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Molecular machinery for vasotocin-dependent transepithelial water movement in amphibians: Aquaporins and Evolution.

Abstract. Amphibians represent the first vertebrates to adapt to terrestrial environments, and are successfully distributed around the world. The ventral skin, kidney, and urinary bladder are important osmoregulatory organs for adult anuran amphibians. Water channel proteins, called aquaporins (AQPs), play key roles in transepithelial water absorption/reabsorption in these organs. At least 43 types of AQPs were identified in anurans; a recent phylogenetic analysis categorized anuran AQPs among 16 classes (AQPO-14, 16). Anuran-specific AQPa2 was assigned to AQP6, then was further subdivided into the ventral skintype (AQP6vs; AQPa2S), whose expression is confined to the ventral skin, and the urinary bladder-type (AQP6ub; AQPa2U), which is basically expressed in the urinary bladder. For the osmoregulatory organs, AQP3 is constitutively located in the basolateral plasma membrane of tight-junctioned epithelial cells. AQP6vs, AQP2 and/or AQP6ub are also expressed in these epithelial cells and are translocated to the apical membrane in response to arginine vasotocin, thereby regulating water absorption/reabsorption. It was suggested recently that two subtypes of AQP6vs contribute to cutaneous water absorption in Ranid species. In addition, AQP5 (AQP5a) and AQP5L (AQP5b) were identified from Xenopus tropicalis Gray, 1864, and AQP5 was localized to the apical membrane of luminal epithelial cells of the urinary bladder in dehydrated Xenopus. This finding suggested that AQP5 may be involved in water reabsorption from this organ under dehydration. Based on the hitherto reported information, we propose models for the evolution of water-absorbing/reabsorbing mechanisms in anuran osmoregulatory organs in association with AQPs.


The extant Amphibia (Lissamphibia) comprises three orders: Anura (frogs and toads), Caudata (salamanders and newts), and Gymnophiona (caecilians) (Kardong, 2012; Frost, 2015). The Anura is the largest order (6,547 species) in the extant Amphibia (7,428 species), and anuran amphibians are successfully distributed around the world, even to forests and arid deserts (Amphibiaweb, 2015). When on dry land, however, most anurans are vulnerable to rapid water loss because their skin has exceptionally low resistance to water evaporation (Lillywhite, 2006). To maintain water homeostasis, anurans have evolved unique physiological mechanisms (Jorgensen, 1997; Bentley, 2002). Most adult anurans do not drink water through their mouth; instead, they absorb water from shallow water sources or moist substrates across the ventral skin (Bentley and Yorio, 1979) (Fig. 1A). In addition, water is reabsorbed from the tubular fluid in the kidney and from stored urine in the urinary bladder (Bentley, 2002; Hillyard et al., 2009) (Fig. 1A). Water movement in these osmoregulatory organs is controlled by the neurohypophyseal hormone, arginine vasotocin (AVT; i.e., the non-mammalian vertebrate counterpart of arginine vasopressin (AVP)) (Bentley, 2002) (Fig. 1B). In addition to AVT, hydrins, or intermediate peptides derived from the AVT precursor, show biological activities (Acher et al., 1997). Hydrin 1 (vasotocinyl-Gly-Lys-Arg) and hydrin 1' (vasotocinyl-Gly-Lys) are synthesized in Xenopus laevis Daudin, 1802, whereas hydrin 2 (vasotocinyl-Gly) is produced in Ranidae and Bufonidae (Rouille et al., 1989; Iwamuro et al., 1993) (Fig. 1B). Hydrins increase water permeability of the ventral skin and urinary bladder, but lack antidiuretic activity in the kidney (Rouille et al., 1995; Acher et al., 1997).

Since their discovery in mammals, water channel proteins, termed aquaporins (AQPs), have been identified in various organisms. Water pathways in the organisms were depicted by the localization of AQPs in the histological architecture. As for anurans, physiological and immunohistological studies unveiled the possible functions of AQP2, AQP3, AQP5, and AQP6 (AQPa2) in the osmoregulatory organs (Kubota et al., 2006; Akabane et al., 2007; Ogushi et al., 2007; Mochida et al., 2008; Finn et al., 2014). It was also revealed that the expression pattern of AQP genes is closely associated with water adaptation strategy in anurans (Ogushi et al., 2010a). In the current review, we describe first the amphibian AQP family and then the osmoregulatory processes and their diversity in anurans, highlighting the physiological roles of AQPs. The evolution of water-absorbing/reabsorbing mechanisms in anuran osmoregulatory organs is also discussed, based on previous and recent findings. Ion transport is also important in amphibian osmoregulation (Macknight et al., 1980; Bentley, 2002; Uchiyama and Konno, 2006; Larsen et al., 2014), but is beyond the scope of this review.

Amphibian AQPs

Classification and molecular phylogeny of deuterostome AQPs were recently advanced by massive Bayesian analysis (Finn et al.,, 2014). According to this analysis, 17 classes of AQPs (AQP0-AQP16) are present in vertebrates and are categorized into four grades; classical AQPs (AQP0-AQP2, AQP4-6, AQP14, and AQP15); aquaglyceroporins (AQP3, AQP7, AQP9, AQP10, and AQP13); AQP8/16; and unorthodox AQPs (AQP11 and AQP0). Notably, it was first thought that classical AQPs conduct only water, whilst aquaglyceroporins transport small, uncharged solutes, such as glycerol and urea, in addition to water (Ishibashi et al., 2009). However, it is now documented that other substrates are conducted via AQPs: anions, such as [NO.sub.3.sup.-] and [I.sup.-]; metalloids, for example, As(III) and SB(III); hydrogen peroxide; and gases, including [CO.sub.2] and [NH.sub.3] (Ishibashi et al., 2009, 2011; Geyer et al., 2013; Bienert and Chaumont, 2014; Kaldenhoff et al., 2014; Mukhopadhyay et al., 2014; Rambow et al., 2014).

As for amphibians, anuran AQPs were divided into 16 classes (AQPO-14, 16) (Finn et al., 2014). Anuran-specific AQPa2 was assigned to AQP6, then further subdivided into the ventral skin-type (AQP6vs; AQPa2S), whose expression is confined to the ventral skin, and the urinary bladder-type (AQP6ub; AQPa2U), which is basically expressed in the urinary bladder (Suzuki et al., 2007; Suzuki and Tanaka, 2009, 2010; Finn et al., 2014; Shibata et al., 2014a) (Table 1; Fig. 2). Two types of AQP5 were designated as AQP5 (AQP5a) and AQP5L (AQP5b) (Finn et al., 2014; Shibata et al., 2014b) (Table 1). In this review, anuran AQPs are described according to the nomenclature of Finn et al. (2014) (Table 1). Among mammalian AQPs (AQPO-14), AQP2 is the only AVP-dependent water channel that facilitates water reabsorption in response to AVP, by translocating from intracellular vesicles to the apical membrane of collecting duct principal cells (Sasaki, 2012; Kortenoeven and Fenton, 2014). This AQP2 translocation is regulated by the phosphorylation states of Ser-256, Ser-261, Ser-264, and Ser-269 in the C-terminal region of AQP2 (Sasaki, 2012; Schey et al., 2013). Of note, Ser-256 serves a key role in AQP2 translocation (Sasaki, 2012; Schey et al., 2013). As for anurans, three types of AQPs: AQP2, AQP6vs, and AQP6ub, are regulated by AVT (Suzuki and Tanaka, 2010) (Fig. 2). Along with six transmembrane domains and two canonical Asn-Pro-Ala motifs, Ser-256 of mammalian AQP2 is conserved in these anuran AQPs (Fig. 2), suggesting the functional importance of this residue. For anuran aquaglyceroporins, AQP3 and AQP9 have been studied in association with cryoprotection during hibernation, as well as in water transport (Zimmerman et al., 2007; Pandey et al., 2010; Mutyam et al., 2011; Hirota et al., 2015). To our knowledge, there is still no report of cloning of aquaporins in urodeles or caecilians.


In many adult anurans, water is absorbed across the ventral skin (Jorgensen, 1997). For terrestrial and arboreal species, the posteroventral area is highly permeable and is referred to as the seat patch or pelvic patch (Hillman et al., 2009; Hillyard et al., 2009). For arboreal Hyla japonica Gunther, 1859, physiological in vitro studies using an Ussing chamber showed that water permeability of the seat patch was increased in response to AVT, hydrin 1, and hydrin 2 (Ogushi et al., 2010b). Isoproterenol (IP), a [beta]-adrenergic receptor agonist, also augmented water permeability of the seat patch, and the response was inhibited by its antagonist, propranolol (PP) (Ogushi et al., 2010b).

The skin consists of the epidermis and dermis, and the epidermis contains four successive layers: the stratum corneum, stratum granulosum, stratum spinosum, and stratum germinativum (Lillywhite, 2006; Kardong, 2012) (Fig. 3A). Tight junctions reside between the keratinized cells of the stratum corneum and between the outermost granular cells (Farquhar and Palade, 1965). However, the keratinized layer is very thin and comprises dead cells with high water permeability. Therefore, it is the outermost granular cell layer, called the first-reacting cell (FRC) layer, which plays a key role in regulating water transport (Voute and Ussing, 1968). For H. japonica, AQP6vs (AQP-h3) and AQP3 (AQP-h3BL) were identified from the seat patch (Tanii et al., 2002; Akabane et al., 2007) (Table 1; Fig. 2). Immunohistochemistry localized AQP6vs mainly to the basolateral plasma membrane and cytoplasm of principal cells in the FRC layer when the skin was not stimulated with AVT (Hasegawa et al., 2003; Ogushi et al., 2010b). AQP6vs was also detected along the whole plasma membrane in the underlying granular cells. Intriguingly, the physiological responses seen in the above-mentioned in vitro experiments were well correlated with the intracellular trafficking of AQP6vs. After stimulation with AVT or IP, AQP6vs was translocated to the apical membrane in the principal cells of the FRC layer (Ogushi et al., 2010b) (Fig. 3B). The amount of AQP6vs located in the basolateral membrane varied between specimens; AQP6vs was scarcely detectable in some cases, but was abundantly observed in others. On the other hand, after treatment with both IP and PP, AQP6vs was detected along the basolateral membrane, as seen in the PP-treated skin (Ogushi et al., 2010b). In contrast, AQP3 remained along the basolateral membrane in the principal cells in any treatment (Fig. 3B). These findings suggest that AQP6vs and AQP3 mediate water absorption across the seat patch in the arboreal species, and, further, that AQP6vs plays a critical role in regulating the water movement (Fig. 3B).


The adult kidney is a mesonephros. The nephrons consist of a glomerulus in the Bowman's capsule and a renal tubule that is divided into the following morphologically distinguishable segments: a neck segment; proximal tubule; thin, intermediate segment; early distal tubule; late distal tubule; and connecting tubule linked to the collecting duct (Uchiyama and Yoshizawa, 2002; Hillman et al., 2009; Kardong, 2012; Larsen et al., 2014) (Fig. 4A). The anuran renal tubule does not have an extended, U-shaped medullary portion similar to Henle's loop in the avian and mammalian kidneys; hence, hyperosmotic urine cannot be produced in the anuran kidney (Bentley, 1998). Nevertheless, the kidney of adult anurans shows significant antidiuretic responses to AVT: a decreased glomerular filtration rate and/or an increased water reabsorption across the renal tubule (Henderson et al., 1972; Pang et al., 1982; Pang, 1983). For arboreal Hyla japonica, AQP2 (AQP-h2K) was expressed in the collecting duct (Ogushi et al., 2007) (Table 1; Fig. 2). AQP2 was located mainly in the cytoplasm and slightly in the apical plasma membrane of the principal cells under hydrated conditions. On the other hand, AQP3 (AQP-h3BL) resided along the basolateral membrane of the collecting duct principal cells (Ogushi et al., 2007) (Fig. 4B). After AVT stimulation, most AQP2s were translocated to the apical membrane, but AQP3 remained along the basolateral membrane (Ogushi et al., 2007) (Fig. 4B). At this state, it is presumed that water moves from the tubular fluid, through the principal cells, to the neighboring interstitium along the osmotic gradient (Fig. 4B), which is produced by reabsorbed solutes such as [Na.sup.+], [Cl.sup.-], and urea (Uchiyama and Konno, 2006; Larsen et al., 2014). Thus, it seems likely that, for anurans, AQP2 plays an important role in AVTdependent water reabsorption in the kidney, as it does in the mammalian kidney (Kortenoeven and Fenton, 2014). However, AQP2 may not be essential in aquatic species because the AQP2 gene is absent from the genome of Xenopus tropicalis (Suzuki and Tanaka, 2009; Shibata et al., 2014b). In addition to AVT, norepinephrine induces glomerular and tubular antidiuresis in the bullfrog kidney (Gallardo et al., 1980; Pang et al., 1982). However, it remains to be elucidated whether IP affects subcellular localization of anuran AQP2 in the collecting duct principal cells.

Urinary bladder

The anuran urinary bladder is a bilobed, thin structure that functions as a water storage organ (Steen, 1929; Macknight et al., 1980). The capacity of the urinary bladder is greatest in fossorial and arboreal species (Hillman et al., 2009), and water reabsorption is observed in response to AVT or hydrins (Iwamuro et al., 1993; Acher et al., 1997). In the tissue structure of the urinary bladder, the mucosal epithelium forms the actual barrier for water movement (Macknight et al., 1980). This epithelium consists of four types of cells: granular cells, mitochondrion-rich cells, goblet (mucous) cells, and basal cells. The granular cells are most prevalent and important for water permeation (Andreoli and Schafer, 1976; Macknight et al., 1980). For Hyla japonica, AQP6ub (AQP-h2) was expressed in the urinary bladder (Hasegawa et al., 2003) (Table 1; Fig. 2). Before AVT stimulation, AQP6ub showed a punctate distribution in the cytoplasm under the apical membrane of the granular cells (Hasegawa et al., 2005). Immunoelectron microscopic analysis localized AQP6ub on the tubular or spherical vesicles of the granular cells (Hasegawa et al., 2005). After AVT stimulation, AQP6ub was translocated to the apical membrane, with possible phosphorylation by protein kinase A (Hasegawa et al., 2005) (Figs. 2, 5). However, AQP3 (AQP-h3BL) was located along the basolateral membrane of the urinary bladder granular cells, irrespective of AVT stimulation (Fig. 5). These data suggest that AQP6ub and AQP3 mediate water reabsorption from the urinary bladder, and that AQP6ub is critical for regulating the water transfer (Fig. 5).

Adaptation of Anurans to Environments

The gene expression pattern of AQP6 seems to be closely associated with the ecological diversification of anurans (Ogushi et al., 2010a; Suzuki and Tanaka, 2010). According to their habitats, anurans can be classified into several groups: e.g., arboreal and terrestrial species, which have expanded their habitats to drier land; semiaquatic species, generally found near permanent water sources; and aquatic species, which dwell in water (Jorgensen, 1997; Hillman et al., 2009). For arboreal Hyla japonica and xeric Incilius (Bufo) alvarius Girard. 1859, AQP6vs and AQP6ub were expressed in the ventral skin and showed similar cellular and subcellular localization (Hasegawa et al., 2003; Shibata et al., 2011). Both AQPs were located in principal cells of the stratum granulosum in the epidermis and were observed in intracellular vesicles or along the plasma membrane during normal hydration. In response to AVT, these AQPs were translocated to the apical membrane in principal cells of the FRC layer (Hasegawa et al., 2003; Ogushi et al., 2010c; Shibata et al., 2011). A similar response was observed when the seat patch of H. japonica was treated with hydrins or IP (Ogushi et al., 2010b). However, for the xeric Anaxyrus (Bufo) punctatus Baird and Girard, 1852, both AQP6vs and AQP6ub were localized to the apical membrane regardless of AVT stimulation (Shibata et al., 2011). For the terrestrial Rhinella marina (Bufo marinus) Linnaeus, 1758, great individual differences were noted, but AVT-induced water transfer was detected in the pectoral skin, ventral pelvic skin, and thigh skin (Ogushi et al., 2010c). Accordingly, the area expressing AQP6 was wider, and both AQP6vs and AQP6ub were detected from the pectoral area to the thigh area (Ogushi et al., 2010c). In response to AVT, these AQP6s also showed translocation from the cytoplasm to the apical membrane in the FRC layer cells (Ogushi et al., 2010c). These lines of evidence suggest that AQP6vs and AQP6ub cooperate to regulate cutaneous water absorption in these arboreal, xeric, and terrestrial species.

For semiaquatic Rana japonica Boulenger, 1879, Pelophylax nigromaculatus (Hallowell, 1861), and Rana (Lithobates) catesbeianus (Shaw, 1802), only AQP6vs was expressed in the ventral skin (Ogushi et al., 2010a, c). However, our recent study revealed the presence of two subtypes of AQP6vs (type 1 (AQPa2S, type a) and type 2 (AQPa2S, type b)) in these Ranid species (Finn et al., 2014; Saitoh et al., 2014) (Table 1). In the Ranid species, in vitro experiments showed that AVT-induced water flow was greatest in the thigh skin, comparable to that of terrestrial Rhinella marina (Ogushi et al., 2010c; Saitoh et al., 2014). For Rana japonica, both AQP6vs subtypes were observed mostly in thigh skin, and they changed their subcellular localization in response to AVT, IP, and PP in a manner similar to Hyla japonica AQP6 in the seat patch (Ogushi et al., 2010b; Saitoh et al., 2014). Hence, in some semiaquatic species, it seems that two subtypes of AQP6vs regulate water absorption across the thigh skin.

In contrast, for aquatic species, ambient water moves into the body along the osmotic gradient (Bentley, 1998). To prevent excessive water influx, the skin of Xenopus laevis shows low water permeability and does not respond to neurohypophyseal peptides (Bentley, 1998, 2002). For X. laevis, albeit at lower levels, AQP6vs (AQP-x3) mRNA was found to be expressed in the ventral skin ranging from the pectoral area to thigh area (Ogushi et al., 2010c) (Table 1). However, AQP6vs protein was not detectable in the skin of this species. Based on its cDNA sequence, X. laevis AQP6vs was predicted to have an extended C-terminus of approximately 10 to 15 amino acids, including Cys-273, compared with other AQP6vs proteins (Ogushi et al., 2010a) (Fig. 2). Functional and immunohistological analyses for X. laevis AQP6vs, mutant AQP6vs, and chimeric AQP suggested that, for X. laevis, AQP6vs gene expression is attenuated at a post-transcriptional step by Cys-273 of AQP6vs and/or Cys-273-coding region in AQP6vs mRNA (Ogushi et al., 2010a).

In aquatic species, AVT does not increase water movement in the urinary bladder (Calamita et al., 1994; Bentley, 2002). Nevertheless, AQP6ub (AQP-x2) protein was detected in the urinary bladder of X. laevis (Shibata et al., 2014a) (Table 1). In contrast to X. laevis AQP6vs, X. laevis AQP6ub did not possess the extended C-terminus (Fig. 2), and oocyte swelling assay showed that this AQP6ub had the ability to transport water, as in Hyla japonica AQP6ub. In the urinary bladder in vivo, X. laevis AQP6ub was localized to the cytoplasm of the luminal granular cells (Shibata et al., 2014a). For H. japonica, in vitro stimulation of the urinary bladder with [10.sup.-8] mol [l.sup.-1] of AVT triggered translocation of AQP6ub from intracellular vesicles to the apical membrane (Hasegawa et al., 2005). However, neither [10.sup.-8] mol [l.sup.-1] of AVT nor [10.sup.-8] mol [l.sup.-1] of hydrins had a clear effect on the subcellular distribution of X. laevis AQP6ub (Shibata et al., 2014a). These results suggest that the poor responsiveness of AQP6ub to neurohypophyseal peptides may be a main cause for the scant water permeability of the urinary bladder in X. laevis.

As mentioned above, for aquatic Xenopus, AQP6 did not appear in the apical membrane in the ventral skin or urinary bladder, presumably helping to prevent water movement into the body. However, in some natural habitats such as arid parts of South Africa, Xenopus survives the dry season by estivating under the mud or hiding in holes, under flat stones, or under roots in the river banks (Amphibiaweb, 2015). For X. tropicalis, AQP5 (AQP5a; AQP-xt5a) was found in the urinary bladder (Shibata et al., 2014b) (Table 1; Fig. 2). When X. tropicalis was kept in water, AQP5 protein was localized to the apical membrane and cytoplasm of a small number of granular cells (Shibata et al., 2014b). On the other hand, after X. tropicalis was dehydrated in the laboratory, the number of AQP5-expressing cells increased and AQP5 was observed in the apical membrane and cytoplasm of most granular cells (Shibata et al., 2014b). These findings suggest that for dehydrated Xenopus, AQP5 may be involved in water reabsorption from the urinary bladder. Thus, AQP5 may contribute to water economizing in Xenopus in the dry season.

Evolution of Molecular Machinery for Water Absorption/Reabsorption

With regard to anuran osmoregulatory organs, different AQPs were basically adopted at the entry site of the transepithelial water transport: AQP6vs in the ventral skin, AQP2 in the kidney, and AQPub in the urinary bladder (Suzuki and Tanaka, 2010). In addition, AQP5 seems to function at the water entry site in the urinary bladder of the dehydrated Xenopus (Shibata et al., 2014b). For the genome of X. tropicalis, aqpovs (aqp-a2s) and aqp6ub (aqp-a2u) form a gene cluster with aqp5 (aqp5a) and aqp5l (aqp5b) in the region between faim2 encoding Fas apoptotic inhibitory molecule 2 and racgapl for Rac GTPase-activating protein 1 (Suzuki and Tanaka, 2010; Finn et al., 2014; Shibata et al., 2014b) (Table 1). In mammalian genomes, Aqp2 is located with Aqp5 and Aqp6 between Faim2 and Racgapl (Shibata et al., 2014b; Cunningham et al., 2015). Because AQP2 is also found in Hyla japonica (Ogushi et al., 2007), it is possible that aqp2 resides with aqp5, aqp6vs, and aqp6ub in the homologous region of the Hyla genome. However, it was recently reported that the genomic region flanked by faim2 and racgapl split in contemporary reptiles and a bird (Finn et al., 2014). Therefore, genomic analysis is necessary to prove the actual arrangement of faim2, aqp2, aqp5, aqp6vs, aqp6ub, and racgapl in H. japonica.

In addition to their close locations in the genome, aqp5, aqp6vs, and aqp6ub comprise a similar four exon-three intron structure in Xenopus tropicalis (Finn et al., 2014; Cunningham et al., 2015). This exon-intron structure is also conserved in H. japonica aqp2 and aqp6vs (unpubl. data). Further, these AQPs have close relationships in the molecular phylogenetic trees (Saitoh et al., 2014; Shibata et al., 2014a). Hence, it seems likely that AQP2, AQP5, and AQP6 had arisen through local gene duplication of a single ancestral AQP gene. Markedly, AQP2-like genes are present between faim2 and racgapl in the coelacanth genome (Finn et al., 2014), and type 2-like AQP (AQP0p) is expressed in the kidney of lungfish Protopterus annectens Owen, 1839 during terrestrial estivation (Konno et al., 2010) (Table 1). These findings suggest that the homolog or ortholog of these fish aqps might have given rise to tetrapod aqp2, aqp5, and aqp6 via gene duplication (Konno et al., 2010; Finn et al., 2014).

The molecular machinery for renal water reabsorption is mandatory for the survival of mammals, and AVP-regulated water reabsorption plays an important role in water economy (Kortenoeven and Fenton, 2014). As for the mammalian kidney, AQP2 is transferred to the apical membrane in collecting duct principal cells in response to AVP, thereby increasing water reabsorption (Sasaki, 2012). Similar molecular machinery involving AVT and AQP2 is seen in the quail and Hyla japonica (Ogushi et al., 2007; Nishimura and Yang, 2013). Further, it is shown that for the estivating lungfish, AVT mRNA expression is dramatically elevated in the hypothalamus, and type 2-like AQP appears along the apical side of the renal late distal tubules, where V2-type AVT receptors are located along the basolateral side (Konno et al., 2010). Given these findings and the data on vertebrate AQPs and osmoregulation, a model is proposed for the evolution of molecular machinery for water absorption/reabsorption involving AQP2, AQP5, and AQP6. Initially, the basic machinery necessary for AVT/AVP-regulated trafficking of AQP2 may have arisen in the kidney before the divergence of the lungfish lineage and higher vertebrate lineage (Konno et al., 2010). Thereafter, the molecular mechanism for AVT-regulated translocation of AQP2 was established in the amphibian kidney and was basically inherited by higher tetrapods such as birds and mammals. The function of AQP2 remains to be reported in reptiles, however. In the anuran amphibian lineage, this mechanism tripled, occurring in the ventral skin and urinary bladder as well as the kidney. AQP6vs and AQP6ub then came to function in the ventral skin and urinary bladder, respectively, instead of AQP2. AQP5 also may have occurred on the apical membrane in the urinary bladder, although its trafficking seems to be regulated via different signaling pathways (Shibata et al., 2014b).

As for the ventral skin, one type of AQP6vs was cloned from Xenopus, Bufo, or Hyla, whilst two subtypes of AQP6vs (types 1 and 2) were identified in Rana (Tanii et al., 2002; Ogushi et al., 2010a; Saitoh et al., 2014) (Table 1). Molecular phylogenetic analysis showed that type 1 from Rana is closely related to the AQP6vs from Xenopus, whereas type 2 from Rana is closer to the AQP6vs from Bufo and Hyla, suggesting that AQP6vs from arboreal and terrestrial species are not the ortholog of AQP6vs from aquatic species (Saitoh et al., 2014). A scenario for the evolution of AQP6 involved in cutaneous water absorption may be that a single gene for AQP6vs (type 1) occurred early in anuran evolution and was inherited by aquatic Pipidae, including Xenopus (Saitoh et al., 2014). In aquatic species, however, AQP6vs is not expressed at the protein level, avoiding excessive water entry into the body. Before the divergence of Ranoidea, another form of AQP6vs (type 2) was generated via gene duplication of the type-1 AQP6vs. Thereafter, both types were inherited by semiaquatic Ranidae, including Rana catesbeiana, and came to contribute to water absorption across the thigh skin. On the other hand, as the common ancestor to terrestrial Bufonidae and arboreal Hylidae advanced into drier environments, AQP6ub began to be expressed in the ventral skin, mediating water absorption in concert with AQP6vs.


In this review, we showed possible basic evolutionary pathways of amphibian AQP2, AQP5, and AQP6 in association with water absorption/reabsorption. Indeed, amphibians have a wide diversity of genome size (0.95-120.1 pg) (Gregory et al., 2007), and it is proposed that polyploidy with epigenetic alterations led to rapid diversity and speciation of anurans (Kawamura, 1984; Becak, 2014). Therefore, it is highly probable that additional gene duplication or deletion of AQP gene(s) occurred in many extant anurans. For a better understanding of the molecular evolution of anuran AQPs, further studies are necessary. For example, whether Ranid AQP6vs types 1 and 2 are caused by local gene duplication or whole genome duplication awaits clarification. In addition, molecular biological studies of urodele and caecilian AQPs, coupled with physiological and histological analyses, would give significant insights into the early evolution of the genes and functions of AQP2, AQP5, and AQP6.

Schematic models were also shown for AVT-regulated trafficking of anuran AQP2 and AQP6. It has been considered that the regulatory mechanisms for water permeability of the tight-junctioned epithelium by the neurohypophyseal hormone are basically common between the anuran osmoregulatory organs and mammalian kidney. Numerous studies were conducted using anuran ventral skin and urinary bladder as model systems, aiming to elucidate the general mechanisms for ion-coupled water movement across the tight epithelium (Macknight et al., 1980; Brown, 1989; Jorgensen, 1997). With respect to AQPs, our immunohistological data suggest that intracellular trafficking of anuran AQP6 is similar to that of mammalian AQP2. In mammals, AVP accumulates AQP2 at the apical membrane mainly by translocation of intracellular subapical storage vesicles, and also by indirect transcytosis from the basolateral membrane (Sasaki, 2012; Yui et al., 2013). Accordingly, our immunocytological analysis of the Hyla urinary bladder showed that intracellular vesicles bearing AQP6ub were translocated to the apical membrane after AVT stimulation (Hasegawa et al., 2005). Although it is not present in all specimens, AQP6 was also detected along the basolateral membrane in the ventral skin of Hyla, Bufo, and Rana (Ogushi et al., 2010b, c; Shibata et al., 2011; Saitoh et al., 2014). In response to AVT, AQP6 occurred abundantly in the apical membrane and partially, or mostly, disappeared from the basolateral membrane in many observations (Hasegawa et al., 2003; Suzuki et al., 2007; Ogushi et al., 2010b, c). Therefore, it is plausible that basolateral AQP6 was delivered to the apical membrane via a transcytotic pathway. Although information about AVT-regulated trafficking is very limited, novel findings may be obtained by applying modern molecular biological techniques to the studies on anuran AQP6; e.g., in-depth analysis of proteome and comparison of the data among anuran ventral skin, urinary bladder, and mammalian renal collecting ducts may lead to a discovery of important molecules.


We are grateful to Dr. Bridget I. Baker, Bath, UK, for critical reading of the manuscript. This work was supported by Grants-in-Aid for Scientific Research (19370024; 20570055) from the Ministry of Education, Culture, Sports, Science and Technology of Japan; a grant from Japan Research Society of Arginine Vasopressin; and a SUNBOR grant from Suntory Institute for Bioorganic Research.

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Integrated Bioscience Section, Graduate School of Science and Technology, Shizuoka University, 836, Ohya, Suruga-ward, Shizuoka-city, Shizuoka 422-8529, Japan

Received 5 February 2015; accepted 4 May 2015.

(*) To whom correspondence should be addressed. E-mail:

Abbreviations: AQP, aquaporin; AQP6ub, urinary bladder-type AQP6; AQP6vs, ventral skin-type AQP6; AQPa2S, ventral skin-type AQPa2; AQPa2U, urinary bladder-type AQPa2; AVP, arginine vasopressin; AVT, arginine vasotocin; FRC, first-reacting cell; IP, isoproterenol; PP, propranolol.

Table 1
Nomenclature of amphibian aquaporins (AQPs)

Present symbol (*)      Previous symbol

AQP2                    AQP-h2K
AQP2-like               AQPOp
AQP3                    AQP-h3BL
AQP5                    AQP5a, AQP-xt5a
AQP5L (AQP5-like)       AQP5b, AQP-xt5b
AQP6                    AQPa2
AQP6ub                  AQPa2U; AQP-h2, AQP-x2
AQP6vs type 1, AQP6vs1  AQPa2S type a; AQP-x3
AQP6vs type 2. AQP6vs2  AQPa2S type b; AQP-h3

(*) Present symbols follow the nomenclature of Finn et al. (2014).
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Author:Suzuki, Masakazu; Shibata, Yuki; Ogushi, Yuji; Okada, Reiko
Publication:The Biological Bulletin
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Date:Aug 1, 2015
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