even a small amount outside the normal range (7.35–7.45). Chiefly, Na+-coupled bicarbonate transporters (NCBT) control intracellular pH in nearly every living cell, maintaining the biological pH required for life. Additionally, NCBTs have important roles to regulate cell volume and maintain salt balance as well as absorption and secretion of acid–base equivalents. Due to their varied tissue expression, NCBTs have roles in pathophysiology, which become apparent in physiologic responses when their expression is reduced or genetically deleted. Variations in physiological pH are seen in a wide variety of conditions, from canonically acid–base related conditions to pathologies not necessarily associated with acid–base dysfunction such as cancer, glaucoma, or various neurological diseases. The membranous location of the SLC4 transporters as well as recent advances in discovering their structural biology makes them accessible and attractive as a druggable target in a disease context. The role of sodium-coupled bicarbonate transporters in such a large array of conditions illustrates the potential of treating a wide range of disease states by modifying function of these transporters, whether that be through inhibition or enhancement.

The regressive evolution of independent lineages often results in convergent phenotypes. Several teleost groups display secondary loss of the stomach, and four gastric genes, atp4a, atp4b, pgc, and pga2 have been co-deleted in agastric (stomachless) fish. Analyses of genotypic convergence among agastric fishes showed that four genes, slc26a9, kcne2, cldn18a, and vsig1, were co-deleted or pseudogenized in most agastric fishes of the four major groups. kcne2 and vsig1 were also deleted or pseudogenized in the agastric monotreme echidna and platypus, respectively. In the stomachs of sticklebacks, these genes are expressed in gastric gland cells or surface epithelial cells. An ohnolog of cldn18 was retained in some agastric teleosts but exhibited an increased non-synonymous substitution when compared with gastric species. These results revealed novel convergent gene losses at multiple loci among the four major groups of agastric fish, as well as a single gene loss in the echidna and platypus. Several teleost groups display secondary loss of the stomach. Analyses of genotypic convergence among agastric fishes showed that four genes, slc26a9, kcne2, cldn18a, and vsig1, are co-deleted or pseudogenized in most agastric fishes.

Na+/Cl- cotransporter 2 (Ncc2 or Slc12a10) is a membrane transport protein that belongs to the electroneutral cation-chloride cotransporter family. The Slc12a10 gene (slc12a10) is widely present in bony vertebrates but is deleted or pseudogenized in birds, some bony fishes, and most mammals. Slc12a10 is highly homologous to Ncc (Slc12a3 or Ncc1); however, there are only a few reports measuring the activity of Slc12a10. In this study, we focused on zebrafish Slc12a10.1 (zSlc12a10.1) and analyzed its activity using Xenopus oocyte electrophysiology. Analysis using Na+-selective microelectrodes showed that intracellular sodium activity (aNai) in zSlc12a10.1 oocytes was significantly decreased in Na+- or Cl--free medium and recovered when Na+ or Cl- was readded to the medium. Similar analysis using a Cl--selective microelectrode showed that intracellular chloride activity (aCli) in zSlc12a10.1 oocytes significantly decreased in Na+- or Cl--free medium and recovered when Na+ or Cl- was readded to the medium. When a similar experiment was performed with a voltage clamp, the membrane current did not change when aNai of zSlc12a10.1 oocytes was decreased in Na+-free medium. Molecular phylogenetic and synteny analyses suggest that gene duplication between slc12a10.2 and slc12a10.3 in zebrafish is a relatively recent event, whereas gene duplication between slc12a10.1 and the ancestral gene of slc12a10.2/slc12a10.3 occurred at least about 2 million years ago. slc12a10 deficiency was observed in species belonging to Ictaluridae, Salmoniformes, Osmeriformes, Batrachoididae, Syngnathiformes, Gobiesociformes, Labriformes, and Tetraodontiformes. These results indicate that zebrafish Slc12a10.1 is an electroneutral Na+/Cl-cotransporter and establish its evolutionary position among various teleost slc12a10 paralogs.NEW & NOTEWORTHY Na+/Cl- cotransporter 2 (Slc12a10; Ncc2) is a protein highly homologous to Ncc (Slc12a3; Ncc1); however, there are only a few reports measuring the activity of Slc12a10. Electrophysiological analysis of Xenopus oocytes expressing zebrafish Slc12a10.1 showed that Slc12a10.1 acts as an electroneutral Na+/Cl-cotransporter. This is the third report on the activity of Slc12a10, following previous reports on Slc12a10 in eels.

Aquaporin (Aqp) 10 is a member of the aquaglyceroporin subfamily of water channels, and human Aqp10 is permeable to solutes such as glycerol, urea, and boric acid. Tetrapods have a single aqp10 gene, whereas ray-finned fishes have paralogs of this gene through tandem duplication, whole-genome duplication, and subsequent deletion. A previous study on Aqps in the Japanese pufferfish Takifugu rubripes showed that one pufferfish paralog, Aqp10.2b, was permeable to water and glycerol, but not to urea and boric acid. To understand the functional differences of Aqp10s between humans and pufferfish from an evolutionary perspective, we analyzed Aqp10s from an amphibian (Xenopus laevis) and a lobe-finned fish (Protopterus annectens) and Aqp10.1 and Aqp10.2 from several ray-finned fishes (Polypterus senegalus, Lepisosteus oculatus, Danio rerio, and Clupea pallasii). The expression of tetrapod and lobe-finned fish Aqp10s and Aqp10.1-derived Aqps in ray-finned fishes in Xenopus oocytes increased the membrane permeabilities to water, glycerol, urea, and boric acid. In contrast, Aqp10.2-derived Aqps in ray-finned fishes increased water and glycerol permeabilities, whereas those of urea and boric acid were much weaker than those of Aqp10.1-derived Aqps. These results indicate that water, glycerol, urea, and boric acid permeabilities are plesiomorphic activities of Aqp10s and that the ray-finned fish-specific Aqp10.2 paralogs have secondarily reduced or lost urea and boric acid permeability.

Aqp10 is an aquaglyceroporin that transports not only water but also uncharged low-molecular-weight compounds. We previously demonstrated the evolution of solute permeability in Aqp10 paralogs and showed that the urea and boric acid permeabilities of Aqp10.2 were much weaker than those of Aqp10.1 and plesiomorphic Aqp10s. However, the molecular mechanism responsible for the weak permeability of Aqp10.2 to urea and boric acid remains unclear. Herein, we present a novel hypothesis that explains the solute selectivity of Aqp10. We deduced the ancestral sequences of Aqp10.1 and Aqp10.2 paralogs via molecular phylogenetic analysis. Constructed structural models of these sequences revealed that both the well known amino acid site at position 3 and the sum of molecular weights of the four amino acid sites in the ar/R region were important for the formation of the Aqp10 selectivity filter. Site-directed mutagenesis revealed that a decrease in the sum of the molecular weights of the four amino acid sites enhanced the Aqp10 permeability to urea and boric acid. Based on this, we proposed a model in which the presence of two or more bulky amino acids in the ar/R region, which increases the sum of the molecular weights of amino acids in the ar/R region, was essential for the formation of a filter that limited urea and boric acid transport. Our results outline the molecular mechanism by which Aqp10.2 acquired a selectivity filter during evolution and provide structural insights into the narrowly tuned filter responsible for the solute selectivity of aquaglyceroporins.Significance Statement In aquaglyceroporins, particularly Aqp10s, the urea and boric acid permeabilities of Aqp10.2 paralogs are much weaker than those of plesiomorphic Aqp10s. Here, we deduced the ancestral sequences of Aqp10.1 and Aqp10.2 via molecular phylogenetic analysis. Structural models of these sequences revealed that the sum of the four amino acid site molecular weights in the ar/R region, if more than one is bulky, contributed to the selectivity filter formation in the pore region. Using site-directed mutagenesis, reduction in the molecular weight of one bulky amino acid residue in the ar/R region restricted urea and boric acid permeability. Therefore, Aqp10.2 acquired a selectivity filter during evolution, and structural differences in this selectivity filter are responsible for the variable solute permeability of aquaglyceroporins.### Competing Interest StatementThe authors have declared no competing interest.

Solute carrier family 26 (Slc26) is a family of anion exchangers with 11 members in mammals (named Slc26a1-a11). Here, we identified a novel member of the slc26 family, slc26a12, located in tandem with slc26a2 in the genomes of several vertebrate lineages. BLAST and synteny analyses of various jawed vertebrate genome databases revealed that slc26a12 is present in coelacanths, amphibians, reptiles, and birds but not in cartilaginous fishes, lungfish, mammals, or ray-finned fishes. In some avian and reptilian lineages such as owls, penguins, egrets, and ducks, and most turtles examined, slc26a12 was lost or pseudogenized. Phylogenetic analysis showed that Slc26a12 formed an independent branch with the other Slc26 members and Slc26a12, Slc26a1 and Slc26a2 formed a single branch, suggesting that these three members formed a subfamily in Slc26. In jawless fish, hagfish have two genes homologous to slc26a2 and slc26a12, whereas lamprey has a single gene homologous to slc26a2. African clawed frogs express slc26a12 in larval gills, skin, and fins. These results show that slc26a12 was present at least before the separation of lobe-finned fish and tetrapods; the name slc26a12 is appropriate because the gene duplication occurred in the distant past.

Marine teleosts ingest large amounts of seawater containing various ions, including 0.4 mM boric acid, which can accumulate at toxic levels in the body. However, the molecular mechanisms by which marine teleosts absorb and excrete boric acid are not well understood. Aquaporins (Aqps) are homologous to the nodulin-like intrinsic protein (NIP) family of plant boric acid channels. To investigate the potential roles of Aqps on boric acid transport across the plasma membrane in marine teleosts, we analyzed the function of Aqps of Japanese pufferfish (Takifugu rubripes) expressed in Xenopus laevis oocytes. Takifugu genome database contains 16 genes encoding the aquaporin family members (aqp0a, aqp0b, aqp1aa, aqp1ab, aqp3a, aqp4a, aqp7, aqp8bb, aqp9a, aqp9b, aqp10aa, aqp10bb, aqp11a, aqp11b, aqp12, and aqp14). When T. rubripes Aqps (TrAqps) were expressed in X. laevis oocytes, a swelling assay showed that boric acid permeability was significantly increased in oocytes expressing TrAqp3a, 7, 8bb, 9a, and 9b. The influx of boric acid into these oocytes was also confirmed by elemental quantification. Electrophysiological analysis using a pH microelectrode showed that these TrAqps increase B(OH)(3) permeability. These results indicate that TrAqp3a, 7, 8bb, 9a, and 9b act as boric acid transport systems, likely as channels, in marine teleosts.

slc12a10 is not a fish-specific gene and is present in a few mammals (e.g., platypus and horse), non-avian reptiles, amphibians, but was pseudogenized or deleted in most mammals (e.g., human, mouse, cat, cow, and rhinoceros), birds, and some ray-finned fishes (pufferfishes).

Boric acid is a vital micronutrient in animals; however, excess amounts are toxic to them. Little is known about whole-body boric acid homeostasis in animals. Seawater (SW) contains 0.4 mM boric acid, and since marine fish drink SW, their urinary system was used here as a model of the boric acid excretion system. We determined that the bladder urine of a euryhaline pufferfish (river pufferfish, Takifugu obscurus) acclimated to fresh water and SW contained 0.020 and 19 mM of boric acid, respectively (a 950-fold difference), indicating the presence of a powerful excretory renal system for boric acid. Slc4a11 is a potential animal homolog of the plant boron transporter BOR1; however, mammalian Slc4a11 mediates H+ (OH-) conductance but does not transport boric acid. We found that renal expression of the pufferfish paralog of Slc4a11, Slc4a11A, was markedly induced after transfer from fresh water to SW, and Slc4a11A was localized to the apical membrane of kidney tubules. When pufferfish Slc4a11A was expressed in Xenopus oocytes, exposure to media containing boric acid and a voltage clamp elicited whole-cell outward currents, a marked increase in pHi, and increased boron content. In addition, the activity of Slc4a11A was independent of extracellular Na+. These results indicate that pufferfish Slc4a11A is an electrogenic boric acid transporter that functions as a B(OH)4- uniporter, B(OH)3-OH- cotransporter, or B(OH)3/H+ exchanger. These observations suggest that Slc4a11A is involved in the kidney tubular secretion of boric acid in SW fish, probably induced by the negative membrane potential and low pH of urine.

Solute carrier 12 (Slc12) is a family of electroneutral cation-coupled chloride (Cl-) cotransporters. Na +/K+/2Cl- (Nkcc) and Na+/ Cl- cotransporters (Ncc) belong to the Nkcc/Ncc subfamily. Human and mouse possess one gene for the Na +/Cl- cotransporter (ncc gene: slc12a3), whereas teleost fishes possess multiple ncc genes, slc12a3 (ncc1) and slc12a10 (ncc2), in addition to their species-specific paralogs. Amphibians and squamates have two ncc genes: slc12a3 (ncc1) and ncc3. However, the evolutionary relationship between slc12a10 and ncc3 remains unresolved, and the presence of slc12a10 (ncc2) in mammals has not been clari-fied. Synteny and phylogenetic analyses of vertebrate genome databases showed that ncc3 is the ortholog of slc12a10, and slc12a10 is present in most ray -finned fishes, coelacanths, amphibians, reptiles, and a few mammals (e.g., platypus and horse) but pseudogenized or deleted in birds, most mammals, and some ray -finned fishes (pufferfishes). This shows that slc12a10 is widely present among bony vertebrates and pseudogenized or deleted independently in multiple lineages. Notably, as com-pared with some fish that show varied slc12a10 tissue expression profile, spotted gar, African clawed frog, red-eared slider turtle, and horse express slc12a10 in the ovaries or premature gonads. In horse tissues, an unexpectedly large number of splicing var-iants for Slc12a10 have been cloned, many of which encode truncated forms of Slc12a10, suggesting that the functional con-straints of horse slc12a10 are weakened, which may be in the process of becoming a pseudogene. Our results elaborate on the evolution of Nkcc/Ncc subfamily of Slc12 in vertebrates.