Molecular characterization of water-selective AQP (EbAQP4) in hagfish: insight into ancestral origin of AQP4

Hagfish (Eptatretus burgeri) are agnathous and are the earliest vertebrates still in existence. Pavement cells adjacent to the mitochondria-rich cells show orthogonal arrays of particles (OAPs) in the gill of hagfish, a known ultrastructural morphology of aquaporin (AQP) in mammalian freeze-replica studies, suggesting that an AQP homolog exists in pavement cells. We therefore cloned water channels from hagfish gill and examined their molecular characteristics. The cloned AQP [E. burgeri AQP4 (EbAQP4)] encodes 288 amino acids, including two NPA motifs and six transmembrane regions. The deduced amino acid sequence of EbAQP4 showed high homology to mammalian and avian AQP4 (rat, 44%; quail, 43%) and clustered with AQP4 subsets by the molecular phylogenetic tree. The osmotic water permeability of Xenopus oocytes injected with EbAQP4 cRNA increased eightfold compared with water-injected controls and was not reversibly inhibited by 0.3 mM HgCl(2). EbAQP4 mRNA expression in the gill was demonstrated by the RNase protection assay; antibody raised against the COOH terminus of EbAQP4 also detected (by Western blot analysis) a major approximately 31-kDa band in the gill. Immunohistochemistry and immunoelectron microscopy showed EbAQP4 localized along the basolateral membranes of gill pavement cells. In freeze-replica studies, OAPs were detected on the protoplasmic face of the split membrane comprising particles 5-6 nm long on the basolateral side of the pavement cells. These observations suggest that EbAQP4 is an ancestral water channel of mammalian AQP4 and plays a role in basolateral water transport in the gill pavement cells.     

The role of aquaporin 3 in teleost fish

The aquaporin isoform, AQP3 has now been identified in a number of different teleost fish species, with additional DNA sequence information on AQP3 genes in further fish species available in genome databases. In zebrafish (Danio rerio), the AQP3 gene is present as two duplicate isoforms resulting from a teleostean fish genome-wide duplication. A further splicoform/isoform has also been identified in rainbow trout (Oncorhynchus mykiss). The identification of these AQP3 isoforms in other fish species is consequently explored. The role of AQP3 in physiological/osmoregulatory processes, in various teleost organs is then described. In teleost gill, AQP3 is expressed in 'chloride' cells, and in some species, in other epithelial cell types, where it may have a number of different functions including the prevention of dehydration. In eel esophagus, immunohistochemistry shows that AQP3 is expressed in surface epithelial cells in the anterior esophagus, but in mucus cells within the epithelium of the posterior esophagus. In eel intestine, AQP3 is found in macrophage-like cells and probably plays no part in osmoregulatory processes. In the rectum, as in the posterior esophagus AQP3 is expressed in mucus cells. In eel kidney, AQP3 is expressed in a subset of renal tubules, and localizes to the apical pole of tubule cells. There is no apparent change in the location or protein abundance of renal AQP3 following the acclimation of eels from freshwater to seawater.     

Expression of aquaporin 3 in gills of the Atlantic killifish (Fundulus heteroclitus): Effects of seawater acclimation

Estuarine fish, such as the Atlantic killifish (Fundulus heteroclitus), are constantly and rapidly exposed to changes in salinity. Although ion transport in killifish gills during acclimation to increased salinity has been studied extensively, no studies have examined the role of aquaglyceroporin 3 (AQP3), a water, glycerol, urea, and ammonia transporter, during acclimation to increased salinity in this sentinel environmental model organism. The goal of this study was to test the hypothesis that transfer from freshwater to seawater decreases AQP3 gene and protein expression in the gill of killifish. Transfer from freshwater to seawater decreased AQP3 mRNA in the gill after 1 day, but had no effect on total gill AQP3 protein abundance as determined by western blot. Quantitative confocal immunocytochemistry confirmed western blot studies that transfer from freshwater to seawater did not change total AQP3 abundance in the gill; however, immunocytochemistry revealed that the amount of AQP3 in pillar cells of secondary lamellae decreased in seawater fish, whereas the amount of AQP3 in mitochondrion rich cells (MRC) in primary filaments of the gill increased in seawater fish. This response of AQP3 expression is unique to killifish compared to other teleosts. Although the role of AQP3 in the gill of killifish has not been completely elucidated, these results suggest that AQP3 may play an important role in the ability of killifish to acclimate to increased salinity.     

Tissue Distribution, Effects of Salinity Acclimation, and Ontogeny of Aquaporin 3 in the Marine Teleost, Silver Sea Bream (Sparus sarba)

The purpose of the present study was to ascertain the tissue-specific expression of the water channel protein, aquaporin 3 (AQP3), during salinity acclimation and larval development of silver sea bream (Sparus sarba). A cDNA fragment encoding aquaporin 3 (aqp3) from silver sea bream gill was cloned and from the deduced amino acid sequence a polyclonal antibody was prepared. AQP3 was found to be present in gill, kidney, liver, brain, heart, and spleen but not in whole blood. The abundance of AQP3 was significantly highest in gills of hypoosmotic (6 ppt) and isoosmotic (12 ppt) acclimated sea bream when compared to seawater (33 ppt) and hypersaline (50 ppt)- acclimated sea bream. Spleen tissue also displayed significantly high levels of AQP3 protein in hypoosmotic and isoosmotic salinities whereas the AQP3 abundance in brain, liver, heart, and kidney remained unchanged across the range of salinities tested. The ontogenetic profile of AQP3 was also investigated from developing sea bream larvae and AQP3 was first detected at 14 days posthatch (dph) and increased steadily up to 28–46 dph. In conclusion, this study has demonstrated that AQP3 expression is modulated in gill and spleen tissue of salinity acclimated sea bream and that it can be detected relatively early during larval development.     

Physiological roles of AQP7 in the kidney: Lessons from AQP7 knockout mice

The aquaporin7 (AQP7) water channel is known to be a member of the aquaglyceroporins, which allow the rapid transport of glycerol and water. AQP7 is abundantly present at the apical membrane of the proximal straight tubules in the kidney. In this paper, we review the physiological functions of AQP7 in the kidney. To investigate this, we generated AQP7 knockout mice. The water permeability of the proximal straight tubule brush border membrane measured by the stopped flow method was reduced in AQP7 knockout mice compared to wild-type mice (AQP7, 18.0+/-0.4 x 10(-3 )cm/s vs. wild-type, 20.0+/-0.3 x 10(-3) cm/s). Although AQP7 solo knockout mice did not show a urinary concentrating defect, AQP1/AQP7 double knockout mice showed reduced urinary concentrating ability compared to AQP1 solo knockout mice, indicating that the contribution of AQP7 to water reabsorption in the proximal straight tubules is physiologically substantial. On the other hand, AQP7 knockout mice showed marked glycerol in their urine (AQP7, 1.7+/-0.34 mg/ml vs. wild-type, 0.005+/-0.002 mg/ml). This finding identified a novel pathway of glycerol reabsorption that occurs in the proximal straight tubules. In two mouse models of proximal straight tubule injury, the cisplatin-induced acute renal failure (ARF) model and the ischemic-reperfusion ARF model, an increase of urine glycerol was observed (pre-treatment, 0.007+/-0.005 mg/ml; cisplatin, 0.063+/-0.043 mg/ml; ischemia, 0.076+/-0.02 mg/ml), suggesting that urine glycerol could be used as a new biomarker for detecting proximal straight tubule injury.     

In silico study of human aquaporin AQP11 and AQP12 channels

AQP11 and AQP12 are the most distantly related paralogs of the aquaporin family in human. They share indeed a low sequence similarity with other aquaporins and exhibit a modified N‐terminal NPA signature motif. Furthermore, they have an anomalous subcellular localization. The AQP11 and AQP12 biological role remains to be fully clarified and their ability to allow transport of water is still debated. We have built accurate 3D‐models for AQP11 and AQP12 and comprehensively compared their sequence and structure to other known aquaporins. In order to investigate whether they appear compatible or not with water permeability, we especially focused on the amino acid composition and electrostatics of their channels, keeping the structure of the low‐water efficiency AQP0 as a reference system. Our analysis points out a possible alternative ar/R site and shows that these aquaporins feature unique residues at key pore‐lining positions that make the shape, composition and electrostatics of their channel peculiar. Such residues can represent pivotal hints to study and explain the AQP11 and AQP12 biological and molecular function.     

Molecular characterization, chromosomal and expression patterns of three aquaglyceroporins (AQP3, 7, 9) from pig

As one subgroup of aquaporin, aquaglyceroporin including AQP3, 7, 9, 10 facilitates glycerol transport as well as water transport. In this study, we cloned the full length coding sequences of porcine (Sus scrofa) AQP3, 7 and 9 and the genomic sequence of AQP3 including 6 exons and 5 introns. Additionally, as a first step toward understanding the regulatory mechanisms of AQP9 in pig, we cloned and analyzed the upstream genomic sequence of the ATG translation initiation codon and found two negative insulin response elements (TGTTTTC and TATTTTG.), glucocorticoid-responsive elements, several CCAAT enhancer binding protein (C/EBP) sites, hepatocyte nuclear factor (HNF) sites, and NF-kappaB sites in this region. Subsequently, semi-quantitative analysis showed that AQP3 selectively expressed in spleen, stomach, kidney and lung. AQP7 and AQP9 were ubiquitously detected in all tissues examined and highly expressed in adipose tissue and liver, respectively. Finally, both AQP3 and AQP7 were assigned to chromosome 10q while AQP9 was mapped to chromosome 1q. This is the first report of molecular characterization of aquaglyceroporin in pig, which provides basic observations useful for future assessing and characterizing the role of aquaglyceroporin.     

Identification of vasopressin-induced genes in AQP2-transfected MDCK cells by suppression subtractive hybridization

Arginine vasopressin (AVP) plays a major role in the modulation of water reabsorption in mammalian kidney. In addition to short-term regulation of aquaporin 2 (AQP2) trafficking, AVP also has long-term effects to regulate the expression of AQP2 in renal collecting duct. However, the detailed mechanism of the long-term effects of AVP in kidney remains to be elucidated. We have searched for genes induced by AVP using the polymerase chain reaction-based suppression subtractive hybridization technique in AVP-responsive AQP2-transfected MDCK cells. We found that the expression of the genes such as VIP17/MAL, annexin II, stimulatory GTP binding protein, tubulin, and mitochondrial ATP synthase was induced by AVP treatment for 4h. These results suggest that AVP might induce the expression of several genes related to the apical targeting of newly synthesized AQP2 as well as that of AQP2 for the long-term modification of water permeability in renal collecting duct.     

Freshening effect on the osmotic response of the Antarctic spiny plunderfish Harpagifer antarcticus

Global warming is having a significant impact around the world, modifying environmental conditions in many areas, including in zones that have been thermally stable for thousands of years, such as Antarctica. Stenothermal sedentary intertidal fish species may suffer due to warming, notably if this causes water freshening from increased freshwater inputs. Acute decreases in salinity, from 33 down to 5, were used to assess osmotic responses to environmental salinity fluctuations in Antarctic spiny plunderfish Harpagifer antarcticus, in particular to evaluate if H. antarcticus is able to cope with freshening and to describe osmoregulatory responses at different levels (haematological variables, muscle water content, gene expression, NKA activity). H. antarcticus were acclimated to a range of salinities (33 as control, 20, 15, 10 and 5) for 1 week. At 5, plasma osmolality and calcium concentration were both at their lowest, while plasma cortisol and percentage muscle water content were at their highest. At the same salinity, gill and intestine Na⁺-K⁺-ATPase (NKA) activities were at their lowest and highest, respectively. In kidney, NKA activity was highest at intermediate salinities (15 and 10). The salinity-dependent NKA mRNA expression patterns differed depending on the tissue. Marked changes were also observed in the expression of genes coding membrane proteins associated with ion and water transport, such as NKCC2, CFTR and AQP8, and in the expression of mRNA for the regulatory hormone prolactin (PRL) and its receptor (PRLr). Our results demonstrate that freshening causes osmotic imbalances in H. antarcticus, apparently due to reduced capacity of both transport and regulatory mechanisms of key organs to maintain homeostasis. This has implications for fish species that have evolved in stable environmental conditions in the Antarctic, now threatened by climate change.     

Amphibian aquaporins and adaptation to terrestrial environments: a review

In many anurans, the pelvic patch of the ventral skin and the urinary bladder are important osmoregulatory organs. Since the discovery of water channel protein, aquaporin (AQP), in mammalian erythrocytes, 17 distinct full sequences of AQP mRNAs have been identified in anurans. Phylogenetic tree of AQP proteins from amphibians and mammals suggested that anuran AQPs can be divided into six types: i.e. types 1, 2, 3, and 5, and anuran-specific types a1 and a2. Among them, two types of anuran AQPs (types 1 and a2) are localized in the skin and urinary bladder by immunohistochemistry. Tree frog type-a2 AQPs, AQP-h2 and AQP-h3, are vasotocin-regulated water channels predominant in the osmoregulatory organs. Both the AQP-h2 and AQP-h3 are expressed at the granular cells underneath the keratinized layer in the pelvic patch, whereas only AQP-h2 is detected at the granular cells in the urinary bladder. In response to vasotocin, both the molecules seem to be translocated from the cytoplasmic pool to the apical plasma membrane of the granular cells. On the other hand, type-1 AQPs, Rana FA-CHIP and Hyla AQP-h1, are detected at the endothelial cells of blood capillaries in frog osmoregulatory organs. These findings suggest that AQP-h2 and AQP-h3 are key players for transepithelial water movement, and that FA-CHIP and AQP-h1 might be important for the transport of absorbed water into the blood flow. Comparative investigation of type-a2 AQPs in anurans further revealed that AQP-h2 and -h3-like molecules might exist at the urinary bladder and the pelvic skin, respectively, in various anurans from aquatic species to arboreal dwellers. AQP-h2-like protein is also detected in the pelvic skin of terrestrial and arboreal species. It is possible that this molecule might have occurred in the pelvic skin as anurans penetrated into drier environments.     

Molecular cloning and expression of aquaporin 1 [correction of aquapolin 1] (AQP1) in dog kidney and erythroblasts

Complementary DNA of the water channel aquaporin 1 (AQP1) was cloned from dog kidney and erythroblasts. The cDNA amplified from mRNA in dog kidney was 816 bp, the same as that in bovines, but longer by 6 bp than that in humans, mice and rats. The 235-bp fragment cDNA amplified from the mRNA in dog erythroblasts, which was differentiated from peripheral blood, was completely identical to the corresponding sequence of cDNA from the dog kidney. Thus, mature red blood cells from dog may have AQP1 in their cell membranes. The amino acid sequence in dog AQP1 was 91-94% identical to that in the other species mentioned above. Dog AQP1 has six predicted transmembrane domains, two NPA motifs, one mercury-sensitive site and four consensus phosphorylation sites, the same as the other species. However, dog and bovine AQP1 have only one N-glycosylation site, while two glycosylation sites were found in human and rodent AQP1. Xenopus oocytes injected with the mRNA of the dog AQP1 exhibited high water permeability in a hyposmotic medium. Thus, dog AQP1 performs water transport the same as in the other species.     

Seasonal effect in expression of AQP1, AQP3 and AQP5 in skin of Murrah buffaloes

Aquaporins are transmembrane protein channels which are known to help the passage of water and solutes across the cell membranes. AQP1, AQP3 and AQP5 are isoforms of aquaporin known to aid in transepithelial water movement. AQP3 is also known to aid in glycerol transport. The present study was conducted to investigate the role of AQP1, AQP3 and AQP5 in thermoregulation of buffaloes by probing the expression of the genes in skin of buffaloes during different season viz. winter, spring and summer. The skin tissue samples were collected from the neck region of Murrah buffaloes (n = 12) and analyzed for gene expression by RT-PCR and immunolocalization. The physiological responses including respiration rate, rectal temperature and neck skin temperature observed during summer were significantly higher than winter and spring seasons. The study revealed the expression of AQP1, AQP3 and AQP5 genes in skin samples. The relative mRNA expressions of AQP1, AQP3 and AQP5 in skin relative to spring season were 1.41 ± 0.47, 1.95 ± 0.22 and 6.77 ± 1.02 folds during summer which were significantly higher than other seasons. The up-regulation of the expression of the studied AQPs were concomitant with the increase in physiological responses including skin temperature and sweating rate during summer. During summer season, AQP1 were mostly immunolocalized in the walls of skin blood capillaries, while AQP3 were observed mostly in the epidermal layer of the skin. The immunolocalization of AQP5 were mostly observed in the secretory glands of skin. The up-regulation of AQP1, AQP3 and AQP5 in skin during summer season indicates their role in thermoregulation of buffaloes.     

Cloning of a new aquaporin (AQP10) abundantly expressed in duodenum and jejunum

A new aquaporin (AQP10) was identified in human small intestine. This gene encoded a 264-amino-acid protein with high sequence identity with AQP3 (53%), 9 (52%), and 7 (43%). These AQPs constitute one subfamily of AQP family that is differentiated from the other subfamily of AQP (AQP0, 1, 2, 4, 5, 6, and 8) by sequence homology. Ribonuclease protection assay and Northern blotting demonstrated almost exclusive expression of AQP10 mRNA in the duodenum and jejunum. In situ hybridization localized it in absorptive jejunal epithelial cells. Xenopus oocytes expressing AQP10 exhibited an increased osmotic water permeability in a mercury-sensitive manner. Although AQP10 belongs to the AQP subfamily, which has been characterized by permeability to water and neutral solutes such as urea and glycerol, it was not permeable to urea nor glycerol. The specific expression of AQP10 suggests its contribution to the water transport in the upper portion of small intestine.     

Expression and localization of aquaporins in the kidney of the musk shrew (Suncus murinus).

Expression and localization of members of the aquaporin (AQP) family (AQP1, 2, 3, 4, and 5) in the kidney of the musk shrew (Suncus murinus) was examined by immunohistochemistry. AQP1 was expressed in the proximal tubules and in the thin limb of the loops of Henle. AQP1 was the only water channel expressed in the proximal nephron examined, indicating that AQP1 may be an independent water transporter in the proximal nephron. AQP2 and AQP5 were localized to the apical cytoplasm of the cortical to medullary collecting duct (CD) cells and AQP3 and AQP4 were localized to the basal aspect of the cortical to medullary CD cells. AQP3 expression was weaker in the cortical cells compared with the medullary cells, whereas AQP4 was strongly positive throughout the CD. These indicate that the CD is the main water reabsorption segment of the nephron and is regulated by AQPs. Indeed, apical water transport of CD cells of the musk shrew may be controlled by both AQP2 and AQP5. The characteristic expression pattern of the AQPs in this animal provides a novel animal model for elucidating the regulation of water reabsorption by AQPs in the mammalian kidney.     

Comparative simulations of aquaporin family: AQP1, AQPZ, AQP0 and GlpF

Molecular dynamics simulations were performed for four members of the aquaporin family (AQP1, AQPZ, AQP0, and GlpF) in the explicit membrane environment. The single-channel water permeability, pf, was evaluated to be GlpF approximately AQPZ > AQP1 > AQP0, while their relative pore sizes were GlpF > AQP1 > AQPZ > AQP0. This relation between pf and pore size indicates that water permeability was determined not only by the channel radius, but also another competing factor. Analysis of water dynamics revealed that this factor was the single-file nature of water transport.     

Physiological roles of AQP7 in the kidney: Lessons from AQP7 knockout mice

The aquaporin7 (AQP7) water channel is known to be a member of the aquaglyceroporins, which allow the rapid transport of glycerol and water. AQP7 is abundantly present at the apical membrane of the proximal straight tubules in the kidney. In this paper, we review the physiological functions of AQP7 in the kidney. To investigate this, we generated AQP7 knockout mice. The water permeability of the proximal straight tubule brush border membrane measured by the stopped flow method was reduced in AQP7 knockout mice compared to wild-type mice (AQP7, 18.0 ± 0.4 × 10⁻³ cm/s vs. wild-type, 20.0 ± 0.3 × 10⁻³ cm/s). Although AQP7 solo knockout mice did not show a urinary concentrating defect, AQP1/AQP7 double knockout mice showed reduced urinary concentrating ability compared to AQP1 solo knockout mice, indicating that the contribution of AQP7 to water reabsorption in the proximal straight tubules is physiologically substantial. On the other hand, AQP7 knockout mice showed marked glycerol in their urine (AQP7, 1.7 ± 0.34 mg/ml vs. wild-type, 0.005 ± 0.002 mg/ml). This finding identified a novel pathway of glycerol reabsorption that occurs in the proximal straight tubules. In two mouse models of proximal straight tubule injury, the cisplatin-induced acute renal failure (ARF) model and the ischemic-reperfusion ARF model, an increase of urine glycerol was observed (pre-treatment, 0.007 ± 0.005 mg/ml; cisplatin, 0.063 ± 0.043 mg/ml; ischemia, 0.076 ± 0.02 mg/ml), suggesting that urine glycerol could be used as a new biomarker for detecting proximal straight tubule injury.