Aquaporin Water Channels in Mammalian Kidney

C H A P T E R

41 Aquaporin Water Channels in Mammalian Kidney Søren Nielsen1, Tae-Hwan Kwon2, Henrik Dimke3, Martin Skott4 and Jørgen Frøkiær5 1

Water and Salt Research Center, Department of Biomedicine, University of Aarhus, Aarhus, Denmark Department of Biochemistry and Cell Biology, School of Medicine, Kyungpook National University, Taegu, Korea 3 Department of Biomedicine, University of Aarhus, Aarhus, Denmark; The Samuel Lunenfeld Research Institute, Mt. Sinai Hospital, Toronto, Ontario, Canada 4 Department of Biomedicine, University of Aarhus, Aarhus, Denmark 5 Water and Salt Research Center, Department of Clinical Physiology, Aarhus University Hospital-Skejby, Aarhus, Denmark

2

INTRODUCTION Water is the most abundant component of all cells, and the ability to absorb and release water is considered a fundamental process of life. Plasma membranes serve as selective barriers that control the solute composition of the cell and regulate the entry of ions, small uncharged solutes, and even water. Epithelial tissues have apical and basolateral plasma membranes that constitute serial barriers that regulate the transepithelial movement of solutes and water, thereby contributing to the homeostasis of multicellular organisms. Identification and characterization of the molecular entities responsible for the function of biologic membranes have been long-standing goals of physiologists; however, the molecular identity of water transporters remained unknown until less than two decades ago. Because water can slowly diffuse through lipid bilayers, all biologic membranes exhibit some degree of water permeability. Nevertheless, observations made in multiple laboratories indicated that specialized membrane water-transport molecules must exist in tissues with distinctively high water permeability (see review 1). For example, the water permeability of red cell membranes is higher than that of many other cell types or artificial lipid bilayers, and the activation

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energy of this process is equivalent to the diffusion of water in solution, Ea ,5 kcal/mol.2 In addition, reversible inhibition by HgCl2 and a subset of organomercurials suggested that the water transporter is a membrane protein (see review 3). Further evidence that a membrane protein is involved in water transport was provided by the observation that some epithelial tissues exhibit changes in water permeability on a timescale that is not compatible with changes in lipid composition. Kidneys are the major determinant of body water and electrolyte composition. Thus, comprehending the mechanisms of water transport is essential to understanding mammalian kidney physiology and water balance. Because of its importance to human health, water permeability has been particularly wellcharacterized in the mammalian kidney (see review 4). Approximately 180 L/day of glomerular filtrate is generated in an average adult human, the majority of this is reabsorbed by the highly water-permeable proximal tubules and descending thin limbs of Henle’s loop. The ascending thin limbs and thick limbs are relatively impermeable to water, and empty into renal distal tubules and ultimately into the collecting ducts. The collecting ducts are extremely important clinically in water-balance disorders, because they are the chief site

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41. AQUAPORIN WATER CHANNELS IN MAMMALIAN KIDNEY

of regulation of water reabsorption. Basal epithelial water permeability in collecting duct principal cells is low, but the water permeability can become exceedingly high when stimulated with vasopressin (also known as ADH, antidiuretic hormone). In this regard, the toad urinary bladder behaves like the collecting duct, and it has served as an important model of vasopressin-regulated water permeability. Stimulation of this epithelium with vasopressin produces an increase in water permeability in the apical membrane, which coincides with the redistribution of intracellular particles to the cell surface.5
8 These particles were believed to contain water channels.

DISCOVERY OF AQUAPORIN-1 (AQP1) The molecular identity of membrane water channels long proved elusive. Attempts to purify water channel proteins from native tissues or to isolate water channel cDNAs by expression cloning, were unproductive (see review 9). This may be explained by the physical characteristics of water, a simple molecule not amenable to chemical modification such as introduction of chemical cross-linking groups or labels. In addition, HgCl2 was known to inhibit membrane water channels, a property potentially useful in the identification of the water channel proteins. However, because the agent reacts with free sulfhydryls in other proteins, its inhibitory effect on water channels is not specific. This circumstance led to the mistaken identification of the band 3 anion exchanger as a molecular water channel.10 In addition, the diffusional permeability of all biologic membranes results in high background permeability, and frustrated efforts to clone cDNAs for water channels by functional expression. The recognized characteristics of membrane water channels led to chance identification of the first known water channel. In the process of isolating the 32 kDa bilayer-spanning polypeptide component of the red cell Rh blood group antigen,11 a 28 kDa polypeptide was partially co-purified.12 Initial studies demonstrated that the 28 kDa polypeptide comprised hydrophobic amino acids and exhibited an unusual detergent solubility, which facilitated purification and biochemical characterization. The 28 kDa polypeptide was found to exist as an oligomeric protein with physical dimensions of a tetramer; a unique N-terminal amino acid sequence was identified13 which permitted cDNA cloning.14 Also of note, radiation inactivation studies of water permeability by renal vesicles yielded a target size of 30 kDa.15 Because the 28 kDa polypeptide was found to be abundant in red cells, renal proximal tubules, and descending thin limbs,12 it was suggested that this protein might be the sought-after water

channel. Although this protein was first known as “CHIP28” (channel-like integral protein of 28 kDa), the need for a functionally relevant name was recognized. The name “aquaporin” was coined. After recognition of related proteins with similar functions, this name was formally proposed for the emerging family of water channels now known as the aquaporins.16 Thus, CHIP28 was designated aquaporin-1 (symbol AQP1). The Human Genome Nomenclature Committee has embraced this nomenclature for all related proteins,17 and presently a total of 13 such related proteins have been identified in mammals. The measurement of the movement of water across cell membranes poses a unique experimental challenge. Unlike ion conductances, which may be measured electrophysiologically, or solute transport, which may be measured with radioactive substrates, transmembrane water movement in cells relies on determination of changes in cell volume in response to an osmotic driving force. The Xenopus laevis oocyte expression system was used to search for water channel RNAs, because these cells are known to exhibit remarkably low membrane water permeability.18,19 Oocytes injected with cRNA encoding AQP1 exhibit remarkably high osmotic water permeability (Pf B200 3 10217 cm/s), causing the cells to swell rapidly and eventually rupture in hyposmotic buffer.20 In contrast, control oocytes not injected with AQP1 cRNA exhibited less than one tenth of this permeability. Oocyte studies demonstrated that AQP1 behaves like the water channels in native cell membranes.20 Osmotically-induced swelling of oocytes expressing AQP1 occurs with a low activation energy, and is reversibly inhibited by HgCl2. Moreover, AQP1 oocytes fail to demonstrate any measurable increase in membrane current. Although these early studies demonstrated only swelling of oocytes, it was predicted that the direction of water flow through AQP1 is determined by the orientation of the osmotic gradient. Thus, AQP1 oocytes swell in hyposmolar buffers but shrink in hyperosmolar buffers.21 To confirm that the interpretation of the oocyte studies was correct, highly purified AQP1 protein from human red cells was reconstituted with pure phospholipids into proteoliposomes, which were compared with simple vesicles (liposomes) by rapid transfer to hyperosmolar buffer.22 These studies permitted determination of the unit water permeability, which had an astonishingly high value (Pf B3 3 109 water molecules subunit2111 sec2111). Moreover, the water permeability is reversibly inhibited by HgCl2, and exhibits low activation energy. Several of these studies have been confirmed by using red cell membranes partially depleted of other proteins,23 and attempts to demonstrate permeation by other small solutes or even

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STRUCTURE AND FUNCTION OF AQUAPORINS

protons showed that AQP1 is water selective.24 Together, these studies indicated that AQP1 is both necessary and sufficient to explain the well-recognized membrane water permeability of the red cell, and suggested that AQP1 or similar proteins could be the longsought-after epithelial water channels of the nephron and collecting ducts.

STRUCTURE AND FUNCTION OF AQUAPORINS General Structure of AQP1 The availability of pure AQP1 protein in milligram quantities and the simple functional assay in oocytes led to rapid advances in the understanding of the molecular structure of AQP1. Hydropathy analysis of the deduced amino acid sequence of AQP1 predicted that the protein resides primarily within the lipid bilayer,14 a feature in agreement with initial studies of red cell AQP1.12,13 As previously described for the homolog major intrinsic protein from lens (MIP, now referred to as AQP0), the polypeptide contains an internal repeat (Figure 41.1), with the N- and C-terminal halves being sequence-related, and each containing the signature motif Asn-Pro-Ala (NPA),25,26 suggesting ancestral gene duplication.14 When evaluated by

A/V45 Colton polymorphism

N42 polylactosaminoglycan

C189 Hg site

A P N

C

A 1

2

3

E 4

5

Extracellular 6 Lipid bilayer Intracellular

B H2N

N P A

D Repeat-2

HOOC

Repeat-1

FIGURE 41.1 Proposed membrane topology of AQP1 subunit. Each molecule subunit spans the plasma membrane six times with the N- and C-termini in the cytoplasmic space. Extracellular and intracellular loops are labeled A
E.32 Loop A contains an attachment site for a polylactosaminoglycan at asparagine-42 and the Co blood group polymorphism at alanine/valine-45.100 The aquaporin signature motif asparagine-proline-alanine (NPA) is present in the loop Band E. Loop E also contains the site of mercury inhibition at cysteine-189. The amino acid sequences of C-termini of the known aquaporins are not conserved, and are sufficiently immunogenic to permit preparation of specific polyclonal antibodies to each aquaporin (see text) (reproduced and modified from ref. [370], with permission).

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hydropathy analysis, six bilayer-spanning domains, five connecting loops (A
E), and intracellular N-and C-termini are predicted. Attenuated total reflectionFTIR (Fourier Transform InfraRed spectroscopy) of highly-purified red cell AQP1 reconstituted into membrane crystals demonstrated a lack of beta structure in AQP1, indicating the existence of tilted alpha helices.27 The two homologous domains equivalent to the N-and C-termini halves of the protein each consist of three transmembrane helices that are thought to be oppositely orientated in the lipid bilayer.28 A system was adapted for analyzing the structure of AQP1 after minimally perturbing the molecule by adding peptide epitopes at various sites. It was important that the epitope did not destroy function, and could be localized to intracellular or extracellular sites with antibodies and by selective proteolysis of intact membranes or inside-out membrane vesicles. These studies demonstrated that loop C resides at the extracellular surface of the oocytes and the intracellular location of loop D as well as the N-and C-termini. Moreover, the obverse symmetry of the N-and C-terminal halves of the molecule was confirmed29 (Figure 41.1).

Loops B and E Loops B and E encompass the two NPA motifs, and are the most conserved regions in the major intrinsic protein family. The loops exhibit significant hydrophobicity, suggesting association with the lipid bilayer.14 Subsequent studies implicated loops B and E as a structural component of the aqueous pathway. Experiments expressing AQP1 in Xenopus oocytes led to the observation that Cys189 in loop E is the site of mercurial inhibition.30,31 Site-directed mutagenesis experiments in oocytes revealed that substitution of Cys189 with a serine residue eliminates HgCl2 sensitivity and increases osmotic water permeability, while substitution with larger amino acids residues prevents facilitated water transport.30 These results suggest that water transport and selectivity in AQP1 is somehow dependent on the steric properties of Cys189. In accordance with the internal repeat theory, Ala73 located in loop B, the equivalent to Cys189 in loop E, was investigated. By creating double mutants expressing Cys189 as a serine and Ala73 as a cysteine, the HgCl2 sensitivity and osmotic water permeability were restored.32 These results suggested that loops B and E were arranged in a symmetrical fashion, and underlined that these loops were functionally essential for water permeability. This line of inquiry lead to the “hourglass” model,32 in which these domains overlap midway between the leaflets of the bilayer, creating a constitutively open, narrow aqueous pathway (Figure 41.2).

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Hemipore-1

Hemipore-2

A

C 1

Structural Analyses and Molecular Dynamics of Aquaporins

C

NPA

2

3

4

5

N

E

P

A

6

A B

D

H2N

HOOC Hourglass C

A 6 1

5 E

2

C P A N A N P A B

3

4

D

H2N

HOOC

FIGURE 41.2 Hourglass model of aquaporin-l (AQP1) structure. Bilayer spans 1
3 (Repeat-l) and spans 4
6 (Repeat-2) are sequencerelated, and are oriented in obverse symmetry. Loop B and loop E are believed to dip into the bilayer and emerge from the same side (top). When folded together, loops B and E form a single aqueous pathway (the “hourglass”), flanked by the site of mercury inhibition at cysteine189 (bottom) (reproduced and modified from ref. [32], with permission).

Tetrameric Organization Early biophysical characterization of AQP1 suggested that the protein formed multisubunit oligomers.13 Rotary and unidirectionally shadowed freeze-fracture electron microscopic analyses of AQP1 protein from human red cell membranes reconstituted in proteoliposomes provided detailed molecular insights. AQP1 had an oligomeric structure, consisting of four subunits surrounding a central depression. These tetrameric structures were also seen in highly water-permeable nephron segments expressing native AQP1.33 Although the oligomerization of AQP1 is still not understood in detail, all studies indicate that the protein is a tetramer composed of functionallyindependent aqueous pores.15,30,32,34

By reconstituting the highly-purified red cell AQP1 into membranes under controlled conditions, membrane crystals were produced with AQP1 in highly uniform lattices. These membranes appeared as flat sheets or as large, resealed vesicles in which the AQP1 protein was found to fully retain water permeability.35 Thus, the opportunity to define the structure of AQP1 in a biologically-active state became possible. Electron microscopic studies by multiple groups permitted the elucidation of the protein at increasing levels of resolution. By performing high-resolution electron microscopic evaluation of negatively-stained membranes at a series of tilts, a three-dimensional view was obtained.36 Image projections revealed the presence of multiple bilayer-spanning domains, and atomic force microscopy further defined the orientation and extramembranous dimensions of AQP1.37 Electron crystallographic analysis of cryopreserved specimens has been undertaken at tilts of up to 60 . These analyses revealed the three-dimensional structure of AQP1 at increasing ˚ .38
46 X-ray crystallographic resolutions, down to 3.8 A analysis has added further information about the structure of AQP1. Using the above method, the structure of AQP1 has been determined down to a resolution of ˚ .47 In contrast to the previous studies, the high 2.2 A resolution enables observation of water molecules in transit through the channel. Based on combined efforts, a detailed picture of AQP1s tertiary structure could be formed. As earlier studies indicated, the AQP1 monomers form a tetrameric cluster. The model shows an extension of the tetramer from the extracellular plane, while the intracellular surfaces form a shallow depression. The N-termini from one monomer is closely situated near the C-termini of the neighboring monomer. The ˚ gap formed by the four monomers narrows from 8.5 A ˚ down to 3.5 A. The residues surrounding the gap are hydrophobic, suggesting an interaction with a hydrophobic molecule.43 So, despite the monomeric formation of a central cavity, the tetrameric structure does not support the transport of water. This is in agreement with earlier observations, suggesting that each monomer is capable of facilitating water transport. ˚ across The dimensions of each monomer are 40 A 47 ˚ and 60 A long, as reported by Sui et al. (Figure 41.3). The monomer is composed of six bilayer-spanning alpha helices surrounding a central density. The C loop located on the extracellular surface connects the N-and C-termini halves of the protein. Part of the central density represents the B and E loops, which appear as two short α-helix structures not permeating the

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FIGURE 41.3 (a)
(b): Structure of the human AQP1 tetramer, viewed from the top (a) and side (b). (c): Structure of the human AQP1 monomer. (d): Model of the central pore region of human AQP1-EM3 structure (pdb ˚ resolution EM potencode 1H6I 371 together with the 3.8 A tial map rendered at 1.0s.43 Several residues critical for AQP1 facilitated water transport are marked (reproduced and modified with permission from ref. [372]).

membrane42 (Figure 41.3). This organization is strikingly similar to the proposed hourglass arrangement of the B and E loops.32 The NPA containing loops are located on opposite sides of the membrane, juxtapositional to each other, interacting through their NPA motifs. The central density is composed of an extracellular and intracellular vestibule, separated by a narrow ˚ pore. The extracellular vestibule is approximately 15 A 47 at its widest. Following the extracellular vestibule towards the lipid bilayer, a decrease in diameter occurs and the pore becomes exceedingly small.43,47 The constriction site (also referred to as the aromatic/arginine ˚ from the beginning of the constriction) is located 20 A extracellular vestibule, and is composed of several highly-conserved residues in conventional aquaporins. ˚ wide, The constriction site is approximately 2.8 A 47 about the diameter of a water molecule. After the ˚ , a region constriction site the pore continues for 20 A 47 termed the “selectivity filter”. The selectivity filter is slightly wider and part of the region is formed by the helical loops B and E.43,47 This locates the asparagine residues in the NPA motifs within the selectivity filter.43,47 Additionally, Cys189 in loop E protrudes into the pore, confirming earlier studies, suggesting that HgCl2 sensitivity was due to steric hindrance of water movement through the pore.32,43,47 Following the pore towards the intracellular vestibule, the diameter ˚ 47 (Figure 41.3). increases again, reaching 15 A Using molecular dynamic simulations, de Groot and Grubmu¨ller obtained time-resolved, atomic resolution

models of water transport through AQP1.48 From the extracellular vestibule, water molecules enter the constriction site, composed of side chains from the aromatic Phe56 and His180, and the charged Arg195. This conformation situates the water molecule, allowing hydrogen bonding with the polar residues, thereby reducing hydrogen bonding between water molecules. In addition, electrostatic repulsion by Arg195 suggests that the constriction site is the main site for exclusion of protons (hydronium ions) and other ions.48 This is further supported by mutagenic analysis of the residues in the constriction site, showing permeation of urea, glycerol, and ammonia in AQP1 when altered. Moreover, replacing Arg195 with a valine residue appears to facilitate proton transport.49 Passing onward through the hydrophobic selectivity filter, exposed polar moieties mainly consisting of backbone carbonyls, lead the water molecules towards the NPA motifs. The water molecule transiently reorientates to bond with the two asparagines residues of the NPA motifs, and is led out of the selectivity filter towards the intracellular vestibule, again by hydrogen bonding with a few selected backbone carbonyls. Hence, the selectivity filter encompassing the highly-conserved NPA motifs appears to serve mainly as a filter for size,48 while the ar/R constriction is a major checkpoint for solute amd ion permeability. The Escherichia coli aquaglyceroporin (GlpF) selectively facilitates glycerol transport over that of water.50 Evaluation of differences between AQP1 and GlpF using molecular dynamic

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simulations revealed a larger selectivity filter in GlpF. Moreover, the preference for glycerol could be explained by what de Groot and Grubmu¨ller describe as a glycerol-mediated “induced fit” gating motion (i.e., glycerol transport serves as the prime mechanism for water exclusion).48 Structural characterizations of crystallized human AQP2 in two-dimensional protein
lipid arrays by atomic force microscopy and electron crystallography have revealed the structure of AQP2 at a resolution of ˚ in the membrane plane.51 As with AQP1, AQP2 4.5 A is found in a tetrameric assembly in the lipid bilayer.51,52 The AQP2 monomer shows structural features similar to those earlier reported for AQP1,51 while structural variation is found around the tetramer’s axis.51,53 The structure of AQP0 has also been determined ˚ .54,55 The water conducdown to a resolution of 2.2 A tance of AQP0 is much lower than that reported for AQP1,56 possibly due to highly-conserved tyrosine residues in AQP0 imposing further constriction on the channel.55 The constriction site is smaller than that in AQP1, and the selectivity filter is also narrower.54,55 Molecular dynamics studies suggest that water movement is facilitated by AQP0 due to thermal motions of certain side chains, although this builds up a free energy barrier, possibly contributing to the lower permeability of AQP0.57 The extensive analyses of the structure of various AQP isoforms have provided detailed insight into the molecular basis for transmembrane water transport. Future studies aimed at defining the distinct structure of other members, including aquaglyceroporins such as AQP7 and AQP9 that appear to be involved in metabolism rather than water transport may provide further insights. Several studies have also been aimed at identifying novel aquaporin blockers. Although some progress has been reported so far, only a few compounds, including related tetraammonium compounds, have shown selective effects on AQPs.58
60 Copper61 and nickel62 also significantly block aquaporins. Over the past five years the overall structural concepts of aquaporins have been confirmed.63
74

DISTRIBUTION OF AQP1 IN KIDNEY AND OTHER TISSUES Well before recognition of its function, the red cell AQP1 protein was known to be expressed at high levels in the proximal convoluted tubules and descending thin limbs of kidney.12 This was confirmed with polyclonal rabbit antiserum,75 and was defined in rat and human kidney with affinity-purified immunoglobulin specific for the N- and C-terminal domains of

FIGURE 41.4 Distribution of aquaporin 1 (AQP1) in kidney. Ultrathin cryosections of rat renal cortex and inner medulla stained with anti-immunogold AQP1. (a): Strong labeling is present over the apical brush border of S3 section of proximal tubule. (b): Strong labeling is present over apical and basolateral membrane of thin descending limb of Henle’s loop (reproduced and modified from ref. [77], with permission).

AQP1.76,77 In all studies, AQP1 was demonstrated to be constitutively present in the apical plasma membranes (i.e., the brush borders), and in basolateral membranes of S2 and S3 segment proximal tubules (Figure 41.4). Quantitative immunoblotting indicated that AQP1 makes up 0.9% of total membrane protein from rat renal cortex and 4% of brush border proteins.77 Enzyme-Linked Immunosorbent Assay (ELISA) measurements of microdissected tubules revealed that proximal tubules contain approximately 20 million copies of AQP1 per cell.78 Additionally, AQP1 is found in the plasma membrane of glomerular mesangial cells in humans, although not in rat.76,77,79 Other immunohistochemical and immunogold electron microscopic studies have demonstrated AQP1 in multiple capillary endothelia throughout the body,80
82 including the renal vasa recta.83 AQP1 is also abundant in peribronchiolar capillary endothelium, where expression is induced by glucocorticoids,80,84 apparently acting through the classic glucocorticoid response elements in the AQP1 gene.85 In addition, AQP1 has been defined in multiple water-permeable epithelia including choroid plexus, peritoneal mesothelial cells,86 fetal

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AQP1 DEFICIENCY

membranes,87 at multiple locations in eye, including ciliary epithelium, lens epithelium, and corneal endothelium,81,88,89 and in hepatobiliary epithelium,90 pancreatic interlobular ducts,91 heart and skeletal muscle,92 gall bladder,93 salivary glands,94 inner ear,95 and several other organs including the nervous system (see review 96). AQP1 has also been localized in tumor cells and their vasculature.97 Developmental expression of AQP1 is complex: transient expression occurs in some tissues before birth; expression in other tissues is subsequent to birth; constitutive life-long expression is found in other tissues.84,98,99

AQP1 DEFICIENCY Humans have been identified who totally lack the AQP1 protein. The human AQP1 gene was localized to chromosome 7p14 and the Co blood group antigens were previously linked to 7p, suggesting a molecular relationship. It was determined that the Co blood group antigen results from an Ala/Val polymorphism at the extracellular surface of red cell AQP1100 (Figure 41.1). Although the International Blood Group Referencing Laboratory in Bristol, England, has detailed phenotyping information on millions of donors worldwide, only six individuals had been shown to lack Co. Most of these Co-null individuals are women who developed anti-Co during pregnancy. Three of these Co-null individuals were found to have mutations in the AQP1 gene.101 Although the exceedingly rare blood group phenotype makes them impossible to match for blood transfusion, it was surprising that none of them exhibited any other obvious severe clinical phenotype. The extreme rarity of the Co-null state may reflect an important developmental role, resulting in reduced fetal survival; however, the frequency of the heterozygous state is unknown. Detailed studies of the urinary concentrating ability of Co-null individuals revealed a marked inability of the individuals to concentrate urine to more than 400 mOsm/l, even in the presence of dehydration or dDAVP treatment, revealing a significant urinary concentrating defect.102 Moreover, Co-null individuals display reduced pulmonary vascular permeability.103 The development of AQP1 gene knockout mice has provided further insights into the role of AQP1 in renal water homeostasis. AQP1-null (AQP12/2) mice appeared moderately polyuric under basal conditions. However, AQP12/2 animals exhibited an extreme degree of polyuria and polydipsia when undergoing water deprivation, including rapid hyperosmolar extracellular fluid volume depletion.104 Additionally, AQP12/2 mice failed to respond appropriately to vasopressin, suggesting that renal water conservation and

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urinary concentration is highly dependent on AQP1 protein.104 Detailed classic in vivo and in vitro physiological evaluation of proximal tubules from the AQP12/2 mice established that transepithelial water permeability was reduced by 80%, and led to an approximate 50% decrease in proximal tubule fluid reabsorption.105 The apparent differences in transepithelial water permeability and proximal tubule fluid reabsorption are likely dependent on the generation of a hypotonic filtrate in proximal tubules of AQP12/2 mice.106 Despite these observations, distal fluid delivery in AQP12/2 remained unchanged, due to a compensatory reduction in single nephron glomerular filtration.105 When blunting the TGF response, and thereby the compensatory reduction in glomerular filtration observed in AQP12/2 mice, ambient urine osmolalities and urinary flow rates appeared no different from normal AQP12/2 mice, probably due to distal tubular compensatory mechanisms.107 The impaired proximal fluid reabsorption observed in AQP12/2 mice, albeit with normal distal fluid delivery, suggests that the polyuric phenotype largely depends on a concentrating defect impairing collecting duct water reabsorption Studies using isolated perfused thin descending limbs have revealed that the osmotic water permeability of the type II thin descending limbs (outer medullary thin descending limbs from long loop nephron) is decreased by 90% relative to wild-type values in kidneys from AQP1 knockout mice.108 Additionally, earlier observations using freeze-fracture electron microscopic techniques showed a remarkably high density of intramembrane particles in the thin descending limb of rat, which has been attributed to the tetrameric assembled AQP1 subunits.33 In the thin descending limb of AQP12/2 mice, the abundance and size of these intramembrane particles was markedly reduced.108 Moreover, the distribution of AQP1 in the vasa recta suggests a role in microvascular exchange, hence affecting urinary concentration. In the presence of a NaCl gradient, osmotic water permeability was almost eliminated in the descending vasa recta of AQP12/2 mice, leading to a predicted reduction in medullary interstitial osmolality and likely an impairment of countercurrent multiplication.109 Additional studies using adenoviral gene delivery restored AQP1 protein expression in the proximal tubule epithelia and renal microvessels of AQP12/2 knockout mice, albeit not in the descending thin limbs. The adenovirustreated mice showed slight restoration of the concentrating defect during water deprivation, probably due to reinsertion of AQP1 water channels in the vasa recta, although urinary concentrating ability was still highly insufficient in comparison to wild-type mice.110 In conclusion, the severe concentrating defect seen in the AQP12/2 mice primarily results from impaired water

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absorption in the thin descending limb, underlining the necessity for a constitutively high water permeability in this segment for a functional countercurrent multiplication system.

ADDITIONAL FUNCTIONS OF AQP1 AQP1 is generally believed to be a constitutively active, water-selective pore. Nonetheless, some observations contradict this. A small degree of permeation by glycerol has been seen in oocytes which may represent opening of an unidentified leak pathway,111 and the biologic significance remains unclear.21 Forskolin was reported to induce a cation current in AQP1expressing oocytes,112 but multiple other scientific groups have failed to reproduce this effect.113 Although small changes in water permeability by oocytes expressing a bovine homolog of AQP1 have also been ascribed to vasopressin and atrial natriuretic peptide, the significance is uncertain.114 Likewise, secretin-induced membrane trafficking has been noted in isolated cholangiocytes115,116; however, this awaits confirmation by immunoelectron microscopy. Permeation of AQP1 by CO2 has also been evaluated. Rates of pH change are about 40% higher in oocytes expressing AQP1117 than in control oocytes. Although the background permeation of lipid bilayers by CO2, as well as oxygen, ammonia, nitric oxide, and other gases, may be high,118 the potential physiological relevance of AQP1 permeation by gases warrants more study (see review 119). Additionally, AQP1 appears to play a role in cell migration.120 AQP12/2 mice implanted with tumor cells show reduced tumor vascularity and growth, thus leading to improved survival in comparison to wild-type mice. Moreover, in vitro analysis of endothelial cells isolated from AQP12/2 mice showed marked impairment in cell migration.120 In primary cultures of proximal tubule cells from AQP12/2 mice, in vitro cell migration was impaired compared to AQP11/1 mice. Furthermore, in an ischemia
reperfusion model of acute renal tubular injury, AQP12/2 mice showed more severe pathological changes, including more prominent tubule degeneration.121 Thus, although the evidence that AQP1 functions as a water channel is incontrovertible, the possibility of yet undiscovered transport functions cannot be excluded.

AQUAPORINS IN KIDNEY The first functional definition of one member of a protein family often prompts a search for related proteins. This has certainly been the case for the

aquaporins, and the homology cloning approach has been undertaken by multiple laboratories whose combined efforts have expanded the aquaporin family membership list. Homology cloning has most frequently been undertaken by using polymerase chain amplification with degenerate oligonucleotide primers.122 Thirteen mammalian aquaporins are now known, and they form at least two subgroups: waterselective channels (orthodox aquaporins) and channels permeated by water, glycerol, and other small molecules (aquaglyceroporins). Given the large potential for confusion, the Human Genome Organization has established an Aquaporin Nomenclature System,117 accessible by internet (http://www.gene.ucl.ac.uk/ nomenclature). Of the known aquaporins, eight are expressed in mammalian kidney (Table 41.1). Soon after AQP1 was discovered to be a water channel, AQP2 was identified in renal collecting duct,123 where it is regulated by vasopressin and is involved in multiple clinical disorders (Table 41.2). AQP3 was identified in kidney and other tissues, and was found to be permeated by glycerol and water.124
126 AQP4, a HgCl2insensitive water channel is most abundantly expressed in brain, and is present in kidney collecting duct in the basolateral plasma membrane of principal and IMCD cells and in other tissues, but it is not inhibited by mercury.127,128 AQP6 was identified at the cDNA level and found to be localized in intracellular vesicles in the collecting duct intercalated cells in the kidney.129
131 AQP7 is permeated by water and glycerol.132,133 First cloned from testis,132 AQP7 is present in segment 3 proximal tubule brush border membranes, where it facilitates glycerol and water transport.134 AQP8 is a HgCl2-sensitive water channel found in intracellular domains of proximal tubule and collecting duct cells135,136; however, its function remains unclear. AQP11 is found in the cytoplasm of renal proximal tubule cells.137 The exact function is not established, although deletion of the AQP11 gene produces a severe phenotype with renal vacuolization and cyst formation.137

Localization and Function of AQP2, AQP3, AQP4, AQP6, AQP7, AQP8, and AQP11 in Kidney The amino acid sequences of the N-and C-termini of the different aquaporins are not closely related, and specific polyclonal antibodies can be raised in rabbits immunized with synthetic peptides conjugated to carrier proteins such as keyhole limpet hemocyanin. As with AQP1, these antibodies have permitted localization of the other aquaporins in kidney by immunocytochemistry, immunoelectron microscopy, and single

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AQUAPORINS IN KIDNEY

TABLE 41.1 Aquaporins in Kidney AQP

Localization (renal)

Subcellular Distribution

Regulation

Localization (extrarenal)

AQP1

S2, S3 segments of proximal tubules

Apical and basolateral plasma membranes

Glucocorticoids (peribronchiolar capillary endothelium)

Multiple tissues, including capillary endothelia, choroids plexus, ciliary and lens epithelium, etc.

AQP2

Collecting duct principal cells

Intracellular vesicles, apical plasma membrane

Vasopressin stimulates short-term exocytosis long-term biosynthesis

Epididymis

AQP3

Collecting duct principal cells

Basolateral plasma membrane

Vasopressin stimulates long-term biosynthesis

Multiple tissues, including airway basal epithelia, conjunctiva, colon

AQP4

Collecting duct principal cells

Basolateral plasma membrane

Dopamine, protein kinase C

Multiple tissues, including central nervous system astroglia, ependyma, airway surface epithelia

AQP6

Collecting duct intercalated

Intracellular vesicles

Rapidly gated

Unknown

AQP7

S3 proximal tubules

Apical plasma membrane

Insulin (adipose tissue)

Multiple tissues, including adipose tissue, testis, and heart

AQP8

Proximal tubule, collecting duct cells

Intracellular domains

Unknown

Multiple tissues, including gastronintestinal tract, testis, and airways

AQP11 Proximal tubule

Intracellular domains

Unknown

Multiple tissues, including liver, testes, and brain

AQP, aquaporin.

tubule microdissection combined with ELISA (Figure 41.5). Three aquaporins (AQP2, AQP3, and AQP4) are expressed in the collecting duct principal cells and the connecting tubule segment, sites where vasopressin regulates epithelial water permeability. AQP2 is located in the apical plasma membrane and small intracellular vesicles of collecting duct principal cells138 (Figure 41.6). ELISA measurements of microdissected rat collecting ducts have revealed that AQP2 is an extremely abundant protein in the collecting duct, with more than six million copies per cell throughout the collecting duct system.139 Additional studies using the single-tubule ELISA method and immunocytochemistry demonstrated co-localization of AQP2 and V2 vasopressin receptor expression on connecting tubule arcades, suggesting that regulated water transport may occur in the arcades of the cortical labyrinth in addition to the collecting duct.139,140 Additionally, AQP2 has been identified in the basolateral membranes of connecting tubules and inner medullary collecting ducts.141 The apical plasma membrane is the rate-limiting barrier for transepithelial water transport across the collecting duct principal cell, and is the site where vasopressin regulates water permeability.142 Localization of AQP2 in the apical plasma membrane suggested that it is the target for vasopressin-regulated collecting duct water permeability (Figure 41.6). This conclusion was firmly established by multiple studies. A direct correlation between AQP2 expression and

collecting duct water permeability has been demonstrated in Brattleboro rats, which lack circulating vasopressin owing to a mutation in the gene encoding vasopressin precursor protein.143 Likewise, human patients with mutations in the AQP2 gene manifest severe vasopressin-resistant nephrogenic diabetes insipidus,144 demonstrating the requirement for AQP2 in collecting duct water transport. Lack of functional AQP2 expression in mice, by generation of AQP2 gene knockouts, produces a severe concentration defect, resulting in early postnatal death.145,146 Moreover, morphological changes including renal medullary atrophy and dilation of the collecting ducts is observed in these mice.145,146 Generation of AQP22/2 knockouts selectively in the collecting ducts, but not in the connecting tubule segments, rescues mice from the lethal phenotype; however, body weight, urinary production, and the response to water deprivation is still severely impaired.145 Additionally, in a tamoxifen-inducible mouse model of AQP2 gene deletion, adult mice present with severe polyuria, a marked urinary concentration defect, and develop mild renal damage.147 Together these studies demonstrate the pivotal role of AQP2 in urinary concentration. AQP3 is localized in a large variety of organs and highly-expressed in the kidney. AQP3 was originally cloned from rat kidney,124
126 and when expressed in Xenopus oocytes it facilitated glycerol transport (discussed below) and increased HgCl2-sensitive osmotic water permeability.148
150 Moreover, channel-gating is

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TABLE 41.2 Water-balance Disorders Associated with Aquaporin Abnormalities Congenital defects Central diabetes insipidus Mutation in gene encoding vasopressin (Brattleboro rat) Nephrogenic diabetes insipidus Mutations in gene encoding V2 receptor (human, X-linked) Mutations in gene encoding aquaporin-2 (human, dominant and recessive) Partial concentration defects Targeted disruption of gene encoding aquaporin-1 (mouse) Targeted disruption of gene encoding aquaporin-4 (mouse) Acquired defects Lithium treatment Hypokalemia Hypercalcemia Postobstructive nephropathy, unilateral or bilateral Conditions with water retention Congestive heart failure Hepatic cirrhosis Nephritic syndrome Pregnancy Other conditions Syndrome of inappropriate vasopressin secretion Primary polydipsia Chronic renal failure Acute renal failure Low-protein diet Age-induced reduction in renal concentration

regulated by protons and copper.61,150 AQP4 was found to be expressed in several tissues with high abundance in the brain. In oocyte expression studies, the protein increased osmotic water permeability in a HgCl2-insensitive manner.127,149 AQP3 and AQP4 are also present in the connecting tubule and collecting duct principal cells; however, the sites where they are expressed do not overlap with the expression of AQP2 in most portions of the collecting duct.140,151
153 AQP3 and AQP4 are restricted to the basolateral plasma membranes of collecting duct principal cells, where they are presumed to permit water entry into the interstitium. Although both are basolateral water channels, they are distributed differently

along the collecting duct system, with the greatest abundance of AQP3 in cortical and outer medullary collecting ducts, and the greatest abundance of AQP4 in inner medullary collecting duct.153 AQP4 is also found in basolateral membranes of proximal tubule S3 segments, although only in mice.154 Using freezefracture electron microscopy, orthogonal arrays of intramembrane particles (OAP) were demonstrated in the basolateral membranes of the proximal tubule S3 segment in AQP41/1, but not in AQP42/2, mice.154 AQP4 is expressed in two splice isoforms, the M1 and M23 splice variants. The M23-AQP4 appears to be the OAP-forming water channel when expressed in LLCPK1 cells. When organized in OAPs, a significantly higher single-channel water permeability coefficient is observed in comparison to the non-AOP forming M1AQP4.141,155 Shuttling of AQP3 or AQP4 is not expected to occur, as the predominant fraction is found in the plasma membrane (short- and long-term regulation of these aquaporins will be discussed later). Sodium restriction or aldosterone infusion in normal and Brattleboro rats greatly increases the abundance of AQP3, while AQP3 abundance is markedly reduced during aldosterone deficiency, suggesting a direct effect of aldosterone on collecting duct AQP3 expression.156 AQP4 appears to be regulated by PKC and dopamine, where stimulation by these factors decreases water permeability in AQP4 transfected cells.157 Deletion of the AQP3 gene induces polyuria and polydipsia and a marked reduction in osmotic water permeability in the basolateral plasma membrane of the cortical collecting duct.148 Moreover, urinary osmolality is reduced in AQP32/2 mice and they fail to respond appropriately to dDAVP, thus presenting with a urinary concentrating defect.148 Targeted disruption of AQP4 in mice results in a 75% reduction in the osmotic water permeability of the inner medullary collecting duct.158 However, phenotypically the AQP42/2 mice appeared grossly normal, presenting with a very mild urinary concentrating defect.159 In double AQP32/2/AQP42/2-knockout mice, concentrating ability was only slightly more impaired than in the AQP32/2 mice.148 It should be noted that the localization of AQP2 in basolateral membranes in both the connecting tubule and inner medullary collecting duct raises the possibility that the observed effect is partly compensated by this mechanism.141 AQP6 vesicles reside in subapical vesicles within intercalated cells of the collecting duct, where it is co-expressed with the V-type H1-ATPase.131,160 AQP6 appears functionally distinct from other known aquaporins. Oocyte expression studies have revealed low water permeability of AQP6 during basal conditions, while in the presence of HgCl2 a rapid increase in water permeability and ion conductance is observed.

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AQUAGLYCEROPORINS

Distal convoluted tubule Connecting tubule Glomerulus AQP2 Water AQP3 Proximal AQP1 AQP4 tubule AQP7 +ADH Water Water Descending thin limb

Water

Cortex Outer stripe Outer Inner stripe medulla

Collecting ducts

Water Ascending thin limb

Inner medulla

+ADH Water

Final urine

FIGURE 41.5 Sites of aquaporin (AQP1) expression in kidney and segmental water-transport function. Proximal nephron: AQP1 is present in apical and basolateral plasma membranes of proximal tubules and thin descending limbs. AQP6 is present in intracellular vesicles of collecting duct intercalated cells. AQP7 is present in proximal tubules. Distal nephron: aquaporins are not present in ascending limbs. Collecting duct: AQP2 is present in intracellular vesicles and apical plasma membranes of principal cells; AQP3 and AQP4 are present in basolateral membranes. AQP6 is present in intracellular vesicles in type A intercalated cells.

Additionally, reductions in pH (below 5.5) quickly and reversibly increase anion conductance and water permeability in AQP6 expressing oocytes.160 Subsequent studies have shown that the channel is permeable to halides, with the highest permeability to NO32,160,161 while the ionic selectivity becomes less specific after the addition of HgCl2.162 Moreover, when Asn60, a residue unique in mammalian AQP6, is converted to glycine, a highly-conserved residue in other mammalian aquaporins, anionic permeability is abolished and osmotic water permeability is increased during basal conditions.163 AQP7 is a member of the aquaglyceroporin family, and was originally cloned from rat testis.132 Additionally, AQP7 is expressed in several organs, including adipose tissue and kidney.132,164 In the kidney, AQP7 localizes to the brush border membrane of the proximal tubule S3 segment.165,166 Expression of AQP7 in Xenopus oocytes showed that AQP7 facilitated the transport of glycerol and urea in addition to water.132 The development of AQP7 knockout mice has aided understanding of the physiological role of AQP7. Glycerol excretion is markedly increased in AQP72/2 mice, suggesting a role for AQP7 in facilitating proximal tubular glycerol transport, and possibly renal gluconeogenesis (discussed below).134 In terms of water transport, AQP72/2 mice experience only a

slight reduction in proximal tubule membrane water permeability, and AQP7/AQP1 double knockout mice showed only a slightly more severe urinary concentrating defect than AQP12/2 mice during water deprivation.134 Additionally, evaluation of the channels’ permeability to urea in AQP72/2 mice revealed no change in urinary urea excretion or urea accumulation in the papilla, indicating a less prominent physiological role in urea transport.134 Expression of AQP7 in Xenopus oocytes increases the permeability to arsenite, thereby providing a possible route for arsenite uptake in mammalian cells167; the physiological role of these observations awaits further investigation. Cloning of the murine AQP8 water channel136,168,169 revealed its presence in many organs, including the kidney.135,136,168,169 In kidney, AQP8 localizes to intracellular domains of proximal tubule and collecting duct cells.135 Expression of AQP8 in Xenopus oocytes increased HgCl2-sensitive osmotic water permeability. Additionally, oocyte expression of AQP8 showed permeability to urea.136 Targeted deletion of the AQP8 gene produced a mild phenotype in AQP82/2 mice, with no obvious changes in renal parameters.170 Subsequent studies have suggested that AQP8 facilitates NH3 transport in Xenopus oocytes171,172 and in ammonia transport-deficient yeast.173 In AQP82/2 mice, ammonia-loading mildly reduced hepatic ammonia accumulation and increased renal ammonia excretion,174 indicating that AQP8 does not play a significant physiological role in facilitated NH3 transport. Recently a new aquaporin was cloned from rat testis, namely AQP11.168 This AQP has only one NPA motif, and shares low similarity with the conventional aquaporins. AQP11 is most abundantly expressed in the testis, kidney, and liver.168 In the kidney, the AQP11 protein localizes to the cytoplasm of the renal proximal tubules.137 While lack of plasma membrane expression of the AQP11 protein in Xenopus oocytes greatly impaired measurements of transport,137 targeted deletion of the AQP11 gene in mice resulted in a severe phenotype with vacuolization and cyst formation in the proximal tubule. The AQP112/2 mice died of renal failure and kidneys were polycystic. The cyst epithelia contained vacuoles, and the AQP112/2 mice also presented with a proximal tubular endosomal acidification defect.137

AQUAGLYCEROPORINS AQP3, AQP7, and AQP9 constitute a subgroup of aquaporins with a broader permeation range that includes glycerol, hence the name “aquaglyceroporins.” AQP3, which transports water and glycerol, was cloned by three groups at the same time.124
126 The

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FIGURE 41.6 Vasopressin-induced trafficking of aquaporin 2 (AQP2) in isolated, perfused rat collecting duct and resulting changes in water permeability. Anti-AQP2 immunogold electron microscopy of ultrathin cryosections of isolated rat collecting duct fixed before exposure to arginine vasopressin (pre-AVP, top left), and after exposure (AVP, bottom left). Note intracellular AQP2 (arrowheads) and plasma membrane AQP2 (small arrows). Quantification of AQP2 labeling density before, during or after vasopressin exposure (top right). Quantification of osmotic water permeability of isolated collecting duct (bottom right) (Nucleus: N; mitochondrion: M; 3 50,000) (reproduced and modified from ref. [182], with permission).

distribution at multiple sites, including the kidney collecting duct principal cells, airway epithelia, skin, urinary bladder, and secretory glands, suggests several functions.151,152,175 AQP3-null mice exhibit a nephrogenic diabetes insipidus phenotype; however, human mutants have not yet been reported. AQP7 is also permeated by water and glycerol, and is localized to the apical brush border of the proximal tubules, especially to the S3 segment. Recent studies have indicated that AQP7 may play a role in glycerol metabolism by showing adaptation to fasting by glycerol transport through AQP7 in adipose tissue.176 AQP72/2 mice have a significantly lower plasma glycerol concentration than wild-type mice, and impaired adipocytic glycerol release in response to a β3 receptor stimulation.176 Moreover, AQP72/2 mice experience severe hypoglycemia, but show normal hepatic gluconeogenesis during fasting, indicative of defective adipocytic glycerol

transport in these mice.176 Glycerol excretion is also markedly increased in AQP72/2 mice, suggesting a role for AQP7 in facilitating proximal tubular glycerol transport.134 AQP9, which is expressed in the liver but not in kidney,177
179 is permeated by a range of solutes including glycerol and urea.180 Recently, it has been demonstrated that expression of AQP9 in liver was induced up to 20-fold in rats fasted for 24
96 hours, and that AQP9 levels gradually declined after re-feeding.181 This indicates that the liver takes up glycerol for gluconeogenesis through AQP9 during starvation.

VASOPRESSIN REGULATION OF KIDNEY AQUAPORINS Of all the aquaporins, AQP2 has invoked the largest interest by nephrologists, because it is expressed

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in principal cells of the renal collecting duct, where it is the primary target for short-term regulation of collecting duct water permeability.182
184 AQP2 and AQP3 are also regulated either directly or indirectly by vasopressin through long-term effects that alter the abundance of these water-channel proteins in collecting duct cells.138,143,151,185
187

Short- and Long-Term Regulation of Water Permeability in the Collecting Duct Two modes of vasopressin-mediated regulation of water permeability have been identified in the renal collecting duct. Both involve regulation of the AQP2 water channel. Short-term regulation is the widely recognized process by which vasopressin rapidly increases water permeability of principal cells by binding to vasopressin V2-receptors at the basolateral membranes, a response measurable within 5 to 30 minutes after increasing peritubular vasopressin concentration.188,189 It is believed that vasopressin, acting through a cyclic adenosine monophosphate (cAMP) cascade, causes intracellular AQP2 vesicles to fuse with the apical plasma membrane, which increases the number of water channels in the apical plasma membrane (Figures 41.6 and 41.7). Long-term regulation of collecting duct water permeability is seen when circulating vasopressin levels are increased for 24 hours or more, resulting in an increase in the maximal water permeability of the collecting duct epithelium.185 This response is a consequence of an increase in the abundance of AQP2 water channels per cell in the collecting duct,138,143 apparently due to increased transcription of the AQP2 gene (Figure 41.7).

Short-Term Regulation of AQP2 by Vasopressin-Induced Trafficking The final concentration of the urine depends on the medullary osmotic gradient built up by the loop of Henle, and the water permeability of the collecting ducts through the cortex and medulla (see review 190). Collecting duct water permeability is regulated by vasopressin, and it has been suspected for many years on the basis of indirect biophysical evidence that the vasopressin-induced increase in water permeability depended on the appearance of specific water channels in the apical plasma membrane of the vasopressin-responsive cells. Molecular actions of vasopressin to increase epithelial water permeability were first demonstrated in the 1950s and early 1960s in the skin and urinary bladder of amphibia.191,192 Direct demonstrations that vasopressin rapidly increases water permeability of isolated

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collecting ducts were made during the next few years.193,194 These studies, undertaken with in vitro preparations, showed that vasopressin applied to the basolateral aspect of the collecting duct cells directly increased transepithelial osmotic water permeability within minutes. Kinetic studies in isolated perfused inner medullary collecting ducts revealed an increase in osmotic water permeability after only 40 seconds of incubation at 37 C, and half of the maximal water permeability was reached within 10 minutes.189,195 cAMP has been implicated in the regulatory process, because direct application of cAMP analogs to the collecting duct was found to mimic the short-term effects of vasopressin.193 Multiple studies with affinity-purified polyclonal antibodies to AQP2 have unequivocally established that AQP2 is specifically involved in vasopressininduced increases of renal collecting duct water permeability. Soon after isolation of the AQP2 cDNA and generation of specific antibodies,123 immunoperoxidase microscopy and immunoelectron microscopy clearly demonstrated that principal cells within renal collecting ducts contain abundant AQP2 in the apical plasma membranes and in subapical vesicles.138,143 These studies strongly supported the “shuttle hypothesis” originally proposed more than a decade earlier.196 This hypothesis proposed that water channels can shuttle between an intracellular reservoir in subapical vesicles and the apical plasma membrane, and that vasopressin alters water permeability by regulating the shuttling process.8 Shuttling of AQP2 was directly demonstrated in isolated perfused tubule studies.182 In these studies, water permeability of isolated perfused collecting ducts was measured before or after stimulation with vasopressin, and the tubules were fixed directly for immunoelectron microscopic examination (Figure 41.6). Vasopressin stimulation resulted in a markedly decreased immunogold labeling of intracellular AQP2, accompanied by a five-fold increase in the appearance of AQP2 immunogold particles in the apical plasma membrane. This redistribution was associated with an increase in osmotic water permeability of similar magnitude. These findings were reproduced in vivo by injecting rats with vasopressin, which also caused redistribution of AQP2 to the apical plasma membrane of collecting duct principal cells.183,197 In contrast to the effect of vasopressin treatment, removal of vasopressin led to a reappearance of AQP2 in intracellular vesicles, and a decline in osmotic water permeability in the isolated perfused collecting duct system182,186 (Figure 41.6). Moreover, the off-set response to vasopressin has been examined in vivo by acute treatment of rats with vasopressin-V2-receptor antagonist198,199 or acute water-loading (to reduce endogenous vasopressin levels200). These treatments (both reducing

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(a)

(b) Microtubule

p p p p

p

p

p

Protein kinase A

Dynein ATP Protein Kinase A cAMP Gαs Recycling?

ATP cAMP Gαs

p p p

AC AQP2

p

AC

Actin Gαs

V2R

Gαs

Myosin-1

p

p p

AQP3 Dynactin

p p p p

p

AQP3

V2R AQP4

AQP4

(d)

(c) AQP2

Exocytic insertion

P-myocin RLC Myocin ? MLCK RLC

AQP3

VAMP-2 NSF Syntaxin-4

AQP3 ATP Protein cAMP Kinase A Gαs Recycling?

CaM

H2O

?

Endocytic retrieval

AQP2

Ca11

?

V2R

Gαs

RyR1 Intracellular Ca++-store

AQP4

(e)

AQP3 Exocytosis

RhoGDI

AQP3

GTP

Protein kinase A ATP

ATP cAMP Gαs Protein kinase A

AC

p

V2R

RhoA

Gαs

AQP2

AQP2 Synthesis

AQP4

cAMP Gαs C-jun/c-fos–CREB-P AP1 CRE AQP2 gene transcription

AC

PDE Gαs

V2R

Nucleus

GDP RhoGDI

V2R AQP4

(f) RhoA

AC

Endocytosis

AQP3 Synthesis

Other receptors

AQP3

FIGURE 41.7 Regulation of AQP2 trafficking (Panels a
e), and expression (Panel F) in collecting duct principal cells. (a):Vasopressin binding to basolateral G-protein-linked V2-receptor stimulates adenylyl cyclase (AC); cyclic adenosine monophosphate (cAMP) activates protein kinase A to phosphorylate AQP2 in intracellular vesicles; phosphorylated AQP2 is exocytosed to the apical plasma membrane, resulting in increased apical membrane water permeability. (b): Overview of cytoskeletal elements, which may be involved in AQP2-trafficking. AQP2containing vesicles may be transported along microtubules by dynein/dynactin. The cortical actin web may act as a barrier to fusion with the plasma-membrane. (c): Intracellular calcium signaling and AQP2-trafficking. Increases in intracellular Ca21 concentration may arise from stimulation of the V2 receptor. The existence and potential role of other receptors and pathways, e.g., VACM-1, in Ca21 mobilization is still uncertain. The downstream targets of the calcium signal are unknown, and conflicting data exist on the importance of a rise in intracellular Ca21 for the hydrosmotic response to vasopressin. (d): Vesicle-targeting receptors and AQP2-trafficking. AQP2 vesicles dock at the apical membrane by association of VAMP-2 with syntaxin-4 targets in the presence of NSF; the exact role of these remains to be established. (e): Changes in the actin cytoskeleton associated with AQP2-trafficking to the plasma membrane. Inactivation of RhoA by phosphorylation and increased formation of RhoARhoGDI complexes seem to control the dissociation of actin fibers seen after vasopressin stimulation. (f): Specifically, cAMP participates in the long-term regulation of AQP2 by increasing the levels of the catalytic subunit of PKA in the nuclei, which is thought to phosphorylate transcription factors such as CREB-P (Cyclic AMP Responsive Element Binding Protein) and C-Jun/c-Fos. Binding of these factors are thought to increase gene transcription of AQP2, resulting in synthesis of AQP2 protein which in turn enters the regulated trafficking system. In parallel, AQP3 and AQP4 synthesis and trafficking to the basolateral plasma membrane takes place. Importantly, AQP2 regulation can be modified by a number of hormones including dopamine, ANP, PGE2, and adrenergic hormones. See text for details (Vasopressin-V2-receptors: V2R; adenylyl cyclase: AC; cAMP and protein kinase A: PKA) (redrawn and modified from ref. [190]).

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vasopressin action) resulted in a prominent internalization of AQP2 from the apical plasma membrane to intracellular subapical vesicles, further underscoring the role of AQP2 trafficking in the regulation of collecting duct water permeability and the reversibility of AQP2 trafficking to the plasma membrane. The quantity of AQP2 in the apical plasma membrane is determined by the relative rates of exocytosis and endocytosis. Both processes occur continuously, and their relative rates are regulated to effect changes in water permeability. Kinetic evidence indicates that vasopressin regulates both endocytosis and exocytosis of apical water channels.195,201 These conclusions are supported by multiple earlier experiments with fluidphase markers, which assessed rates of internalization of the apical plasma membrane.202
204 Intravenous injection of horseradish peroxidase was followed by uptake of the material into intracellular vesicles and multivesicular bodies by collecting duct principal cells.202 When compared with normal rats, the rate of uptake by vasopressin-deficient Brattleboro rats was much lower; however, this difference was lost after administration of vasopressin, presumably reflecting an effect of vasopressin in accelerating exocytosis, followed by a secondary increase in the rate of endocytosis to re-establish the steady-state. These observations were supported by studies with fluorescein-labeled dextrans,203 which confirmed that vasopressin enhances the rate of endocytosis of the apical plasma membrane under steady-state conditions. In apparent contrast, removal of vasopressin from isolated perfused rabbit cortical collecting ducts also was accompanied by increased endocytosis of horseradish peroxidase from the lumen, and it was suggested that water channels would also be internalized at an increased rate.204 Similar results were obtained in isolated rat inner medullary collecting duct, which showed that vasopressin washout was associated with increased appearance of albumin-gold or cationic ferritin in small apical vesicles and multivesicular bodies.205 Presumably this response reflects a direct effect of vasopressin to decrease the intrinsic rate of endocytosis and is reversed on vasopressin removal. Therefore, it appears that two factors affect the rate of endocytosis of the apical plasma membrane of collecting duct principal cells: (1) vasopressin directly decelerates endocytosis by unknown mechanisms; and (2) vasopressin indirectly accelerates the rate of endocytosis by stimulating exocytosis and increasing the total amount of apical plasma membrane. The latter effect is believed to be slower than the former, resulting in a biphasic decline in water permeability on vasopressin removal.195,201 Other studies have demonstrated that apical endocytosis is most rapid

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at about 3 to 5 minutes after vasopressin washout from isolated perfused inner medullary collecting ducts,205 corresponding to the period of the most rapid decrease in transepithelial water permeability.195,201 Thus, the results taken together strongly suggest that vasopressin directly regulates both endocytosis and exocytosis of AQP2. Presumably the net increase in water permeability seen in response to vasopressin is due both to direct stimulation of exocytosis and to direct inhibition of endocytosis. Several groups have now successfully reconstituted the AQP2 delivery system using cultured cells transfected with either wild-type AQP2 or AQP2 tagged with a marker protein or a fluorescent protein.206
211 Using such cultured cells stably transfected with AQP2, it has been shown that AQP2 trafficking occurs from vesicles to the apical plasma membrane, albeit in some cases to the basolateral plasma membrane, as well as retrieval and subsequent trafficking back to the surface upon repeated stimulation. This recycling of AQP2 also occurs in LLC-PK1 cells in the continued presence of cycloheximide, preventing de novo AQP2 synthesis. Despite remaining uncertainties, a working model for collecting duct water permeability permits molecular insight into this process (Figure 41.7). The short-term effects of vasopressin-enhanced water permeability are explained by the redistribution of AQP2 to the apical plasma membrane in collecting duct principal cells. Acting through the V2 receptor, vasopressin stimulates adenylyl cyclase; the elevated levels of intracellular cAMP activate protein kinase A (PKA), causing a redistribution of AQP2 from intracellular vesicles to the apical plasma membrane by inducing exocytosis of AQP2 vesicles, and by inhibiting endocytosis of AQP2 from the apical membrane. The net increase of AQP2 in the apical plasma membrane increases the water permeability. In the pre-antidiuretic state, the apical membrane is the ratelimiting barrier for transepithelial water transport, because AQP3 and AQP4 are abundant in the basolateral plasma membrane. Thus, increasing the water permeability of the apical plasma membrane by insertion of AQP2 water channels results in an increase in transepithelial osmotic water permeability. Whereas the general model is now well-established, the molecular details explaining how an elevation in intracellular cAMP levels regulates trafficking of AQP2 to the apical plasma membrane and back remain uncertain. Serine at position 256, in the carboxyl-terminal tail of AQP2, is apparently a substrate for protein kinase A, and regulation of exocytosis and endocytosis of AQP2 is expected to involve phosphorylation and dephosphorylation of AQP2,208,212,213 and of ancillary proteins involved in the trafficking processes. This

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question will likely be a major focus of future research.

Signal Transduction Pathways Involved in Vasopressin Regulation of AQP2 Trafficking The signal transduction pathways have been described thoroughly in previous reviews.190,214
217 cAMP levels in collecting duct principal cells are increased by binding of vasopressin to V2-receptors.218,219 The synthesis of cAMP by adenylate cyclase is stimulated by a V2-receptor coupled heterotrimeric GTP-binding protein, Gs. Gs interconverts between an inactive GDP-bound form and an active GTP-bound form, and the vasopressin-V2-receptor complex catalyzes the exchange of GTP for bound GDP on the ’-subunit of Gs. This causes release of the ’-subunit, Gs’-GTP, which subsequently binds to adenylate cyclase, thereby increasing cAMP production. Protein kinase A is a multimeric protein which is activated by cAMP, and consists in its inactive state of two catalytic subunits and two regulatory subunits. When cAMP binds to the regulatory subunits, these dissociate from the catalytic subunits, resulting in activation of the kinase activity of the catalytic subunits. AQP2 contains a consensus site for PKA phosphorylation (RRQS) in the cytoplasmic COOH terminus at Ser256.123 Recent studies using 32P labeling or using an antibody specific for phosphorylated AQP2 showed a very rapid phosphorylation of AQP2 (within 1 minute) in response to vasopressin-treatment of slices of the kidney papilla.220 This is compatible with the timecourse of vasopressin-stimulated water permeability of kidney collecting ducts.189 As described above, PKAinduced phosphorylation of AQP2 apparently does not change the water conductance of AQP2 significantly. Importantly, it was recently demonstrated that vasopressin or forskolin treatment failed to induce translocation of AQP2 when Ser256 was substituted by an alanine (S256A) in contrast to a significant regulated trafficking of wild-type AQP2 in LLC-PK1 cells.208 A parallel study by Sasaki and colleagues also demonstrated the lack of cAMP-mediated exocytosis of mutated (S256A) AQP2 transfected into LLC-PK1 cells.221 Thus, these studies indicate a specific role of PKA-induced phosphorylation of AQP2 for regulated trafficking. To explore this further, an antibody was designed that exclusively recognizes AQP2 which is phosphorylated in the PKA consensus site (Ser256). In normal rats phosphorylated AQP2 (Ser256) is present in intracellular vesicles and apical plasma membrane, indicating that it is constitutively phosphorylated even in low circulating vasopressin states.222,223 In contrast, phosphorylated AQP2 is mainly located in

intracellular vesicles in Brattleboro rats, as shown by immunocytochemistry and immunoblotting using membrane fractionation.222 Importantly, dDAVP treatment of Brattleboro rats caused a marked redistribution of phosphorylated AQP2 (Ser256) to the apical plasma membrane, which is in agreement with an important role of PKA phosphorylation in this trafficking.222 Conversely, treatment with V2-receptor antagonist induced a marked decrease in the abundance of phosphorylated AQP2 (Ser256),222 likely due to reduced PKA activity and/or increased dephosphorylation of AQP2, e.g., by increased phosphatase activity. Moreover, the necessity for the Ser256 phosphorylation in AQP2-regulated urine concentration is underlined by the recent identification of this site as being spontaneously mutated in congenital progressive hydronephrosis (cph).224 The spontaneous conversion of the serine phosphorylated site (Ser 256) to leucine in the cytoplasmic tail of the AQP2 protein in cph-mice prevents its phosphorylation at Ser256, and inhibits the subsequent accumulation of AQP2 on the apical membrane of the collecting duct principal cells. This causes polydipsia, polyuria, and a severe urinary concentration defect leading to hydronephrosis and obstructive nephropathy in these mice. Recently, three previously unknown phosphorylation sites in the C-terminus of AQP2 were identified (S261, S264, and S269) by using liquid chromatography coupled to mass spectrometry neutral loss scanning.225 During basal conditions, AQP2 phosphorylated on Ser261 is approximately 24-fold more abundant than Ser256 phosphorylated AQP2. During administration of dDAVP a decrease in Ser261 phosphorylation was observed, while AQP2 phosphorylated on Ser256, Ser264, and Ser269 increased.225,226 It is clear that phosphorylation of AQP2 is required for cell surface expression; however, it is still unclear how phosphorylation of AQP2 (S256, S261, S264, S269) induces apical trafficking of AQP2. One possibility is that phosphorylation of AQP2 directly influences an interaction between AQP2containing vesicles and the cell cytoskeleton, microtubules or accessory cross-linking proteins. Indeed, S256 is important for a direct interaction of AQP2 with 70 kDa heat-shock proteins.227 Alternatively, phosphorylation could prevent endocytosis of AQP2, leading to accumulation at the cell surface. Prostaglandin E2 (PGE2) has been known to inhibit vasopressin-induced water permeability by reducing cAMP levels. Zelenina et al.228 investigated the effect of PGE2 on PKA phosphorylation of AQP2 in rat kidney papilla, and the results suggest that the action of prostaglandins are associated with retrieval of AQP2 from the plasma membrane, but this appears to be independent of AQP2 phosphorylation by PKA.228 In contrast, a recent study revealed the effect of selective

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E-prostanoid receptor agonists on the vasopressinindependent targeting of AQP2.229 In vitro, PGE2 and selective agonists for the E-prostanoid receptors EP2 or EP4 all increased trafficking and AQP2 phosphorylation (Ser264) in MDCK cells. In vivo, a V2R antagonist caused a severe urinary concentrating defect in rats, which was greatly alleviated by treatment with a selective agonist for the EP2 receptor.229 Further studies are still needed on the effects of PGE2 on urine concentration. Phosphorylation of AQP2 by other kinases, e.g., protein kinase C or casein kinase II, may potentially participate in regulation of AQP2 trafficking. Angiotensin II-induced activation of protein kinase C could be, at least in part, involved in the AQP2 trafficking/expression.207,230,231 S256 is also a substrate for Golgi casein kinase 2, which is required for the Golgi transition of AQP2.232 Additionally, the phosphatase inhibitor okadaic acid induces increased AQP2 expression at the cell surface, even in the presence of the PKA inhibitor H89 and S256A AQP2 mutants (that lack S256 phosphorylation) which are able to accumulate at the cell surface after treatment with cholesterol-depleting agents.233 Phosphorylation of other cytoplasmic or vesicular regulatory proteins may also be involved. These issues remain to be investigated directly.

Involvement of the Cytoskeleton and Ca21 in AQP2 Trafficking The cytoskeleton has been shown to be involved in AQP2 trafficking in the kidney collecting duct. In particular, the microtubular network has been implicated in this process, since chemical disruption of microtubules inhibits the increase in permeability both in the toad bladder and in the mammalian collecting duct.234,235 Thus, AQP2 vesicles may be transported along microtubules on their way to the apical plasma membrane. The microtubule-based motor protein dynein and the associated protein Arp1, which is part of the protein complex dynactin, were found by immunoblotting to be among the proteins associated with AQP2 vesicles from rat inner medulla. Immunoelectron microscopy further supported the presence of both AQP2 and dynein on the same vesicles.236 Moreover, both vanadate (a non-specific inhibitor of ATPases) and EHNA (a specific inhibitor of dynein) inhibit the antidiuretic response in toad bladder237,238 and kidney collecting duct.239 Thus, it is likely that dynein may drive the microtubule-dependent delivery of AQP2containing vesicles to the apical plasma membrane. Actin filaments are also involved in the hydrosmotic response.240
245 Recently evidence was provided that the myosin light chain kinase (MLCK) pathway,

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through calmodulin-mediated calcium activation of MLCK, leads to phosphorylation of myosin regulatory light chain and non-muscle myosin 2 motor activity.246 Studies in isolated perfused rat inner medullary collecting ducts showed the MLCK inhibitors ML-7 and ML-9 reduce the vasopressin-induced increase in water permeability,246 indicating that MLCK may be a downstream target for the vasopressin-induced Ca21 signal. The intracellular Ca21 concentration has been shown to increase upon stimulation of isolated perfused rat inner medullary collecting ducts with vasopressin or dDAVP.247 These observations have been followed by a number of studies on the role of Ca21 in the vasopressin-induced increase in water permeability. Vasopressin or 8
4-chlorophenylthio-cAMP induced a marked increase in intracellular [Ca21] in isolated perfused IMCD, and BABTA blocked the rise in intracellular [Ca21] and co-contaminantly led to inhibition of the vasopressin-induced increase in tubular water permeability.248 Moreover, blocking calmodulin with W7 or trifluoperazine also inhibited the effect of vasopressin on tubular water permeability, and inhibited the vasopressin-induced AQP2 plasma membrane targeting in primary cultured IMCD cells.248 Removing Ca21 from both bath and lumen in isolated perfused tubules did not affect the vasopressin-induced Ca21 signal, indicating that Ca21 was released from an intracellular source. This was further supported by the observations that ryanodine inhibited the Ca21 signal in perfused IMCDs and inhibited AQP2 accumulation in the plasma membrane in primary cultured IMCD cells. In addition, RyR1 ryanodine receptor was localized to rat IMCD by immunohistochemistry.248 Ryanodine receptors are generally known to mediate a positive feedback and release Ca21 from intracellular stores in response to an initial rise in intracellular [Ca21]. Thus, it is likely that another mechanism is responsible for the initialization of the rise in intracellular [Ca21]. Further studies revealed that vasopressin or dDAVP elicited oscillations in intracellular [Ca21] in isolated perfused rat IMCD.249 The results on isolated perfused IMCDs indicate that an intracellular increase in [Ca21] is an obligate component of the vasopressin response leading to increased AQP2 expression in the apical plasma membrane. However, it should be mentioned that there is a discrepancy between the results from primary cultured IMCD cells250 and isolated perfused IMCD tubules248 with regard to the role of intracellular [Ca21] in vasopressin-induced AQP2 trafficking. Consistent with this, Lorenz et al.250 demonstrated that AQP2 shuttling is evoked neither by AVP-dependent increase of [Ca21] nor by AVP-independent increase of [Ca21] in primary cultured IMCD cells from rat kidney, although clamping of intracellular [Ca21] below resting levels inhibits AQP2 exocytosis. Further studies are

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required to clarify this discrepancy, which may be related to altered expression levels of vasopressin receptors and/or AQP2 or other elements in the AQP2 trafficking system.

Mechanism of AQP2 Trafficking by Targeting Receptors The mechanism by which AQP2 vesicles are targeted to the apical plasma membrane and the mechanism by which cAMP controls docking and fusion of vesicles is a current area of active investigation. Considerable insight into this problem has been obtained from previous studies of regulated exocytosis of synaptic vesicles, which involve the actions of multiple proteins.251,252 Vesicle-targeting receptors (often referred to as “SNAREs”) are believed to induce specific interaction of vesicles with membrane sites. Vesicle-targeting receptors associated chiefly with translocating vesicles are known as “VAMPs” (vesicle associated membrane proteins, also referred to as “synaptobrevins”) and “synaptotagmins.” Two other families of membrane proteins are believed to serve as receptors in target membranes: the “syntaxins” and SNAP-25 homologs. Several of these SNAREs have been found in the renal collecting duct.253
260 Syntaxins are 30 to 40 kDa integral membrane proteins. Syntaxins have a one bilayer-spanning domain near the C-terminus, so the majority of the protein resides in the cytoplasm. Syntaxins are widely distributed among mammalian tissues. It has been established that syntaxin-4 is expressed in the mammalian collecting duct by studies using polyclonal antibodies raised to conjugated peptides specific for individual syntaxins, and this has been confirmed by reverse transcription polymerase chain reaction.258 Immunolocalization studies have demonstrated that syntaxin-4 is predominantly located in the apical plasma membrane of collecting duct principal cells, where AQP2 is targeted in response to vasopressin. VAMPs are believed to induce specific docking of vesicles by interacting directly with syntaxins in the target plasma membrane (Figure 41.7). Although their primary amino acid sequences are not related to syntaxins, VAMPs also have a single bilayer-spanning domain near the C-terminus, and the majority of the protein resides in the cytoplasm. Three VAMP isoforms were initially identified261: VAMP-1; VAMP-2; and VAMP-3 (also referred to as “cellubrevin”), and subsequently several additional homologs have been cloned.262 VAMP-2 and VAMP-3 have been localized in principal cells of renal collecting duct.253
260 Doublelabeling immunoelectron microscopy revealed that AQP2 and VAMP-2 reside in the same intracellular

vesicles in collecting duct principal cells.260 Furthermore, AQP2 vesicles isolated by differential centrifugation were found to contain VAMP-2.254,257 At this time, several putative vesicle-targeting proteins are known to reside in collecting duct principal cells. VAMP-2 resides in AQP2 intracellular vesicles, and syntaxin-4 resides in the apical plasma membrane. In vitro binding assays have documented that VAMP-2 binds syntaxin-4 with high affinity, but VAMP-2 has not been shown to interact with syntaxin-3 or syntaxin2.263,264 Thus, VAMP-2 and syntaxin-4 are likely participants in the targeting of AQP2 vesicles to the apical plasma membrane; however, a functional role for these targeting proteins remains to be formally demonstrated. A third SNARE protein, SNAP-23, has also been identified in principal cells of the collecting duct.255 Although considered a target-membrane SNARE (t-SNARE), SNAP-23 is present both in AQP2containing vesicles and in the apical plasma membrane of principal cells. VAMP-2, syntaxin-4, and SNAP-23 may potentially form a complex with the N-ethylmaleimide-sensitive factor (NSF). Finally, synaptotagmin VIII, a calcium-insensitive homolog, was identified in collecting duct principal cells,256 and may potentially also play a role in vesicle targeting. Recently, LC-MS/MS-based proteomic analysis of immunoisolated AQP2-containing intracellular vesicles from rat inner medullary collecting duct revealed that AQP2-containing vesicles are heterogeneous, and that intracellular AQP2 resides chiefly in endosomes, transGolgi network, and rough endoplasmic reticulum.265 Vasopressin-stimulated exocytosis of AQP2 vesicles involves several steps including: (1) translocation of vesicles from a diffuse distribution throughout the cell to the apical region of the cell; (2) translocation of AQP2 across the apical part of the cell composed of a dense filamentous actin network; (3) priming of vesicles for docking; (4) docking of vesicles; and (5) fusion of vesicles with the apical plasma membrane. Theoretically, each of these steps could be a target for regulation by vasopressin. If the SNARE proteins are involved in the vasopressin-induced trafficking of AQP2 to the apical plasma membrane, the regulatory mechanism might involve selective phosphorylation of one of the SNARE proteins or an ancillary protein that binds to them, possibly via protein kinase A or calmodulin-dependent kinase II. Although the SNARE proteins are recognized as potential targets for phosphorylation,266
268 there is presently no evidence for phosphorylation in the collecting duct. As noted above, however, AQP2 itself appears to be a target for PKA-mediated phosphorylation at a serine in position 256.212 The phosphorylation does not modify the single channel water permeability of AQP2,213 and instead the phosphorylation is believed to play a critical role in

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regulation of AQP2 trafficking to the plasma membrane.208,221 The establishment of a role for these SNARE proteins in regulated trafficking of AQP2 will most likely depend on the preparation of targeted knockouts for each of these genes in the collecting duct.

Long-Term Regulation of Aquaporin Expression It has been known for more than 40 years that urinary concentrating ability is regulated through a long-term conditioning effect of sustained increases in vasopressin levels exerted over a period of days.269 The physiological and molecular basis for long-term conditioning of urinary concentrating ability is still being investigated. Water restriction causes a large, stable increase in water permeability of inner medullary collecting ducts dissected from rat kidneys and perfused in vitro.185 Thus, water permeability of the tubules from water-restricted rats became five-fold greater than that measured in well-hydrated rats by a mechanism independent of the short-term action of vasopressin. Other studies confirmed that water restriction enhances the vasopressin-independent water permeability of collecting ducts,270,271 and increases vasopressin-stimulated water permeability.272 This contrasts with the lack of an observed increase in urea permeability in water-restricted rats.185 These studies provided the firm conclusion that restriction of water intake for 24 hours or longer leads to a marked increase in the water-reabsorbing capacity of the renal collecting duct epithelium. Further studies with anti-peptide antibodies have documented that the conditioned increase in water permeability is associated with increased expression of aquaporin proteins in collecting duct principal cells. Specifically, the increased water permeability that occurs in response to water restriction is accompanied by significant increases in the levels of AQP2 and AQP3 proteins in rat collecting ducts.138,151,195 Water restriction for 24 to 48 hours resulted in an approximately five-fold increase in AQP2 protein in the rat renal inner medulla when measured by immunoblotting138,195 and by immunostaining.199 This increase paralleled the increase in water permeability in response to water restriction.185 A direct role for vasopressin in AQP2 expression has been established by multiple studies. Continuous infusion of arginine vasopressin into Brattleboro rats for 5 days resulted in a three-fold increase in AQP2 expression and a three-fold increase in water permeability of inner medullary collecting ducts.143 When the time-course of AQP2 expression in inner medullary collecting duct was studied, it was found that 5 days of vasopressin infusion were required to reach maximal AQP2 levels in Brattleboro rats.139

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Vasopressin is believed to be essential for evoking AQP2 expression, because Brattleboro rats totally lack circulating vasopressin fail to exhibit increased expression of AQP2 protein after long-term water restriction.273 Moreover, administration of a selective V2-receptor antagonist blocked the increase in AQP2 expression usually seen with thirsting in normal rats,199 whereas administration of the V2-selective agonist dDAVP (1-desamino-8-D-arginine vasopressin) to rats elicited a large increase in renal AQP2 mRNA274 and AQP2 protein abundance.275 The question of hyperosmolar induction of AQP2 during thirsting has been raised; however, five-day infusion of arginine vasopressin increased AQP2 comparably in renal cortex and medulla. This indicates that the vasopressininduced expression of AQP2 is not critically dependent on increased osmolality or ionic strength in the tissue surrounding the collecting ducts.187 Thus, it has been clearly established that the major increase in AQP2 water channel expression elicited by restriction of water intake is due to vasopressin binding to the V2 receptor in collecting duct principal cells. However, studies of the mechanism of escape from the antidiuretic effects of vasopressin275,276 make it clear that AQP2 expression is regulated by other factors in addition to vasopressin (see later). AQP3 protein expression also has been found to parallel AQP2 expression. A marked increase in AQP3 protein expression was observed in rats in response to restriction of water intake151 and after a five-day infusion of arginine vasopressin.187 These conditions did not lead to observed changes in AQP1 or AQP4 protein expression.153,187 Thus, it is concluded that long-term circulation of high vasopressin levels is associated with an adaptive increase in maximal water permeability in collecting ducts, apparently due a selective increase in the expression of both AQP2 and AQP3 in the principal cells of the collecting duct. However, immunoelectron microscopy has demonstrated that AQP3 is predominantly present in the basolateral plasma membranes with little labeling of intracellular vesicles.151 This suggests that AQP3 is not regulated by vesicular trafficking (in contrast to the findings with AQP2182). Moreover, there are several examples where there is a decoupling of AQP2 and AQP3 expression, suggesting that other factors in addition to vasopressin may regulate one or other of the aquaporins. This is seen in conditions such as hepatic cirrhosis,277 vasopressinescape,275 low-protein diet,278 and restriction of NaCl intake.156 The adaptive changes in AQP2 and AQP3 expression are believed to be due to transcriptional regulation of these genes. Increased AQP2 mRNA was found in the inner medullae of water-restricted rats,279 and in response to infusion of dDAVP.274,275 All of these

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studies indicate that the long-term upregulation of AQP2 expression in the rat kidney is due to elevation of the levels of AQP2 mRNA. Vasopressin-induced increase in AQP2 mRNA expression could reflect either increased gene transcription or reduced transcript degradation. Examination of the 30 -untranslated region of the AQP2 mRNA has not revealed the presence of recognizable mRNA stabilization motifs, and it is presumed that vasopressin directly increases AQP2 gene transcription; however, no direct evidence supports this presumption. Transcriptional regulation would most likely occur through the vasopressin-induced increases in intracellular cyclic AMP that activates protein kinase A in collecting duct cells. Examination of the 50 -flanking region of the AQP2 gene revealed a putative cAMPresponse element (CRE), which may play a role in the vasopressin-induced increase in AQP2 expression.280 Attempts to demonstrate expression of AQP2 promoter-luciferase reporter constructs have been undertaken in cultured renal epithelial cells and suggest that the putative CRE can drive increased expression of the AQP2 gene; however, the inability of the cell line to express AQP2 protein limits the interpretation.281
283 As with AQP2, AQP3 mRNA levels were induced by infusion with the selective V2 agonist dDAVP.275 Examination of the 50 -flanking DNA of AQP3 failed to reveal a CRE; however, Sp1 and AP2 cis-regulatory elements are present,284 and these are known to be associated with cyclic AMP-mediated transcriptional regulation of other genes. A previous study revealed that AQP2 is ubiquitinated with one UbLys63-linked poly-Ub chain at K270 of AQP2 and lysosomal degradation was extensive for ubiquitinated AQP2.285 The degradation pathways, therefore, balance the abundance of proteins which play an important role in body water homeostasis.186

DYSREGULATION OF RENAL AQUAPORINS IN WATER BALANCE DISORDERS In a variety of conditions renal water handling is disturbed. Over the past decade, the role of changes in the expression and/or function of aquaporins have been investigated in a range of these conditions, including genetic defects or acquired defects showing a decreased renal responsiveness to vasopressin (acquired nephrogenic diabetes insipidus). The importance of aquaporins playing an essential role for regulation of renal water balance has been established (Table 41.2), and the following text represents extracted and updated information reported in previous reviews by the authors or other investigators.190,286
288

URINARY CONCENTRATING DEFECTS Inherited Diabetes Insipidus There are two significant inherited forms of diabetes insipidus (DI): central and nephrogenic. In central (or neurogenic) DI, there is a defect of vasopressin production. Central DI is rarely hereditary in man, but usually occurs as a consequence of head trauma or diseases in the hypothalamus or pituitary gland. The Brattleboro rat provides an excellent model of this condition. These animals have a total or near-total lack of vasopressin production.289,290 Consequently, Brattleboro rats have substantially decreased expression levels of vasopressin-regulated AQP2 compared with the parent strain (Long Evans), and the AQP2 deficit was reversed by chronic vasopressin infusion, suggesting that patients lacking vasopressin are likely to have decreased AQP2 expression.143 The subsequent work showing that expression of AQP3 is also regulated by vasopressin implies that the expression levels of these water channels will also be decreased in patients with CDI. The most important denominator is the deficiency of AQP2 trafficking to the apical membrane. These deficits are likely to be the most important causes of the polyuria from which these patients suffer, which will be reversed by desmopressin treatment. The second form of DI is called nephrogenic diabetes insipidus (NDI), and is caused by the inability of the kidney to respond to vasopressin stimulation. The most common hereditary cause (in 95% of cases) is an X-linked disorder associated with mutations of the V2 vasopressin receptor (AVPR2) making the collecting duct cells insensitive to vasopressin.291 Although there is no direct evidence, it is likely that this form of NDI will be associated with decreased expression of AQP2, since the cells are unable to respond to circulating vasopressin.292 This will compound the lack of AQP2 trafficking. Consistent with this, urinary AQP2 levels are very low in patients with X-linked NDI.196,293 However, since the amount of AQP2 in the urine appears to be determined largely by the response of the collecting duct cells to vasopressin294 rather than their content of AQP2, the data must be interpreted with caution with respect to predicting AQP2 expression levels. More rarely (in 5% of cases), congenital diabetes insipidus (CNDI) is inherited in an autosomal recessive fashion which is not due to mutations in the gene encoding the V2 vasopressin receptor, but these patients have been found to possess mutations in both alleles of the AQP2 gene, and the sites of point mutations were observed at functionally important sites in the water transport pathway.144,295
298 Since these patients manifest a particularly severe form of diabetes insipidus, the critical role of AQP2 in renal water conservation was

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established. It is believed that these mutations lead to abnormal intracellular routing of the expressed protein.299 More than 155 mutations in the vasopressin receptor gene have been recognized to cause CNDI. Functional analysis has been carried out for over 79 of these mutations.300 The analyses have revealed four different types of mutant receptors. The most common is impaired intracellular trafficking, which is seen in up to 70% of cases.300 The remaining types include reduced ligand-binding capacity, failure to generate cAMP, and defects in the synthesis of stable mRNA.301 Only five of the 155 known mutations cause partial CNDI (D85N, R104C, G201D, P322S, and S329R). Recently, a different kindred was identified with a dominant form of nephrogenic diabetes insipidus, and biochemical evaluations revealed that the mutation in the cytoplasmic C-terminus blocked the trafficking of AQP2 vesicles to the cell surface.302 Thus, intracellular trafficking of vesicles containing products of the mutant allele and the product of the normal allele may be misdirected. It is not known if these individuals suffer a more or less severe clinical defect, and recently it was shown that the ligand-binding and signal transduction capability are dependent on localization of the amino acid variation, suggesting that striking divergences at the level of receptor functionality may thus underlie similar clinical phenotypes in CNDI.303
305

Acquired Nephrogenic Diabetes Insipidus Acquired abnormalities of AQP2 expression have been implicated in multiple clinical disorders associated with abnormal water balance (Table 41.2). The role of vasopressin-regulated AQP2 has been established in a number of rat models with acquired NDI. In many of these conditions the kidney is unable to handle water, due to an impaired responsiveness to vasopressin; in the following text the most important of these condtions are described.

Lithium-Induced NDI Lithium is a major therapeutic agent used to treat bipolar disorder (also referred to as “manic depressive disease”) which affects approximately 1% of the US population.306 However, lithium treatment is associated with a variety of renal side-effects, including nephrogenic diabetes insipidus (NDI, i.e., a pronounced vasopressin-resistant polyuria and inability to concentrate urine),307,308 increased urinary sodium excretion,309 and distal renal tubular acidosis.310 Patients who have been treated with lithium manifest a slow recovery of urinary concentrating ability when treatment is discontinued. In rats treated with lithium

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for 4 weeks, AQP2 and AQP3 levels were progressively reduced to approximately 5% of levels in control rats.307,308 AQP2 downregulation was paralleled by a progressive development of severe polyuria.307,308 Quantitative immunoelectron microscopy of AQP2 labeling in the inner medullary collecting duct principal cells showed that there was a reduction of AQP2 in the apical plasma membrane, as well as in the basolateral plasma membrane and intracellular vesicles. Reduction in AQP2 (and AQP3) expression may be induced by a lithium-dependent impairment in the production of cAMP in collecting duct principal cells,309,311 indicating that inhibition of cAMP production may in part be responsible for the reduction in AQP2 expression, as well as the inhibition of targeting to the plasma membrane in response to lithium treatment. This is consistent with the presence of a cAMPresponsive element in the 50 -untranslated region of the AQP2 gene,281,282 and with the demonstration that mice with inherently low cAMP levels have low expression of AQP2 (DI1/1 severe mouse).312 Lithium treatment is also associated with a concomitant increase in urinary sodium excretion, which is likely to play a role in the polyuria. It has also been demonstrated that chronic lithium treatment induces a marked decrease in protein abundance of epithelial sodium channel β- and γ-ENaC in the cortex and outer medulla, whereas the other renal sodium transporters upstream from the connecting tubule are unchanged.313 This was also revealed by immunocytochemistry showing an almost complete absence of β-ENaC and γ-ENaC labeling in cortical and outer medullary collecting duct. This suggests a reduced responsiveness to aldosterone and vasopressin in these specific renal tubule segments, and that dysregulation of ENaC subunits is likely to play a role in the development of natriuresis and partly in the decreased urinary concentrating ability in rats with lithiuminduced NDI.314 Additional studies have identified proteins with altered abundance in the inner medullary collecting ducts (IMCD) of lithium-treated rats and their possible cellular function.315,316 Moreover, it has been shown that prostaglandins, in lithium-treated mpkCCD cells, dcreases APQ2 protein stability by increasing its lysosomal degradation, indicating that in vivo paracrine-produced prostaglandins might have a role in lithium-induced NDI via this mechanism.317

Hypokalemia and Hypercalcemia Hypokalemia and hypercalcemia are also associated with polyuria due to a vasopressin-resistant urinary concentrating defect. Rat models of these conditions are valuable tools to study the molecular defects. Treating rats with a potassium-deficient diet for 4 days

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induced a significant hypokalemia which was associated with downregulation of AQP2 expression and polyuria.318 Likewise, hypercalcemia induced in rats by oral treatment for 7 days with dihydrotachysterol produced a urinary concentrating defect and polyuria which was also associated with downregulaion of AQP2.319,320 Thus, both hypokalemia and hypercalcemia are associated with downregulation of AQP2 expression, and immunolocalization studies of AQP2 demonstrated similar features. In addition to the downregulation of AQP2, expression of Na-K-2Cl co-transporter in the thick ascending limb was decreased in both conditions,321,322 suggesting that reduced sodium and chloride reabsorption in the thick ascending limb, and hence decreased medullary hyperosmolality, could also partly contribute to the polyuria and decreased urinary concentration in hypokalemia and hypercalcemia. Thus, these studies in part describe the underlying molecular defects involved in the development of the polyuria in these conditions.

Urinary Tract Obstruction Another serious condition seen in both children and adults is urinary tract obstruction which is associated with complex changes in renal function involving marked alterations in both glomerular and tubular function and bilateral urinary tract obstruction (BUO) which may result in long-term impairment of the ability to concentrate urine.323 BUO for 24 hours in rats is associated with markedly reduced expression of AQP2, AQP3, AQP4, and AQP1.324
327 In addition, BUO is associated with marked downregulation of key sodium transporters and urea transporters.328 Following release of the obstruction, there is a marked polyuria, during which period AQP2 and AQP3 levels remain downregulated up to two week after release, providing an explanation at the molecular level for the observed post-obstructive polyuria. In a number of studies BUO has been demonstrated to be associated with COX2 induction and cellular infiltration of the renal medulla.292,329
331 Using specific COX2 inhibition to rats subjected to BUO it was demonstrated that this treatment prevents downregulation of AQP2 and several sodium transporters located to the proximal tubule and mTAL.329,330 Also, increased expression and activity of the P2Y2, EP1, and EP3 receptors have been suggested to play important roles in post-obstructive polyuria.332 Moreover, specific inhibition of the AT1 receptor in rats subjected to BUO prevented AVP2R downregulation of NaPi2 in the PT, BSC-1 in the mTAL, and AQP2/pS256-AQP2 in the CD three days after release of BUO,134,333 confirming that the renin
angiotensin system plays an important role for

the pathophysiological changes in urinary tract obstruction. In contrast to BUO conditions, unilateral ureteral obstruction is not associated with changes in the absolute excretion of sodium and water since the nonobstructed kidney compensates for the reduced ability of the obstructed kidney to excrete solutes.328,334 These studies demonstrated a profound downregulation of AQP1, AQP2, AQP3, and AQP4, and pAQP2 levels in the obstructed kidney, suggesting that 1ocal factors play a major role.

Renal Failure Renal failure, both acute and chronic, is associated with polyuria and a urinary concentrating defect, and in both cases there is a wide range of glomerular and tubular abnormalities that contribute to the overall renal dysfunction. Ischemia and reperfusion (I/R)induced experimental acute renal failure (ARF) in rats is a model that is widely used. In this model there are structural alterations in the renal tubule, in association with an impaired urinary concentration. ARF is associated with defects both in collecting duct water reabsorption and proximal tubule water reabsorption, as well as defects in solute handling.335
337 Using the isolated tubule microperfusion model, it was demonstrated that water reabsorption in the proximal tubule and cortical collecting duct was significantly reduced following ischemia,335 and no differences in either basal or vasopressin-induced cAMP levels in outer or inner medulla in rats with ARF were demonstrated,338 supporting the view that there are defects in collecting duct water reabsorption. Consistent with these findings, it was demonstrated that AQP2 and AQP3 expression in the collecting duct, as well as AQP1 expression in the proximal tubule, are significantly reduced in response to ARF.339,340 The decreased levels of aquaporins were associated with impaired urinary concentration in rats with both oliguric or nonoliguric ARF. The reduced expression of AQP1
3 and the reduced urinary concentration capacity was significantly prevented by co-treatment with alpha-melanocyte-stimulating hormone (α-MSH), which is an anti-inflammatory cytokine that inhibits both neutrophil and nitric oxide pathways.340 This finding indicates that decreased levels of aquaporins in both the proximal tubule and collecting duct in postischemic kidneys may play a significant role in the impaired urinary concentration. Recently it was also demonstrated that hemorrhagic shock-induced acute renal failure is associated with decreased expression of collecting duct water channel AQP2 and AQP3,341 and that erythropoietin treatment (single or combined with

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α-MSH) in rats with I/R-induced ARF, which is known to prevent caspase-3, -8, and -9 activation in vivo and reduces apoptotic cell death,342 prevents or reduces the urinary concentrating defects and downregulation of AQP expression levels.341 Consistent with this, it was demonstrated that NF-kappaB activation is of importance for the downregulation of AQP2 channel and vasopressin V2-receptor expression during sepsis in a cecal ligation and puncture (CLP) mouse model of ARF, and that NF-kappaB inhibition ameliorates sepsis-induced ARF in the CLP model.343 Patients with advanced chronic renal failure (CRF) have urine which remains hypotonic to plasma despite the administration of supramaximal doses of vasopressin.344 This vasopressin-resistant hyposthenuria specifically implies abnormalities in collecting duct water reabsorption in CRF patients. Recent observations demonstrated virtual absence of V2 receptor mRNA in the inner medulla of CRF rat kidneys,345 providing evidence for significant defects in the collecting duct water permeability. Consistent with these observations, it has shown both decreased collecting duct water channel AQP2 and AQP3 expression and a vasopressin-resistant downregulation of AQP2 in a 5/6 nephrectomy-induced CRF rat model.346

WATER RETENTION Congestive Heart Failure Retention of sodium and water is a common and clinically important complication of congestive heart failure (CHF). Two studies have examined the changes in renal AQP expression in rats with CHF induced by ligation of the left coronary artery347,348 to test if upregulation of AQP2 expression and targeting may play a role in the water retention in CHF. Both studies demonstrated that renal water retention in severe CHF in rats is associated with dysregulation of AQP2 in the renal collecting duct principal cells, involving both an increase in the AQP2 expression and a marked redistribution of AQP2 to the apical plasma membrane.347,348 Rats with severe CHF had significantly elevated left ventricular end-diastolic pressures (LVEDP), and had reduced plasma sodium concentrations.347 Immunoblotting revealed a three-fold increase in AQP2 expression compared with sham operated animals. These changes were associated with elevated LVEDP or hyponatremia, since animals with normal LVEDP and plasma sodium did not have increased AQP2 levels compared with sham operated controls.347 Furthermore, this study showed an increased plasma membrane targeting, providing an explanation for the increased permeability of the collecting duct and an

1427

increase in water reabsorption. This may provide an explanation for excess free water retention in severe CHF, and for the development of hyponatremia. In parallel, the other study showed upregulation of both AQP2 protein and AQP2 mRNA levels in kidney inner medulla and cortex in rats with CHF.348 These rats had significantly decreased cardiac output and, importantly, increased plasma vasopressin levels. Furthermore, in this study administration of V2 antagonist OPC 31260 was associated with a significant increase in diuresis, a decrease in urine osmolality, a rise in plasma osmolality, and a significant reduction in AQP2 protein and AQP2 mRNA levels compared with untreated rats with CHF. Consistent with this, treatment of V2-receptor antagonist in human patients and rats with heart failure is associated with a doserelated increase in water excretion and a decrease in urinary AQP2 excretion.349,350 Moreover, it has been shown in patients with CHF that treatment with furosemide increases the urinary excretion of AQP2, free water clearance, and sodium exceretion.351

Hepatic Cirrhosis Hepatic cirrhosis is another chronic condition associated with water retention.352 It has been suggested that an important pathophysiological factor in the impaired ability to excrete water could be increased levels of plasma vasopressin. However, unlike CHF, the changes in expression of AQP2 protein levels vary considerably between different experimental models of hepatic cirrhosis. Several studies have examined the changes in renal AQP expression in rats with cirrhosis induced by common bile duct ligation (CBDL).277,353,354 The rats displayed impaired vasopressin-regulated water reabsorption despite normal plasma vasopressin levels. Consistent with this, semiquantitative immunoblotting showed a significant decrease in AQP2 expression in rats with hepatic cirrhosis.277,353 In addition, the expression levels of AQP3 and AQP4 were downregulated in CBDL rats. This may predict a reduced water permeability of the collecting duct in this model277; hence, renal water reabsorption in the collecting duct is decreased in rats with compensated liver cirrhosis. In contrast, Fujita et al.274 demonstrated that hepatic cirrhosis induced by intraperitoneal administration of carbon tetrachloride (CCl4) was associated with a significant increase in both AQP2 protein levels and AQP2 mRNA expression. Interestingly, AQP2 mRNA expression correlated with the amount of ascites, suggesting that AQP2 may play a role in the abnormal water retention followed by the development of ascites in hepatic cirrhosis.355 In a different model of CCl4-induced cirrhosis, using CCl4 inhalation, AQP2

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expression was not increased.356 There was, however, evidence for increased trafficking of AQP2 to the plasma membrane, consistent with the presence of elevated levels of vasopressin in the plasma. Interestingly, there was a marked increase in AQP3 expression that is likely to be due to increased vasopressin levels. The pattern of increased AQP3 expression without upregulation of AQP2 is consistent with previous findings observed in the vasopressin escape,276 suggesting that the lack of increase in AQP2 expression could be a result of a normal compensatory response related to the escape phenomenon. Although the explanation for the differences between cirrhosis induced by CBDL and CCl4 administration remains to be determined, it is well-known that the dysregulation of body water balance depends on the severity of cirrhosis.357
360 CBDL results in a compensated cirrhosis characterized by peripheral vasodilation and increased cardiac output, whereas cirrhosis induced by 12 weeks of CCl4 administration may be associated with the late state of decompensated liver cirrhosis characterized by sodium retention, edema, and ascites.358,359,361 Thus, the downregulation of AQP2 observed in milder forms of cirrhosis (i.e., in a compensated stage without water retention) may represent a compensatory mechanism to prevent development of water retention. In contrast, the increased levels of vasopressin seen in severe “noncompensated” cirrhosis with ascites may induce an inappropriate upregulation of AQP2 that would in turn participate in the development of water retention. Recent studies have shown a prominent role for AQP1 in angiogenesis, fibrosis, and portal hypertension after bile duct ligation in wild-type and AQP1 knockout mice.362

share similarities with the downregulation of AQP2 in water-loaded dDAVP-treated rats that escape from the action of vasopressin.275,276

SIADH and Vasopressin Escape Hyponatremia, defined as a serum sodium less than 135 mmol/L, is one of the most commonly encountered electrolyte disorders of clinical medicine.366 The predominant cause of hyponatremia is an inappropriate secretion of vasopressin relative to serum osmolality or the “syndrome of inappropriate antidiuretic hormone secretion” (SIADH).367 SIADH occurs most frequently in association with vascular, infectious or neoplastic abnormalities in the lung or central nervous system. In an experimental rat model of SIADH it was shown that AQP2 mRNA expression and AQP2 protein expression were increased in the collecting duct.274 Thus, increased expression of AQP2 in the collecting duct accounts for the water retention and hyponatremia in SIADH. The degree of hyponatremia is limited by a process that counters the water-retaining action of vasopressin, namely “vasopressin escape.” Vasopressin escape is characterized by a sudden increase in urine volume with a decrease in urine osmolality independent of high circulating vasopressin levels. The onset of escape coincided temporally with a marked decrease in renal AQP2 protein, as measured by immunoblotting, as well as decreased mRNA expression, as assessed by Northern blotting.276 In contrast to AQP2, there were no decreases in renal expression of AQP1, AQP3, and AQP4.276 These results suggest that escape from vasopressin-induced antidiuresis is attributable, at least in part, to a selective vasopressin-independent decrease in AQP2 expression in the renal collecting duct.368,369

Experimental Nephrotic Syndrome Nephrotic syndrome is characterized by extracellular volume expansion with excessive renal salt and water reabsorption. The underlying mechanisms of salt and water retention are poorly-understood; however, they can be expected to be associated with dysregulation of solute transporters and water channels.359 In contrast to congestive heart failure and liver cirrhosis, a marked downregulation of AQP2 and AQP3 expression was demonstrated in rats with PAN-induced and adriamycin-induced nephrotic syndrome.363
365 The reduced expression of collecting duct water channels could represent a physiologically appropriate response to extracellular volume expansion. The signal transduction involved in this process is not clear, but circulating vasopressin levels are high in rats with PAN-induced nephrotic syndrome. Thus, the marked downregulation of AQP2 in experimental nephrotic syndrome may

Acknowledgments Support for this chapter was partially provided by funds from the Danish National Research Foundation, The European Commission, the Danish Medical Research Council, Novo Nordic Foundation, Karen Elise Jensen Foundation and the National Institutes of Health. Portions of the text were previously published in abridged form and are reproduced and modified with permission.122 Moreover, the authors gratefully acknowledge the major contributions from Peter Agre and Mark Knepper, who together with SN authored a previous version of this chapter.

References [1] Finkelstein A. Water movement through lipid bilayers, pores, and plasma membranes: theory and reality. New York: John Wiley & Sons; 1987. [2] Solomon AK. Characterization of biological membranes by equivalent pores. J Gen Physiol 1968;51(Suppl).

III. FLUID AND ELECTROLYTE REGULATION AND DYSREGULATION

1429

REFERENCES

[3] Macey RI. Transport of water and urea in red blood cells. Am J Physiol 1984;246:C195
203. [4] Knepper MA, Wade JB, Terris J, Ecelbarger CA, Marples D, Mandon B, et al. Renal aquaporins. Kidney Int 1996;49:1712
7. [5] Bourguet J, Chevalier J, Hugon JS. Alterations in membraneassociated particle distribution during antidiuretic challenge in frog urinary bladder epithelium. Biophys J 1976;16:627
39. [6] Brown D, Orci L. Vasopressin stimulates formation of coated pits in rat kidney collecting ducts. Nature 1983;302:253
5. [7] Kachadorian WA, Wade JB, DiScala VA. Vasopressin: induced structural change in toad bladder luminal membrane. Science 1975;190:67
9. [8] Wade JB, Stetson DL, Lewis SA. ADH action: evidence for a membrane shuttle mechanism. Ann N.Y Acad Sci 1981;372: 106
17. [9] Agre P, Preston GM, Smith BL, Jung JS, Raina S, Moon C, et al. The archetypal molecular water channel. Am J Physiol 1993;265: F463
76. [10] Solomon AK, Chasan B, Dix JA, Lukacovic MF, Toon MR, Verkman AS. The aqueous pore in the red cell membrane: band 3 as a channel for anions, cations, nonelectrolytes, and water. Ann NY Acad Sci 1983;414:97
124. [11] Agre P, Saboori AM, Asimos A, Smith BL. Purification and partial characterization of the Mr 30,000 integral membrane protein associated with the erythrocyte Rh(D) antigen. J Biol Chem 1987;262:17497
503. [12] Denker BM, Smith BL, Kuhajda FP, Agre P. Identification, purification, and partial characterization of a novel Mr 28,000 integral membrane protein from erythrocytes and renal tubules. J Biol Chem 1988;263:15634
42. [13] Smith BL, Agre P. Erythrocyte Mr 28,000 transmembrane protein exists as a multisubunit oligomer similar to channel proteins. J Biol Chem 1991;266:6407
15. [14] Preston GM, Agre P. Isolation of the cDNA for erythrocyte integral membrane protein of 28 kilodaltons: member of an ancient channel family. Proc Natl Acad Sci USA 1991;88:11110
4. [15] Van Hoek AN, Hom ML, Luthjens LH, de J, Dempster JA, van Os CH. Functional unit of 30 kDa for proximal tubule water channels as revealed by radiation inactivation. J Biol Chem 1991;266:16633
5. [16] Agre P, Sasaki S, Chrispeels MJ. Aquaporins: a family of water channel proteins. Am J Physiol 1993;265:F461. [17] Agre P. Molecular physiology of water transport: aquaporin nomenclature workshop. Mammalian aquaporins. Biol Cell 1997;89:255
7. [18] Fischbarg J, Kuang KY, Vera JC, Arant S, Silverstein SC, Loike J, et al. Glucose transporters serve as water channels. Proc Natl Acad Sci USA 1990;87:3244
7. [19] Zhang RB, Logee KA, Verkman AS. Expression of mRNA coding for kidney and red cell water channels in Xenopus oocytes. J Biol Chem 1990;265:15375
8. [20] Preston GM, Carroll TP, Guggino WB, Agre P. Appearance of water channels in Xenopus oocytes expressing red cell CHIP28 protein. Science 1992;256:385
7. [21] Meinild AK, Klaerke DA, Zeuthen T. Bidirectional water fluxes and specificity for small hydrophilic molecules in aquaporins 0
5. J Biol Chem 1998;273:32446
51. [22] Zeidel ML, Ambudkar SV, Smith BL, Agre P. Reconstitution of functional water channels in liposomes containing purified red cell CHIP28 protein. Biochemistry 1992;31:7436
40. [23] Van Hoek AN, Verkman AS. Functional reconstitution of the isolated erythrocyte water channel CHIP28. J Biol Chem 1992;267:18267
9. [24] Zeidel ML, Nielsen S, Smith BL, Ambudkar SV, Maunsbach AB, Agre P. Ultrastructure, pharmacologic inhibition, and transport

[25]

[26]

[27]

[28]

[29]

[30]

[31]

[32]

[33]

[34]

[35]

[36]

[37]

[38]

[39]

[40]

[41]

[42]

[43]

selectivity of aquaporin channel-forming integral protein in proteoliposomes. Biochemistry 1994;33:1606
15. Pao GM, Wu LF, Johnson KD, Hofte H, Chrispeels MJ, Sweet G, et al. Evolution of the MIP family of integral membrane transport proteins. Mol Microbiol 1991;5:33
7. Wistow GJ, Pisano MM, Chepelinsky AB. Tandem sequence repeats in transmembrane channel proteins. Trends Biochem Sci 1991;16:170
1. Cabiaux V, Oberg KA, Pancoska P, Walz T, Agre P, Engel A. Secondary structures comparison of aquaporin-1 and bacteriorhodopsin: a fourier transform infrared spectroscopy study of two-dimensional membrane crystals. Biophys J 1997;73:406
17. Reizer J, Reizer A, Saier Jr MH. The MIP family of integral membrane channel proteins: sequence comparisons, evolutionary relationships, reconstructed pathway of evolution, and proposed functional differentiation of the two repeated halves of the proteins. Crit Rev Biochem Mol Biol 1993;28:235
57. Preston GM, Jung JS, Guggino WB, Agre P. Membrane topology of aquaporin CHIP. Analysis of functional epitope-scanning mutants by vectorial proteolysis. J Biol Chem 1994;269:1668
73. Preston GM, Jung JS, Guggino WB, Agre P. The mercurysensitive residue at cysteine 189 in the CHIP28 water channel. J Biol Chem 1993;268:17
20. Zhang R, Van Hoek AN, Biwersi J, Verkman AS. A point mutation at cysteine 189 blocks the water permeability of rat kidney water channel CHIP28k. Biochemistry 1993;32:2938
41. Jung JS, Preston GM, Smith BL, Guggino WB, Agre P. Molecular structure of the water channel through aquaporin CHIP. The hourglass model. J Biol Chem 1994;269:14648
54. Verbavatz JM, Brown D, Sabolic I, Valenti G, Ausiello DA, Van Hoek AN, et al. Tetrameric assembly of CHIP28 water channels in liposomes and cell membranes: a freeze-fracture study. J Cell Biol 1993;123:605
18. Shi LB, Skach WR, Verkman AS. Functional independence of monomeric CHIP28 water channels revealed by expression of wild-type mutant heterodimers. J Biol Chem 1994;269:10417
22. Walz T, Smith BL, Zeidel ML, Engel A, Agre P. Biologically active two-dimensional crystals of aquaporin CHIP. J Biol Chem 1994;269:1583
6. Walz T, Smith BL, Agre P, Engel A. The three-dimensional structure of human erythrocyte aquaporin CHIP. EMBO J 1994;13:2985
93. Walz T, Tittmann P, Fuchs KH, Muller DJ, Smith BL, Agre P, et al. Surface topographies at subnanometer-resolution reveal asymmetry and sidedness of aquaporin-1. J Mol Biol 1996;264:907
18. Cheng A, Van Hoek AN, Yeager M, Verkman AS, Mitra AK. Three-dimensional organization of a human water channel. Nature 1997;387:627
30. Jap BK, Li H. Structure of the osmo-regulated H2O-channel, ˚ resolution. J Mol Biol AQP-CHIP, in projection at 3.5 A 1995;251:413
20. Li H, Lee S, Jap BK. Molecular design of aquaporin-1 water channel as revealed by electron crystallography. Nat Struct Mol Biol 1997;4:263
5. Mitra AK, Van Hoek AN, Wiener MC, Verkman AS, Yeager M. The CHIP28 water channel visualized in ice by electron crystallography. Nat Struct Biol 1995;2:726
9. Mitsuoka K, Murata K, Walz T, Hirai T, Agre P, Heymann JB, ˚ resolution reveals et al. The structure of aquaporin-1 at 4.5-A short [alpha]-helices in the center of the monomer. J Struct Biol 1999;128:34
43. Murata K, Mitsuoka K, Hirai T, Walz T, Agre P, Heymann JB, et al. Structural determinants of water permeation through aquaporin-1. Nature 2000;407:599
605.

III. FLUID AND ELECTROLYTE REGULATION AND DYSREGULATION

1430

41. AQUAPORIN WATER CHANNELS IN MAMMALIAN KIDNEY

[44] Ren G, Cheng A, Reddy V, Melnyk P, Mitra AK. Threedimensional fold of the human AQP1 water channel determined ˚ resolution by electron crystallography of twoat 4 A dimensional crystals embedded in ice. J Mol Biol 2000;301: 369
87. [45] Ren G, Reddy VS, Cheng A, Melnyk P, Mitra AK. Visualization of a water-selective pore by electron crystallography in vitreous ice. Proc Natl Acad Sci USA 2001;98:1398
403. [46] Walz T, Hirai T, Murata K, Heymann JB, Mitsuoka K, Fujiyoshi Y, et al. The three-dimensional structure of aquaporin-1. Nature 1997;387:624
7. [47] Sui H, Han BG, Lee JK, Walian P, Jap BK. Structural basis of water-specific transport through the AQP1 water channel. Nature 2001;414:872
8. [48] de Groot BL, Grubmuller H. Water permeation across biological membranes. mechanism and dynamics of aquaporin-1 and GlpF. Science 2001;294:2353
7. [49] Beitz E, Wu B, Holm LM, Schultz JE, Zeuthen T. Point mutations in the aromatic/arginine region in aquaporin 1 allow passage of urea, glycerol, ammonia, and protons. Proc Nat Acad Sci 2006;103:269
74. [50] Maurel C, Reizer J, Schroeder JI, Chrispeels MJ, Saier Jr. MH. Functional characterization of the Escherichia coli glycerol facilitator, GlpF, in Xenopus oocytes. J Biol Chem 1994;269:11869
72. [51] Schenk AD, Werten PJL, Scheuring S, de Groot BL, Muller SA, ˚ structure of human AQP2. J Mol Stahlberg H, et al. The 4.5 A Biol 2005;350:278
89. [52] Werten PJL, Hasler L, Koenderink JB, Klaassen CHW, de Grip WJ, Engel A, et al. Large-scale purification of functional recombinant human aquaporin-2. FEBS Lett 2001;504:200
5. [53] Fotiadis D, Suda K, Tittmann P, Jeno P, Philippsen A, Muller DJ, et al. Identification and structure of a putative Ca21-binding domain at the C terminus of AQP1. J Mol Biol 2002;318: 1381
94. [54] Gonen T, Sliz P, Kistler J, Cheng Y, Walz T. Aquaporin-0 membrane junctions reveal the structure of a closed water pore. Nature 2004;429:193
7. [55] Harries WEC, Akhavan D, Miercke LJW, Khademi S, Stroud ˚ resoluRM. The channel architecture of aquaporin 0 at a 2.2-A tion. Proc Nat Acad Sci 2004;101:14045
50. [56] Mulders SM, Preston GM, Deen PMT, Guggino WB, Os CH, Agre P. Water channel properties of major intrinsic protein of lens. J Biol Chem 1995;270:9010
6. [57] Han BG, Guliaev AB, Walian PJ, Jap BK. Water transport in AQP0 aquaporin. Molecular dynamics studies. J Mol Biol 2006;360:285
96. [58] Brooks HL, Regan JW, Yool AJ. Inhibition of Aquaporin-1 water permeability by tetraethylammonium: involvement of the loop E pore region. Mol Pharmacol 2000;57:1021
6. [59] Detmers FJM, de Groot BL, Muller EM, Hinton A, Konings IBM, Sze M, et al. Quaternary ammonium compounds as water channel blockers. Specificity, potency, and site of action. J Biol Chem 2006;281:14207
14. [60] Verkman AS. Applications of aquaporin inhibitors. Drug News Perspect 2001;14:412. [61] Zelenina M, Tritto S, Bondar AA, Zelenin S, Aperia A. Copper inhibits the water and glycerol permeability of aquaporin-3. J Biol Chem 2004;279:51939
43. [62] Zelenina M, Bondar AA, Zelenin S, Aperia A. Nickel and extracellular acidification inhibit the water permeability of human aquaporin-3 in lung epithelial cells. J Biol Chem 2003;278: 30037
43. [63] Agemark M, Kowal J, Kukulski W, Norden K, Gustavsson N, Johanson U, et al. Reconstitution of water channel function and

[64]

[65]

[66]

[67]

[68]

[69]

[70]

[71]

[72]

[73]

[74] [75]

[76]

[77]

[78]

[79]

[80]

[81]

[82]

2D-crystallization of human aquaporin 8. Biochim Biophys Acta 2012;1818:839
50. Berthaud A, Manzi J, Perez J, Mangenot S. Modeling detergent organization around aquaporin-0 using small angle X-ray scattering. J Am Chem Soc 2012;134(24):10080
8. Crane JM, Rossi A, Gupta T, Bennett JL, Verkman AS. Orthogonal array formation by human aquaporin-4: examination of neuromyelitis optica-associated aquaporin-4 polymorphisms. J Neuroimmunol 2011;236:93
8. Fischer G, Kosinska-Eriksson U, ponte-Santamaria C, Palmgren M, Geijer C, Hedfalk K, et al. Crystal structure of a yeast aquaporin at 1.15 angstrom reveals a novel gating mechanism. PLoS Bio 2009;7:e1000130. Fujiyoshi Y. Electron crystallography for structural and functional studies of membrane proteins. J Electron Microsc (Tokyo) 2011;60(Suppl. 1):S149
59. Ho JD, Yeh R, Sandstrom A, Chorny I, Harries WE, Robbins ˚ and RA, et al. Crystal structure of human aquaporin 4 at 1.8 A its mechanism of conductance. Proc Natl Acad Sci USA 2009;106:7437
42. Horsefield R, Norden K, Fellert M, Backmark A, TornrothHorsefield S, Terwisscha van Scheltinga AC, et al. Highresolution x-ray structure of human aquaporin 5. Proc Natl Acad Sci USA 2008;105:13327
32. Jiang Y. Expression and functional characterization of NPA motif-null aquaporin-1 mutations. IUBMB Life 2009;61: 651
7. Strand L, Moe SE, Solbu TT, Vaadal M, Holen T. Roles of aquaporin-4 isoforms and amino acids in square array assembly. Biochemistry 2009;48:5785
93. Wolburg H, Wolburg-Buchholz K, Fallier-Becker P, Noell S, Mack AF. Structure and functions of aquaporin-4-based orthogonal arrays of particles. Int Rev Cell Mol Biol 2011;287:1
41. Wree D, Wu B, Zeuthen T, Beitz E. Requirement for asparagine in the aquaporin NPA sequence signature motifs for cation exclusion. FEBS J 2011;278:740
8. Yakata K, Tani K, Fujiyoshi Y. Water permeability and characterization of aquaporin-11. J Struct Biol 2011;174:315
20. Sabolic I, Valenti G, Verbavatz JM, Van Hoek AN, Verkman AS, Ausiello DA, et al. Localization of the CHIP28 water channel in rat kidney. Am J Physiol 1992;263:C1225
33. Maunsbach AB, Marples D, Chin E, Ning G, Bondy C, Agre P, et al. Aquaporin-1 water channel expression in human kidney. J Am Soc Nephrol 1997;8:1
14. Nielsen S, Smith B, Christensen EI, Knepper MA, Agre P. CHIP28 water channels are localized in constitutively waterpermeable segments of the nephron. J Cell Biol 1993;120: 371
83. Maeda Y, Smith BL, Agre P, Knepper MA. Quantification of aquaporin-CHIP water channel protein in microdissected renal tubules by fluorescence-based ELISA. J Clin Invest 1995;95: 422
8. Bedford JJ, Leader JP, Walker RJ. Aquaporin expression in normal human kidney and in renal disease. J Am Soc Nephrol 2003;14:2581
7. King LS, Nielsen S, Agre P. Aquaporin-1 water channel protein in lung: ontogeny, steroid-induced expression, and distribution in rat. J Clin Invest 1996;97:2183
91. Nielsen S, Smith BL, Christensen EI, Agre P. Distribution of the aquaporin CHIP in secretory and resorptive epithelia and capillary endothelia. Proc Natl Acad Sci USA 1993;90:7275
9. Schnitzer JE, Oh P. Aquaporin-1 in plasma membrane and caveolae provides mercury-sensitive water channels across lung endothelium. Am J Physiol 1996;270:H416
22.

III. FLUID AND ELECTROLYTE REGULATION AND DYSREGULATION

REFERENCES

[83] Pallone TL, Kishore BK, Nielsen S, Agre P, Knepper MA. Evidence that aquaporin-1 mediates NaCl-induced water flux across descending vasa recta. Am J Physiol 1997;272:F587
96. [84] King LS, Nielsen S, Agre P. Aquaporins in complex tissues. I. Developmental patterns in respiratory and glandular tissues of rat. Am J Physiol 1997;273:C1541
8. [85] Moon C, King LS, Agre P. Aqp1 expression in erythroleukemia cells: genetic regulation of glucocorticoid and chemical induction. Am J Physiol 1997;273:C1562
70. [86] Lai, KN., Li, FG., Yui, LH., Tang, S., Tsang, AWL., Chan, DTM, et al., Expression of aquaporin-1 in human peritoneal mesothelial cells and its upregulation by glucose in vitro. J Am Soc Nephrol 12:1036
45. [87] Mann SE, Ricke EA, Yang BA, Verkman AS, Taylor RN. Expression and localization of aquaporin 1 and 3 in human fetal membranes. Am J Obstet Gynecol 2002;187:902
7. [88] Hasegawa H, Lian SC, Finkbeiner WE, Verkman AS. Extrarenal tissue distribution of CHIP28 water channels by in situ hybridization and antibody staining. Am J Physiol 1994;266:C893
903. [89] Stamer WD, Snyder RW, Smith BL, Agre P, Regan JW. Localization of aquaporin CHIP in the human eye: implications in the pathogenesis of glaucoma and other disorders of ocular fluid balance. Invest Ophthalmol Vis Sci 1994;35:3867
72. [90] Roberts SK, Yano M, Ueno Y, Pham L, Alpini G, Agre P, et al. Cholangiocytes express the aquaporin CHIP and transport water via a channel-mediated mechanism. Proc Natl Acad Sci USA 1994;91:13009
13. [91] Ko SBH, Naruse S, Kitagawa M, Ishiguro H, Furuya S, Mizuno N, et al. Aquaporins in rat pancreatic interlobular ducts. AJPGastrointest Liver Physiol 2002;282:G324
31. [92] Au CG, Cooper ST, Lo HP, Compton AG, Yang N, Wintour EM, et al. Expression of aquaporin 1 in human cardiac and skeletal muscle. J Mol Cell Cardiol 2004;36:655
62. [93] Calamita G, Ferri D, Bazzini C, Mazzone A, Botta G, Liquori G, et al. Expression and subcellular localization of the AQP8 and AQP1 water channels in the mouse gall-bladder epithelium. Biol Cell 2005;97(6):415
23. [94] Gresz V, Kwon TH, Hurley PT, Varga G, Zelles T, Nielsen S, et al. Identification and localization of aquaporin water channels in human salivary glands. AJP-Gastrointest Liver Physiol 2001;281:G247
54. [95] Huang D, Chen P, Chen S, Nagura M, Lim DJ, Lin X. Expression patterns of aquaporins in the inner ear: evidence for concerted actions of multiple types of aquaporins to facilitate water transport in the cochlea. Hear Res 2002;165:85
95. [96] Takata K, Matsuzaki T, Tajika Y. Aquaporins: water channel proteins of the cell membrane. Prog Histochem Cytochem 2004;39:1
83. [97] Endo M, Jain RK, Witwer B, Brown D. Water channel (aquaporin 1) expression and distribution in mammary carcinomas and glioblastomas. Microvasc Res 1999;58:89
98. [98] Bondy C, Chin E, Smith BL, Preston GM, Agre P. Developmental gene expression and tissue distribution of the CHIP28 water-channel protein. Proc Natl Acad Sci USA 1993;90:4500
4. [99] Smith BL, Baumgarten R, Nielsen S, Raben D, Zeidel ML, Agre P. Concurrent expression of erythroid and renal aquaporin CHIP and appearance of water channel activity in perinatal rats. J Clin Invest 1993;92:2035
41. [100] Smith BL, Preston GM, Spring FA, Anstee DJ, Agre P. Human red cell aquaporin CHIP. I. Molecular characterization of ABH and Colton blood group antigens. J Clin Invest 1994;94:1043
9.

1431

[101] Preston GM, Smith BL, Zeidel ML, Moulds JJ, Agre P. Mutations in aquaporin-1 in phenotypically normal humans without functional CHIP water channels. Science 1994;265: 1585
7. [102] King LS, Choi M, Fernandez PC, Cartron JP, Agre P. Defective urinary concentrating ability due to a complete deficiency of aquaporin-1. N Engl J Med 2001;345:175
9. [103] King LS, Nielsen S, Agre P, Brown RH. Decreased pulmonary vascular permeability in aquaporin-1-null humans. Proc Natl Acad Sci 2002;99:1059
63. [104] Ma T, Yang B, Gillespie A, Carlson EJ, Epstein CJ, Verkman AS. Severely impaired urinary concentrating ability in transgenic mice lacking aquaporin-1 water channels. J Biol Chem 1998;273:4296
9. [105] Schnermann J, Chou CL, Ma T, Traynor T, Knepper MA, Verkman AS. Defective proximal tubular fluid reabsorption in transgenic aquaporin-1 null mice. Proc Natl Acad Sci USA 1998;95:9660
4. [106] Vallon V, Verkman AS, Schnermann J. Luminal hypotonicity in proximal tubules of aquaporin-1-knockout mice. AJP-Renal Physiol 2000;278:F1030
3. [107] Hashimoto S, Huang Y, Mizel D, Briggs J, Schnermann J. Compensation of proximal tubule malabsorption in AQP1deficient mice without TGF-mediated reduction of GFR. Acta Physiol Scand 2004;181:455
62. [108] Chou CL, Knepper MA, Hoek AN, Brown D, Yang B, Ma T, et al. Reduced water permeability and altered ultrastructure in thin descending limb of Henle in aquaporin-1 null mice. J Clin Invest 1999;103:491
6. [109] Pallone TL, Edwards A, Ma T, Silldorff EP, Verkman AS. Requirement of aquaporin-1 for NaCl-driven water transport across descending vasa recta. J Clin Invest 2000;105:215
22. [110] Yang B, Tonghui MA, Dong JY, Verkman AS. Partial correction of the urinary concentrating defect in aquaporin-1 null mice by adenovirus-mediated gene delivery. Human Gene Therapy 2000;11:567
75. [111] Abrami L, Tacnet F, Ripoche P. Evidence for a glycerol pathway through aquaporin 1 (CHIP28) channels. Pflugers Arch 1995;430:447
58. [112] Yool AJ, Stamer WD, Regan JW. Forskolin stimulation of water and cation permeability in aquaporin 1 water channels. Science 1996;273:1216
8. [113] Agre P, Lee MD, Devidas S, Guggino WB. Aquaporins and ion conductance. Science 1997;275:1490. [114] Patil RV, Saito I, Yang X, Wax MB. Expression of aquaporins in the rat ocular tissue. Exp Eye Res 1997;64:203
9. [115] Marinelli RA, Pham L, Agre P, LaRusso NF. Secretin promotes osmotic water transport in rat cholangiocytes by increasing aquaporin-1 water channels in plasma membrane. Evidence for a secretin-induced vesicular translocation of aquaporin-1. J Biol Chem 1997;272:12984
8. [116] Marinelli RA, Tietz PS, Pham LD, Rueckert L, Agre P, LaRusso NF. Secretin induces the apical insertion of aquaporin-1 water channels in rat cholangiocytes. AJP-Gastrointest Liver Physiol 1999;276:G280
6. [117] Nakhoul NL, Davis BA, Romero MF, Boron WF. Effect of expressing the water channel aquaporin-1 on the CO2 permeability of Xenopus oocytes. Am J Physiol 1998;274:C543
8. [118] Reuss L. Focus on “Effect of expressing the water channel aquaporin-1 on the CO2 permeability of Xenopus oocytes”. Am J Physiol 1998;274:C297
8. [119] Cooper GJ, Zhou Y, Bouyer P, Grichtchenko II, Boron WF. Transport of volatile solutes through AQP1. J Physiol Online 2002;542:17
29.

III. FLUID AND ELECTROLYTE REGULATION AND DYSREGULATION

1432

41. AQUAPORIN WATER CHANNELS IN MAMMALIAN KIDNEY

[120] Saadoun S, Papadopoulos MC, Hara-Chikuma M, Verkman AS. Impairment of angiogenesis and cell migration by targeted aquaporin-1 gene disruption. Nature 2005;434:786
92. [121] Hara-Chikuma M, Verkman AS. Aquaporin-1 facilitates epithelial cell migration in kidney proximal tubule. J Am Soc Nephrol 2006;17:39
45. [122] Agre P, Bonhivers M, Borgnia MJ. The aquaporins, blueprints for cellular plumbing systems. J Biol Chem. 1998;273: 14659
62. [123] Fushimi K, Uchida S, Hara Y, Hirata Y, Marumo F, Sasaki S. Cloning and expression of apical membrane water channel of rat kidney collecting tubule. Nature 1993;361:549
52. [124] Echevarria M, Windhager EE, Tate SS, Frindt G. Cloning and expression of AQP3, a water channel from the medullary collecting duct of rat kidney. Proc Natl Acad Sci USA 1994;91:10997
1001. [125] Ishibashi K, Sasaki S, Fushimi K, Uchida S, Kuwahara M, Saito H, et al. Molecular cloning and expression of a member of the aquaporin family with permeability to glycerol and urea in addition to water expressed at the basolateral membrane of kidney collecting duct cells. Proc Natl Acad Sci USA 1994;91:6269
73. [126] Ma T, Frigeri A, Hasegawa H, Verkman AS. Cloning of a water channel homolog expressed in brain meningeal cells and kidney collecting duct that functions as a stilbene-sensitive glycerol transporter. J Biol Chem 1994;269:21845
9. [127] Hasegawa H, Ma T, Skach W, Matthay MA, Verkman AS. Molecular cloning of a mercurial-insensitive water channel expressed in selected water-transporting tissues. J Biol Chem. 1994;269:5497
500. [128] Jung JS, Bhat RV, Preston GM, Guggino WB, Baraban JM, Agre P. Molecular characterization of an aquaporin cDNA from brain: candidate osmoreceptor and regulator of water balance. Proc Natl Acad Sci USA 1994;91:13052
6. [129] Ma T, Frigeri A, Skach W, Verkman AS. Cloning of a novel rat kidney cDNA homologous to CHIP28 and WCH-CD water channels. Biochem Biophys Res Commun 1993;197: 654
9. [130] Ma T, Yang B, Kuo WL, Verkman AS. cDNA cloning and gene structure of a novel water channel expressed exclusively in human kidney: evidence for a gene cluster of aquaporins at chromosome locus 12q13. Genomics 1996;35:543
50. [131] Yasui M, Kwon TH, Knepper MA, Nielsen S, Agre P. Aquaporin-6. An intracellular vesicle water channel protein in renal epithelia. Proc Natl Acad Sci 1999;96:5808
13. [132] Ishibashi K, Kuwahara M, Gu Y, Kageyama Y, Tohsaka A, Suzuki F, et al. Cloning and functional expression of a new water channel abundantly expressed in the testis permeable to water, glycerol, and urea. J Biol Chem. 1997;272:20782
6. [133] Kuriyama H, Kawamoto S, Ishida N, Ohno I, Mita S, Matsuzawa Y, et al. Molecular cloning and expression of a novel human aquaporin from adipose tissue with glycerol permeability. Biochem Biophys Res Commun 1997;241:53
8. [134] Sohara E, Rai T, Miyazaki Ji, Verkman AS, Sasaki S, Uchida S. Defective water and glycerol transport in the proximal tubules of AQP7 knockout mice. AJP-Renal Physiol 2005;289: F1195
200. [135] Elkjar ML, Nejsum LN, Gresz V, Kwon TH, Jensen UB, Frokiar J, et al. Immunolocalization of aquaporin-8 in rat kidney, gastrointestinal tract, testis, and airways. AJP-Renal Physiol 2001;281:F1047
57. [136] Ma T, Yang B, Verkman AS. Cloning of a novel water and urea-permeable aquaporin from mouse expressed strongly in colon, placenta, liver, and heart. Biochem Biophys Res Commun 1997;240:324
8.

[137] Morishita Y, Matsuzaki T, Hara-chikuma M, Andoo A, Shimono M, Matsuki A, et al. Disruption of aquaporin-11 produces polycystic kidneys following vacuolization of the proximal tubule. Mol Cell Biol 2005;25:7770
9. [138] Nielsen S, DiGiovanni SR, Christensen EI, Knepper MA, Harris HW. Cellular and subcellular immunolocalization of vasopressin- regulated water channel in rat kidney. Proc Natl Acad Sci USA 1993;90:11663
7. [139] Kishore BK, Terris JM, Knepper MA. Quantitation of aquaporin-2 abundance in microdissected collecting ducts: axial distribution and control by AVP. Am J Physiol 1996;271: F62
70. [140] Coleman RA, Wu DC, Liu J, Wade JB. Expression of aquaporins in the renal connecting tubule. AJP-Renal Physiol 2000;279: F874
83. [141] Christensen BM, Wang W, Frokiar J, Nielsen S. Axial heterogeneity in basolateral AQP2 localization in rat kidney: effect of vasopressin. AJP-Renal Physiol 2003;284:F701
17. [142] Flamion B, Spring KR. Water permeability of apical and basolateral cell membranes of rat inner medullary collecting duct. Am J Physiol 1990;259:F986
99. [143] DiGiovanni SR, Nielsen S, Christensen EI, Knepper MA. Regulation of collecting duct water channel expression by vasopressin in Brattleboro rat. Proc Natl Acad Sci USA 1994;91:8984
8. [144] Deen PM, Verdijk MA, Knoers NV, Wieringa B, Monnens LA, van Os CH, et al. Requirement of human renal water channel aquaporin-2 for vasopressin-dependent concentration of urine. Science 1994;264:92
5. [145] Rojek A, Fuchtbauer EM, Kwon TH, Frokiar J, Nielsen S. Severe urinary concentrating defect in renal collecting ductselective AQP2 conditional-knockout mice. Proc Natl Acad Sci 2006;103(15):6037
42. [146] Yang B, Gillespie A, Carlson EJ, Epstein CJ, Verkman AS. Neonatal mortality in an aquaporin-2 knock-in mouse model of recessive nephrogenic diabetes insipidus. J Biol Chem 2001;276:2775
9. [147] Yang B, Zhao D, Qian L, Verkman AS. Mouse model of inducible nephrogenic diabetes insipiduc produced by floxed aquaporin-2 gene deletion. AJP-Renal Physiol 2006;291(2): F465
72. [148] Ma T, Song Y, Yang B, Gillespie A, Carlson EJ, Epstein CJ, et al. Nephrogenic diabetes insipidus in mice lacking aquaporin-3 water channels. Proc Natl Acad Sci 2000;97: 4386
91. [149] Yang B, Verkman AS. Water and glycerol permeabilities of aquaporins 1
5 and MIP determined quantitatively by expression of epitope-tagged constructs in Xenopus oocytes. J Biol Chem 1997;272:16140
6. [150] Zeuthen T, Klaerke DA. Transport of water and glycerol in aquaporin 3 is gated by H1. J Biol Chem 1999;274:21631
6. [151] Ecelbarger CA, Terris J, Frindt G, Echevarria M, Marples D, Nielsen S, et al. Aquaporin-3 water channel localization and regulation in rat kidney. Am J Physiol 1995;269:F663
72. [152] Frigeri A, Gropper MA, Turck CW, Verkman AS. Immunolocalization of the mercurial-insensitive water channel and glycerol intrinsic protein in epithelial cell plasma membranes. Proc Natl Acad Sci USA 1995;92:4328
31. [153] Terris J, Ecelbarger CA, Marples D, Knepper MA, Nielsen S. Distribution of aquaporin-4 water channel expression within rat kidney. Am J Physiol 1995;269:F775
85. [154] van Hoek AN, Ma T, Yang B, Verkman AS, Brown D. Aquaporin-4 is expressed in basolateral membranes of proximal tubule S3 segments in mouse kidney. AJP-Renal Physiol 2000;278:F310
6.

III. FLUID AND ELECTROLYTE REGULATION AND DYSREGULATION

REFERENCES

[155] Silberstein C, Bouley R, Huang Y, Fang P, Pastor-Soler N, Brown D, et al. Membrane organization and function of M1 and M23 isoforms of aquaporin-4 in epithelial cells. AJP-Renal Physiol 2004;287:F501
11. [156] Kwon TH, Nielsen J, Masilamani S, Hager H, Knepper MA, Frokiar J, et al. Regulation of collecting duct AQP3 expression: response to mineralocorticoid. AJP-Renal Physiol 2002;283: F1403
21. [157] Zelenina M, Zelenin S, Bondar AA, Brismar H, Aperia A. Water permeability of aquaporin-4 is decreased by protein kinase C and dopamine. AJP-Renal Physiol 2002;283:F309
18. [158] Chou CL, Ma T, Yang B, Knepper MA, Verkman AS. Fourfold reduction of water permeability in inner medullary collecting duct of aquaporin-4 knockout mice. Am J Physiol 1998;274: C549
54. [159] Ma T, Yang B, Gillespie A, Carlson EJ, Epstein CJ, Verkman AS. Generation and phenotype of a transgenic knockout mouse lacking the mercurial-insensitive water channel aquaporin-4. J Clin Invest 1997;100:957
62. [160] Yasui M, Hazama A, Kwon TH, Nielsen S, Guggino WB, Agre P. Rapid gating and anion permeability of an intracellular aquaporin. Nature 1999;402:184
7. [161] Ikeda M, Beitz E, Kozono D, Guggino WB, Agre P, Yasui M. Characterization of aquaporin-6 as a nitrate channel in mammalian cells. Requirement of pore-lining residue threonine 63. J Biol Chem 2002;277:39873
9. [162] Hazama A, Kozono D, Guggino WB, Agre P, Yasui M. Ion permeation of AQP6 water channel protein. Single-channel recordings after Hg21 activation. J Biol Chem 2002;277: 29224
30. [163] Liu K, Kozono D, Kato Y, Agre P, Hazama A, Yasui M. From the cover. Conversion of aquaporin 6 from an anion channel to a water-selective channel by a single amino acid substitution. Proc Natl Acad Sci 2005;102:2192
7. [164] Kishida K, Kuriyama H, Funahashi T, Shimomura I, Kihara S, Ouchi N, et al. Aquaporin adipose, a putative glycerol channel in adipocytes. J Biol Chem 2000;275:20896
902. [165] Ishibashi K, Imai M, Sasaki S. Cellular localization of aquaporin 7 in the rat kidney. Nephron Exp Nephrology 2000;8:252
7. [166] Nejsum LN, Elkjaer M-L, Hager H, Frokiaer J, Kwon TH, Nielsen S. Localization of aquaporin-7 in rat and mouse kidney using RT-PCR, immunoblotting, and immunocytochemistry. Biochem Biophys Res Commun 2000;277:164
70. [167] Liu Z, Shen J, Carbrey JM, Mukhopadhyay R, Agre P, Rosen BP. Arsenite transport by mammalian aquaglyceroporins AQP7 and AQP9. Proc Natl Acad Sci 2002;99:6053
8. [168] Ishibashi K, Kuwahara M, Kageyama Y, Tohsaka A, Marumo F, Sasaki S. Cloning and functional expression of a second new aquaporin abundantly expressed in testis. Biochem Biophys Res Commun 1997;237:714
8. [169] Koyama Y, Yamamoto T, Tani T, Nihei K, Kondo D, Funaki H, et al. Expression and localization of aquaporins in rat gastrointestinal tract. Am J Physiol 1999;276:C621
7. [170] Yang B, Song Y, Zhao D, Verkman AS. Phenotype analysis of aquaporin-8 null mice. AJP-Cell Physiol 2005;288:C1161
70. [171] Holm LM, Jahn TP, Moller AL, Schjoerring JK, Ferri D, Klaerke DA, et al. NH3 and NH41 permeability in aquaporinexpressing Xenopus oocytes. Pflugers Arch 2005;450:415
28. [172] Liu KF, Nagase HF, Huang CG, Calamita G, Agre P. Purification and functional characterization of aquaporin-8. Biol Cell 2006;98(3):153
61. [173] Jahn TP, Moller ALB, Zeuthen T, Holm LM, Klaerke DA, Mohsin B, et al. Aquaporin homologues in plants and mammals transport ammonia. FEBS Lett 2004;574:31
6.

1433

[174] Yang B, Zhao D, Solenov E, Verkman AS. Evidence from knockout mice against physiologically significant aquaporin-8 facilitated ammonia transport. AJP-Cell Physiol 2006;291(3): C417
423. [175] Nielsen S, King LS, Christensen BM, Agre P. Aquaporins in complex tissues. II. Subcellular distribution in respiratory and glandular tissues of rat. AJP-Cell Physiol 1997;273:C1549
61. [176] Maeda N, Funahashi T, Hibuse T, Nagasawa A, Kishida K, Kuriyama H, et al. Adaptation to fasting by glycerol transport through aquaporin 7 in adipose tissue. Proc Natl Acad Sci 2004;101:17801
6. [177] Ishibashi K, Kuwahara M, Gu Y, Tanaka Y, Marumo F, Sasaki S. Cloning and functional expression of a new aquaporin (AQP9) abundantly expressed in the peripheral leukocytes permeable to water and urea, but not to glycerol. Biochem Biophys Res Commun 1998;244:268
74. [178] Ko SB, Uchida S, Naruse S, Kuwahara M, Ishibashi K, Marumo F, et al. Cloning and functional expression of rAOP9L a new member of aquaporin family from rat liver. Biochem Mol Biol Int 1999;47:309
18. [179] Tsukaguchi H, Weremowicz S, Morton CC, Hediger MA. Functional and molecular characterization of the human neutral solute channel aquaporin-9. AJP-Renal Physiol 1999;277: F685
96. [180] Tsukaguchi H, Shayakul C, Berger UV, Mackenzie B, Devidas S, Guggino WB, et al. Molecular characterization of a broad selectivity neutral solute channel. J Biol Chem 1998;273: 24737
43. [181] Carbrey JM, Gorelick-Feldman DA, Kozono D, Praetorius J, Nielsen S, Agre P. Aquaglyceroporin AQP9. Solute permeation and metabolic control of expression in liver. Proc Natl Acad Sci 2003;100:2945
50. [182] Nielsen S, Chou CL, Marples D, Christensen EI, Kishore BK, Knepper MA. Vasopressin increases water permeability of kidney collecting duct by inducing translocation of aquaporin-CD water channels to plasma membrane. Proc Natl Acad Sci USA 1995;92:1013
7. [183] Sabolic I, Katsura T, Verbavatz JM, Brown D. The AQP2 water channel: effect of vasopressin treatment, microtubule disruption, and distribution in neonatal rats. J Membr Biol 1995;143: 165
75. [184] Yamamoto T, Sasaki S, Fushimi K, Ishibashi K, Yaoita E, Kawasaki K, et al. Vasopressin increases AQP-CD water channel in apical membrane of collecting duct cells in Brattleboro rats. AJP-Cell Physiol 1995;268:C1546
51. [185] Lankford SP, Chou CL, Terada Y, Wall SM, Wade JB, Knepper MA. Regulation of collecting duct water permeability independent of cAMP-mediated AVP response. Am J Physiol 1991;261: F554
66. [186] Lee YJ, Lee JE, Choi HJ, Lim JS, Jung HJ, Baek MC, et al. E3 ubiquitin-protein ligases in rat kidney collecting duct: response to vasopressin stimulation and withdrawal. Am J PhysiolRenal Physiol 2011;301:F883
96. [187] Terris J, Ecelbarger CA, Nielsen S, Knepper MA. Long-term regulation of four renal aquaporins in rats. Am J Physiol 1996;271:F414
22. [188] Kuwahara M, Verkman AS. Pre-steady-state analysis of the turn-on and turn-off of water permeability in the kidney collecting tubule. J Membr Biol 1989;110:57
65. [189] Wall SM, Han JS, Chou CL, Knepper MA. Kinetics of urea and water permeability activation by vasopressin in rat terminal IMCD. Am J Physiol 1992;262:F989
98. [190] Nielsen S, Frokiar J, Marples D, Kwon TH, Agre P, Knepper MA. Aquaporins in the Kidney. From molecules to medicine. Physiol Rev 2002;82:205
44.

III. FLUID AND ELECTROLYTE REGULATION AND DYSREGULATION

1434

41. AQUAPORIN WATER CHANNELS IN MAMMALIAN KIDNEY

[191] Hays RM, Leaf A. Studies on the movement of water through the isolated toad bladder and its modification by vasopressin. J Gen Physiol 1962;45:905
19. [192] Koefoed-Johnsen V, Ussing HH. The contributions of diffusion and flow to the passage of D2O through living membranes; effect of neurohypophyseal hormone on isolated anuran skin. Acta Physiol Scand 1953;28:60
76. [193] Grantham JJ, Burg MB. Effect of vasopressin and cyclic AMP on permeability of isolated collecting tubules. Am J Physiol 1966;211:255
9. [194] Morgan T, Berliner RW. Permeability of the loop of Henle, vasa recta, and collecting duct to water, urea, and sodium. Am J Physiol 1968;215:108
15. [195] Nielsen S, Knepper MA. Vasopressin activates collecting duct urea transporters and water channels by distinct physical processes. Am J Physiol 1993;265:F204
13. [196] Kanno K, Sasaki S, Hirata Y, Ishikawa S, Fushimi K, Nakanishi S, et al. Urinary excretion of aquaporin-2 in patients with diabetes insipidus. N Eng J Med 1995;332:1540
5. [197] Marples D, Knepper MA, Christensen EI, Nielsen S. Redistribution of aquaporin-2 water channels induced by vasopressin in rat kidney inner medullary collecting duct. Am J Physiol 1995;269:C655
64. [198] Christensen BM, Marples D, Jensen UB, Frokiaer J, SheikhHamad D, Knepper M, et al. Acute effects of vasopressin V2receptor antagonist on kidney AQP2 expression and subcellular distribution. AJP-Renal Physiol 1998;275:F285
97. [199] Hayashi M, Sasaki S, Tsuganezawa H, Monkawa T, Kitajima W, Konishi K, et al. Expression and distribution of aquaporin of collecting duct are regulated by vasopressin V2 receptor in rat kidney. J Clin Invest 1994;94:1778
83. [200] Saito T, Ishikawa SE, Sasaki S, Fujita N, Fushimi K, Okada K, et al. Alteration in water channel AQP-2 by removal of AVP stimulation in collecting duct cells of dehydrated rats. AJPRenal Physiol 1997;272:F183
91. [201] Knepper MA, Nielsen S. Kinetic model of water and urea permeability regulation by vasopressin in collecting duct. Am J Physiol 1993;265:F214
24. [202] Brown D, Weyer P, Orci L. Vasopressin stimulates endocytosis in kidney collecting duct principal cells. Eur J Cell Biol 1988;46:336
41. [203] Lencer WI, Brown D, Ausiello DA, Verkman AS. Endocytosis of water channels in rat kidney: cell specificity and correlation with in vivo antidiuresis. Am J Physiol 1990; 259:C920
32. [204] Strange K, Willingham MC, Handler JS, Harris Jr HW. Apical membrane endocytosis via coated pits is stimulated by removal of antidiuretic hormone from isolated, perfused rabbit cortical collecting tubule. J Membr Biol 1988;103:17
28. [205] Nielsen S, Muller J, Knepper MA. Vasopressin- and cAMPinduced changes in ultrastructure of isolated perfused inner medullary collecting ducts. Am J Physiol 1993;265:F225
38. [206] Deen PM, van Aubel RA, Van Lieburg AF, van Os CH. Urinary content of aquaporin 1 and 2 in nephrogenic diabetes insipidus. J Am Soc Nephrol 1996;7:836
41. [207] Katsura T, Ausiello DA, Brown D. Direct demonstration of aquaporin-2 water channel recycling in stably transfected LLCPK1 epithelial cells. AJP-Renal Physiol 1996;270:F548
53. [208] Katsura T, Gustafson CE, Ausiello DA, Brown D. Protein kinase A phosphorylation is involved in regulated exocytosis of aquaporin-2 in transfected LLC-PK1 cells. Am J Physiol 1997;272:F817
22. [209] Katsura T, Verbavatz J, Farinas J, Ma T, Ausiello DA, Verkman AS, et al. Constitutive and regulated membrane expression of aquaporin 1 and aquaporin 2 water channels in stably

[210]

[211]

[212]

[213]

[214]

[215]

[216]

[217]

[218]

[219]

[220]

[221]

[222]

[223]

[224]

[225]

[226]

[227]

transfected LLC-PK1 epithelial cells. Proc Natl Acad Sci 1995;92:7212
6. Valenti G, Frigeri A, Ronco PM, D’Ettorre C, Svelto M. Expression and functional analysis of water channels in a stably AQP2-transfected human collecting duct cell line. J Biol Chem 1996;271:24365
70. Frische S, Kwon TH, Frokiaer J, Nielsen S. Aquaporin-2 trafficking. In: Boles E, Kramer R, editors. Molecular mechanisms controlling transmembrane transport. Berlin: Springer; p. 353
77. Kuwahara M, Fushimi K, Terada Y, Bai L, Marumo F, Sasaki S. cAMP-dependent phosphorylation stimulates water permeability of aquaporin-collecting duct water channel protein expressed in Xenopus oocytes. J Biol Chem 1995;270:10384
7. Lande MB, Jo I, Zeidel ML, Somers M, Harris Jr. HW. Phosphorylation of aquaporin-2 does not alter the membrane water permeability of rat papillary water channel-containing vesicles. J Biol Chem 1996;271:5552
7. Knepper M, Nielsen S, Chou CL, DiGiovanni, SR, Mechanism of vasopressin action in the renal collecting duct. Semin Nephrol 14:302
21. Kwon TH, Nielsen J, Moller HB, Fenton RA, Nielsen S, Frokiaer J. Aquaporins in the kidney. Handb Exp Pharmacol 2009;190:95
132. Moeller HB, Olesen ET, Fenton RA. Regulation of the water channel aquaporin-2 by posttranslational modification. AJPRenal Physiol 2011;300:F1062
73. Nedvetsky PI, Tamma G, Beulshausen S, Valenti G, Rosenthal W, Klussmann E. Regulation of aquaporin-2 trafficking. Handb Exp Pharmacol 2009;190:133
57. Edwards RM, Jackson BA, Dousa TP. ADH-sensitive cAMP system in papillary collecting duct: effect of osmolality and PGE2. Am J Physiol 1981;240:F311
8. Kurokawa K, Massry SG. Interaction between catecholamines and vasopressin on renal medullary cyclic AMP of rat. Am J Physiol 1973;225:825
9. Nishimoto G, Zelenina M, Li D, Yasui M, Aperia A, Nielsen S, et al. Arginine vasopressin stimulates phosphorylation of aquaporin-2 in rat renal tissue. Am J Physiol 1999;276:F254
9. Fushimi K, Sasaki S, Marumo F. Phosphorylation of serine 256 is required for cAMP-dependent regulatory exocytosis of the aquaporin-2 water channel. J Biol Chem 1997;272:14800
4. Christensen BM, Zelenina M, Aperia A, Nielsen S. Localization and regulation of PKA-phosphorylated AQP2 in response to V (2)-receptor agonist/antagonist treatment. Am J Physiol Renal Physiol 2000;278:F29
42. Moeller HB, Praetorius J, Rutzler MR, Fenton RA. Phosphorylation of aquaporin-2 regulates its endocytosis and protein
protein interactions. Proc Natl Acad Sci USA 2010;107:424
9. McDill BW, Li SZ, Kovach PA, Ding L, Chen F. Congenital progressive hydronephrosis (cph) is caused by an S256L mutation in aquaporin-2 that affects its phosphorylation and apical membrane accumulation. Proc Natl Acad Sci 2006;103:6952
7. Hoffert JD, Pisitkun T, Wang G, Shen RF, Knepper MA. Quantitative phosphoproteomics of vasopressin-sensitive renal cells. Regulation of aquaporin-2 phosphorylation at two sites. Proc Natl Acad Sci 2006;103:7159
64. Hoffert JD, Fenton RA, Moeller HB, Simons B, Tchapyjnikov D, McDill BW, et al. Vasopressin-stimulated increase in phosphorylation at Ser269 potentiates plasma membrane retention of aquaporin-2. J Biol Chem 2008;283:24617
27. Lu HAJ, Sun TX, Matsuzaki T, Yi XH, Eswara J, Bouley R, et al. Heat shock protein 70 interacts with aquaporin-2 and regulates its trafficking. J Biol Chem 2007;282:28721
32.

III. FLUID AND ELECTROLYTE REGULATION AND DYSREGULATION

REFERENCES

[228] Zelenina M, Christensen BM, Palmer J, Nairn AC, Nielsen S, Aperia A. Prostaglandin E(2) interaction with AVP: effects on AQP2 phosphorylation and distribution. Am J Physiol Renal Physiol 2000;278:F388
94. [229] Olesen ET, Rutzler MR, Moeller HB, Praetorius HA, Fenton RA. Vasopressin-independent targeting of aquaporin-2 by selective E-prostanoid receptor agonists alleviates nephrogenic diabetes insipidus. Proc Natl Acad Sci USA 2011;108: 12949
54. [230] Kwon TH, Nielsen J, Knepper MA, Frokiaer J, Nielsen S. Angiotensin II AT1 receptor blockade decreases vasopressininduced water reabsorption and AQP2 levels in NaClrestricted rats. AJP-Renal Physiol 2005;288:F673
84. [231] Lee YJ, Song IK, Jang KJ, Nielsen J, Frokiaer J, Nielsen S, et al. Increased AQP2 targeting in primary cultured IMCD cells in response to angiotensin II through AT1 receptor. Am J Physiol-Renal Physiol 2007;292:F340
50. [232] Procino G, Carmosino M, Marin O, Brunanti AM, Contri A, Pinna LA, et al. Ser-256 phosphorylation dynamics of aquaporin 2 during maturation from the endoplasmic reticulum to the vesicular compartment in renal cells. FASEB J 2003;17(13): 1886
8. [233] Lu H, Sun TX, Bouley R, Blackburn K, McLaughlin M, Brown D. Inhibition of endocytosis causes phosphorylation (S256)-independent plasma membrane accumulation of AQP2. AJP-Renal Physiol 2004;286:F233
43. [234] Phillips ME, Taylor A. Effect of nocodazole on the water permeability response to vasopressin in rabbit collecting tubules perfused in vitro. J Physiol 1989;411:529
44. [235] Phillips ME, Taylor A. Effect of colcemid on the water permeability response to vasopressin in isolated perfused rabbit collecting tubules. J Physiol 1992;456:591
608. [236] Marples D, Schroer TA, Ahrens N, Taylor A, Knepper MA, Nielsen S. Dynein and dynactin colocalize with AQP2 water channels in intracellular vesicles from kidney collecting duct. Am J Physiol 1998;274:F384
94. [237] de Sousa RC, Grosso A. Vanadate blocks cyclic AMP-induced stimulation of sodium and water transport in amphibian epithelia. Nature 1979;279:803
4. [238] Marples D, Barber B, Taylor A. Effect of a dynein inhibitor on vasopressin action in toad urinary bladder. J Physiol 1996;490 (Pt 3):767
74. [239] Shaw S, Marples D. N-ethylmaleimide causes aquaporin-2 trafficking in the renal inner medullary collecting duct by direct activation of protein kinase A. Am J Physiol Renal Physiol 2005;288:F832
9. [240] Dibona DR. Cytoplasmic involvement in ADH-mediated osmosis across toad urinary bladder. Am J Physiol 1983;245: C297
307. [241] Ding GH, Franki N, Condeelis J, Hays RM. Vasopressin depolymerizes F-actin in toad bladder epithelial cells. Am J Physiol 1991;260:C9
16. [242] Kachadorian WA, Ellis SJ, Muller J. Possible roles for microtubules and microfilaments in ADH action on toad urinary bladder. Am J Physiol 1979;236:F14
20. [243] Muller J, Kachadorian WA. Aggregate-carrying membranes during ADH stimulation and washout in toad bladder. Am J Physiol 1984;247:C90
8. [244] Pearl M, Taylor A. Actin filaments and vasopressin-stimulated water flow in toad urinary bladder. Am J Physiol 1983;245: C28
39. [245] Wade JB, Kachadorian WA. Cytochalasin B inhibition of toad bladder apical membrane responses to ADH. Am J Physiol. 1988;255:C526
30.

1435

[246] Chou CL, Christensen BM, Frische S, Vorum H, Desai RA, Hoffert JD, et al. Non-muscle myosin II and myosin light chain kinase are downstream targets for vasopressin signaling in the renal collecting duct. J Biol Chem 2004;279:49026
35. [247] Star RA, Nonoguchi H, Balaban R, Knepper MA. Calcium and cyclic adenosine monophosphate as second messengers for vasopressin in the rat inner medullary collecting duct. J Clin Invest 1988;81:1879
88. [248] Chou CL, Yip KP, Michea L, Kador K, Ferraris JD, Wade JB, et al. Regulation of aquaporin-2 trafficking by vasopressin in the renal collecting duct. Roles of ryanodine-sensitive Ca21 stores and calmodulin. J Biol Chem 2000;275:36839
46. [249] Yip KP. Coupling of vasopressin-induced intracellular Ca21 mobilization and apical exocytosis in perfused rat kidney collecting duct. J Physiol 2002;538:891
9. [250] Lorenz D, Krylov A, Hahm D, Hagen V, Rosenthal W, Pohl P, et al. Cyclic AMP is sufficient for triggering the exocytic recruitment of aquaporin-2 in renal epithelial cells. EMBO Rep 2003;4:88
93. [251] Bajjalieh SM, Scheller RH. The biochemistry of neurotransmitter secretion. J Biol Chem 1995;270:1971
4. [252] Sollner T, Whiteheart SW, Brunner M, Erdjument-Bromage H, Geromanos S, Tempst P, et al. SNAP receptors implicated in vesicle targeting and fusion. Nature 1993;362:318
24. [253] Franki N, Macaluso F, Schubert W, Gunther L, Hays RM. Water channel-carrying vesicles in the rat IMCD contain cellubrevin. Am J Physiol 1995;269:C797
801. [254] Harris Jr HW, Zeidel ML, Jo I, Hammond TG. Characterization of purified endosomes containing the antidiuretic hormonesensitive water channel from rat renal papilla. J Biol Chem 1994;269:11993
2000. [255] Inoue T, Nielsen S, Mandon B, Terris J, Kishore BK, Knepper MA. SNAP-23 in rat kidney: colocalization with aquaporin-2 in collecting duct vesicles. Am J Physiol 1998;275:F752
60. [256] Kishore BK, Wade JB, Schorr K, Inoue T, Mandon B, Knepper MA. Expression of synaptotagmin VIII in rat kidney. Am J Physiol 1998;275:F131
42. [257] Liebenhoff U, Rosenthal W. Identification of Rab3-, Rab5a- and synaptobrevin II-like proteins in a preparation of rat kidney vesicles containing the vasopressin-regulated water channel. FEBS Lett 1995;365:209
13. [258] Mandon B, Chou CL, Nielsen S, Knepper MA. Syntaxin-4 is localized to the apical plasma membrane of rat renal collecting duct cells: possible role in aquaporin-2 trafficking. J Clin Invest 1996;98:906
13. [259] Mandon B, Nielsen S, Kishore BK, Knepper MA. Expression of syntaxins in rat kidney. Am J Physiol 1997;273:F718
30. [260] Nielsen S, Marples D, Birn H, Mohtashami M, Dalby NO, Trimble M, et al. Expression of VAMP-2-like protein in kidney collecting duct intracellular vesicles. Colocalization with aquaporin-2 water channels. J Clin Invest 1995;96: 1834
44. [261] Sudhof TC, De CP, Niemann H, Jahn R. Membrane fusion machinery: insights from synaptic proteins. Cell 1993;75:1
4. [262] Advani RJ, Bae HR, Bock JB, Chao DS, Doung YC, Prekeris R, et al. Seven novel mammalian SNARE proteins localize to distinct membrane compartments. J Biol Chem 1998;273: 10317
24. [263] Calakos N, Bennett MK, Peterson KE, Scheller RH. Protein
protein interactions contributing to the specificity of intracellular vesicular trafficking. Science 1994;263:1146
9. [264] Pevsner J, Hsu SC, Braun JE, Calakos N, Ting AE, Bennett MK, et al. Specificity and regulation of a synaptic vesicle docking complex. Neuron 1994;13:353
61.

III. FLUID AND ELECTROLYTE REGULATION AND DYSREGULATION

1436

41. AQUAPORIN WATER CHANNELS IN MAMMALIAN KIDNEY

[265] Barile M, Pisitkun T, Yu MJ, Chou CL, Verbalis MJ, Shen RF, et al. Large scale protein identification in intracellular aquaporin-2 vesicles from renal inner medullary collecting duct. Mol Cell Proteomics 2005;4:1095
106. [266] Foster LJ, Yeung B, Mohtashami M, Ross K, Trimble WS, Klip A. Binary interactions of the SNARE proteins syntaxin-4, SNAP23, and VAMP-2 and their regulation by phosphorylation. Biochemistry 1998;37:11089
96. [267] Risinger C, Bennett MK. Differential phosphorylation of syntaxin and synaptosome-associated protein of 25 kDa (SNAP25) isoforms. J Neurochem 1999;72:614
24. [268] Shimazaki Y, Nishiki T i, Omori A, Sekiguchi M, Kamata Y, Kozaki S, et al. Phosphorylation of 25-kDa synaptosomeassociated protein. Possible involvement in protein kinase cmediated regulation of neurotransmitter release. J Biol Chem 1996;271:14548
53. [269] Jones RV, De Wardener HE. Urine concentration after fluid deprivation or pitressin tannate in oil. Br Med J 1956;1 (4961):271
4. [270] Han JS, Maeda Y, Ecelbarger C, Knepper MA. Vasopressinindependent regulation of collecting duct water permeability. Am J Physiol 1994;266:F139
46. [271] Wade JB, Nielsen S, Coleman RA, Knepper MA. Long-term regulation of collecting duct water permeability: freezefracture analysis of isolated perfused tubules. Am J Physiol 1994;266:F723
30. [272] Flamion B, Spring KR, Abramow M. Adaptation of inner medullary collecting duct to dehydration involves a paracellular pathway. Am J Physiol 1995;268:F53
63. [273] Chou CL, DiGiovanni SR, Mejia R, Nielsen S, Knepper MA. Oxytocin as an antidiuretic hormone. I. Concentration dependence of action. Am J Physiol 1995;269:F70
7. [274] Fujita N, Ishikawa SE, Sasaki S, Fujisawa G, Fushimi K, Marumo F, et al. Role of water channel AQP-CD in water retention in SIADH and cirrhotic rats. Am J Physiol 1995;269: F926
31. [275] Ecelbarger CA, Nielsen S, Olson BR, Murase T, Baker EA, Knepper MA, et al. Role of renal aquaporins in escape from vasopressin-induced antidiuresis in rat. J Clin Invest 1997;99:1852
63. [276] Ecelbarger CA, Chou CL, Lee AJ, DiGiovanni SR, Verbalis JG, Knepper MA. Escape from vasopressin-induced antidiuresis: role of vasopressin resistance of the collecting duct. Am J Physiol 1998;274:F1161
6. [277] Fernandez-Llama P, Turner R, Dibona G, Knepper MA. Renal expression of aquaporins in liver cirrhosis induced by chronic common bile duct ligation in rats. J Am Soc Nephrol 1999;10:1950
7. [278] Sands JM, Naruse M, Jacobs JD, Wilcox JN, Klein JD. Changes in aquaporin-2 protein contribute to the urine concentrating defect in rats fed a low-protein diet. J Clin Invest 1996;97:2807
14. [279] Ma T, Hasegawa H, Skach WR, Frigeri A, Verkman AS. Expression, functional analysis, and in situ hybridization of a cloned rat kidney collecting duct water channel. Am J Physiol 1994;266:C189
97. [280] Uchida S, Sasaki S, Fushimi K, Marumo F. Isolation of human aquaporin-CD gene. J Biol Chem 1994;269:23451
5. [281] Hozawa S, Holtzman EJ, Ausiello DA. cAMP motifs regulating transcription in the aquaporin 2 gene. Am J Physiol 1996;270: C1695
702. [282] Matsumura Y, Uchida S, Rai T, Sasaki S, Marumo F. Transcriptional regulation of aquaporin-2 water channel gene by cAMP. J Am Soc Nephrol 1997;8:861
7.

[283] Yasui M, Zelenin SM, Celsi G, Aperia A. Adenylate cyclasecoupled vasopressin receptor activates AQP2 promoter via a dual effect on CRE and AP1 elements. Am J Physiol 1997;272: F443
50. [284] Inase N, Fushimi K, Ishibashi K, Uchida S, Ichioka M, Sasaki S, et al. Isolation of human aquaporin 3 gene. J Biol Chem 1995;270:17913
6. [285] Kamsteeg EJ, Hendriks G, Boone M, Konings IBM, Oorschot V, van der Sluijs P, et al. Short-chain ubiquitination mediates the regulated endocytosis of the aquaporin-2 water channel. Proc Natl Acad Sci 2006;103:18344
9. [286] Agre P, King LS, Yasui M, Guggino WB, Ottersen OP, Fujiyoshi Y, et al. Aquaporin water channels
from atomic structure to clinical medicine. J Physiol Online 2002;542:3
16. [287] Nielsen S, Knepper MA, Kwon T-H, Frokiaer J. Regulation of water balance. Urine concentration and dilution. In: Schrier RW, editor. Diseases of the Kidney and Urinary Tract. Philadelphia, Lippincott Willialms & Wilkins; 2004. p. 109
34. [288] Robben JH, Knoers NV, Deen PM. Cell biological aspects of the vasopressin type-2 receptor and aquaporin 2 water channel in nephrogenic diabetes insipidus. AJP-Renal Physiol 2006;291: F257
70. [289] Babey M, Kopp P, Robertson GL. Familial forms of diabetes insipidus: clinical and molecular characteristics. Nat Rev Endocrinol 2011;7:701
14. [290] Schmale H, Ivell R, Breindl M, Darmer D, Richter D. The mutant vasopressin gene from diabetes insipidus (Brattleboro) rats is transcribed but the message is not efficiently translated. EMBO J 1984;3(13):3289
93. [291] Bichet DG. Vasopressin receptors in health and disease. Kidney Int 1996;49:1706
11. [292] Jensen AM, Bae EH, Fenton RA, Noregaard R, Nielsen S, Kim SW, et al. Angiotensin II regulates V2 receptor and pAQP2 during ureteral obstruction. Am J Physiol-Renal Physiol 2009;296:F127
34. [293] Deen PM, van Aubel RA, Van Lieburg AF, van Os CH. Urinary content of aquaporin 1 and 2 in nephrogenic diabetes insipidus. J Am Soc Nephrol 1996;7(6):836
41. [294] Wen H, Frokiaer J, Kwon TH, Nielsen S. Urinary excretion of aquaporin-2 in rat is mediated by a vasopressin-dependent apical pathway. J Am Soc Nephrol 1999;10:1416
29. [295] Moon SS, Kim HJ, Choi YK, Seo HA, Jeon JH, Lee JE, et al. Novel mutation of aquaporin-2 gene in a patient with congenital nephrogenic diabetes insipidus. Endocrine J 2009;56: 905
10. [296] Mulders SM, Knoers NV, Van Lieburg AF, Monnens LA, Leumann E, Wuhl E, et al. New mutations in the AQP2 gene in nephrogenic diabetes insipidus resulting in functional but misrouted water channels. J Am Soc Nephrol 1997;8:242
8. [297] Oksche A, Moller A, Dickson J, Rosendahl W, Rascher W, Bichet DG, et al. Two novel mutations in the aquaporin-2 and the vasopressin V2 receptor genes in patients with congenital nephrogenic diabetes insipidus. Hum Genet 1996;98:587
9. [298] Van Lieburg AF, Verdijk MA, Knoers VV, van Essen AJ, Proesmans W, Mallmann R, et al. Patients with autosomal nephrogenic diabetes insipidus homozygous for mutations in the aquaporin 2 water-channel gene. Am J Hum Genet 1994;55: 648
52. [299] Deen PM, Croes H, van Aubel RA, Ginsel LA, van Os CH. Water channels encoded by mutant aquaporin-2 genes in nephrogenic diabetes insipidus are impaired in their cellular routing. J Clin Invest 1995;95:2291
6. [300] Morello JP, Salahpour A, Petaja-Repo UE, Laperriere A, Lonergan M, Arthus MF, et al. Association of calnexin with

III. FLUID AND ELECTROLYTE REGULATION AND DYSREGULATION

1437

REFERENCES

[301]

[302]

[303]

[304]

[305]

[306] [307]

[308]

[309]

[310]

[311]

[312]

[313]

[314]

[315]

[316]

wild type and mutant AVPR2 that cause nephrogenic diabetes insipidus. Biochemistry 2001;40:6766
75. Knoers NV, van Os CH. Molecular and cellular defects in nephrogenic diabetes insipidus. Curr Opin Nephrol Hypertens 1996;5:353
8. Mulders SM, Bichet DG, Rijss JP, Kamsteeg EJ, Arthus MF, Lonergan M, et al. An aquaporin-2 water channel mutant which causes autosomal dominant nephrogenic diabetes insipidus is retained in the Golgi complex. J Clin Invest 1998;102:57
66. Faerch M, Christensen JH, Corydon TJ, Kamperis K, de ZF, Gregersen N, et al. Partial nephrogenic diabetes insipidus caused by a novel mutation in the AVPR2 gene. Clin Endocrinol(Oxf) 2008;68:395
403. Faerch M, Christensen JH, Rittig S, Johansson JO, Gregersen N, de ZF, et al. Diverse vasopressin V2 receptor functionality underlying partial congenital nephrogenic diabetes insipidus. AJP-Renal Physiol 2009;297:F1518
25. Faerch M, Corydon TJ, Rittig S, Christensen JH, Hertz JM, Jendle J. Skewed X-chromosome inactivation causing diagnostic misinterpretation in congenital nephrogenic diabetes insipidus. Scand J Urol Nephrol 2010;44:324
30. Timmer RT, Sands JM. Lithium intoxication. J Am Soc Nephrol 1999;10:666
74. Kwon T-H, Laursen UH, Marples D, Maunsbach AB, Knepper MA, Frokiaer J, et al. Altered expression of renal AQPs and Na (1) transporters in rats with lithium-induced NDI. Am J Physiol Renal Physiol 2000;279:F552
64. Marples D, Christensen S, Christensen EI, Ottosen PD, Nielsen S. Lithium-induced downregulation of aquaporin-2 water channel expression in rat kidney medulla. J Clin Invest 1995;95:1838
45. Christensen S, Kusano E, Yusufi AN, Murayama N, Dousa TP. Pathogenesis of nephrogenic diabetes insipidus due to chronic administration of lithium in rats. J Clin Invest 1985;75:1869
79. Kim YH, Kwon TH, Christensen BM, Nielsen J, Wall SM, Madsen KM, et al. Altered expression of renal acid
base transporters in rats with lithium-induced NDI. AJP-Renal Physiol 2003;285:F1244
57. Wilting I, Baumgarten R, Movig KL, Laarhoven J, Apperloo AJ, Nolen WA, et al. Urine osmolality, cyclic AMP and aquaporin-2 in urine of patients under lithium treatment in response to water loading followed by vasopressin administration. Eur J Pharmacol 2007;566:50
7. Frokiaer J, Marples D, Valtin H, Morris JF, Knepper MA, Nielsen S. Low aquaporin-2 levels in polyuric DI1/1 severe mice with constitutively high cAMP-phosphodiesterase activity. AJP-Renal Physiol 1999;276:F179
90. Nielsen J, Kwon TH, Praetorius J, Kim YH, Frokiaer J, Knepper MA, et al. Segment-specific ENaC downregulation in kidney of rats with lithium-induced NDI. AJP-Renal Physiol 2003;285: F1198
209. Christensen BM, Zuber AM, Loffing J, Stehle JC, Deen PM, Rossier BC, et al. ENaC-mediated lithium absorption promotes nephrogenic diabetes insipidus. J Am Soc Nephrol 2011;22: 253
61. Nielsen J, Hoffert JD, Knepper MA, Agre P, Nielsen S, Fenton RA. Proteomic analysis of lithium-induced nephrogenic diabetes insipidus. Mechanisms for aquaporin 2 downregulation and cellular proliferation. Proc Natl Acad Sci 2008;105:3634
9. Nielsen J, Kwon TH, Christensen BM, Frokiaer J, Nielsen S. Dysregulation of renal aquaporins and epithelial sodium

[317]

[318]

[319]

[320]

[321]

[322]

[323]

[324]

[325]

[326]

[327]

[328]

[329]

[330]

[331]

[332]

channel in lithium-induced nephrogenic diabetes insipidus. Seminars Nephrol 2008;28(3):227
44. Kortenoeven ML, Schweer H, Cox R, Wetzels JF, Deen PM. Lithium reduces aquaporin-2 transcription independent of prostaglandins. Am J Physiol-Cell Physiol 2012;302: C131
40. Marples D, Frokiaer J, Dorup J, Knepper MA, Nielsen S. Hypokalemia-induced downregulation of aquaporin-2 water channel expression in rat kidney medulla and cortex. J Clin Invest 1996;97:1960
8. Earm JH, Christensen BM, Frokiaer J, Marples D, Han JS, Knepper MA, et al. Decreased aquaporin-2 expression and apical plasma membrane delivery in kidney collecting ducts of polyuric hypercalcemic rats. J Am Soc Nephrol 1998;9: 2181
93. Sands JM, Flores FX, Kato A, Baum MA, Brown EM, Ward DT, et al. Vasopressin-elicited water and urea permeabilities are altered in IMCD in hypercalcemic rats. AJP-Renal Physiol 1998;274:F978
85. Elkjar ML, Kwon TH, Wang W, Nielsen J, Knepper MA, Frokiar J, et al. Altered expression of renal NHE3, TSC, BSC-1, and ENaC subunits in potassium-depleted rats. AJP-Renal Physiol 2002;283:F1376
88. Wang W, Li C, Kwon TH, Miller RT, Knepper MA, Frokiaer J, et al. Reduced expression of renal Na1 transporters in rats with PTH-induced hypercalcemia. AJP-Renal Physiol 2004;286: F534
45. Zeidel M, Pirtskhalaishvili G. Urinary tract obstruction. In: Brenner BM, editor. The kidney. Philadelphia: Saunders; 2004. p. 1867
94. Frokiaer J, Marples D, Knepper MA, Nielsen S. Bilateral ureteral obstruction downregulates expression of vasopressinsensitive AQP-2 water channel in rat kidney. Am J Physiol 1996;270:F657
68. Kim SW, Cho SH, Oh BS, Yeum CH, Choi KC, Ahn KY, et al. Diminished renal expression of aquaporin water channels in rats with experimental bilateral ureteral obstruction. J Am Soc Nephrol 2001;12:2019
28. Li C, Wang W, Kwon TH, Isikay L, Wen JG, Marples D, et al. Downregulation of AQP1, -2, and -3 after ureteral obstruction is associated with a long-term urine-concentrating defect. AJPRenal Physiol 2001;281:F163
71. Stodkilde L, Norregaard R, Fenton RA, Wang G, Knepper MA, Frokiar J. Bilateral ureteral obstruction induces early downregulation and redistribution of AQP2 and phosphorylated AQP2. Am J Physiol-Renal Physiol 2011;301:F226
35. Li C, Wang W, Knepper MA, Nielsen S, Frokiar J. Downregulation of renal aquaporins in response to unilateral ureteral obstruction. AJP-Renal Physiol 2003;284:F1066
79. Cheng X, Zhang H, Lee HL, Park JM. Cyclooxygenase-2 inhibitor preserves medullary aquaporin-2 expression and prevents polyuria after ureteral obstruction. J Urol 2004. Norregaard R, Jensen BL, Li C, Wang W, Knepper MA, Nielsen S, et al. COX-2 inhibition prevents downregulation of key renal water and sodium transport proteins in response to bilateral ureteral obstruction. AJP-Renal Physiology 2005;289: F322
33. Norregaard R, Jensen BL, Topcu SO, Diget M, Schweer H, Knepper MA, et al. COX-2 activity transiently contributes to increased water and NaCl excretion in the polyuric phase after release of ureteral obstruction. Am J Physiol-Renal Physiol 2007;292:F1322
33. Zhang Y, Kohan DE, Nelson RD, Carlson NG, Kishore BK. Potential involvement of P2Y2 receptor in diuresis of

III. FLUID AND ELECTROLYTE REGULATION AND DYSREGULATION

1438

[333]

[334]

[335]

[336]

[337]

[338]

[339]

[340]

[341]

[342]

[343]

[344]

[345]

[346]

[347]

[348]

[349]

41. AQUAPORIN WATER CHANNELS IN MAMMALIAN KIDNEY

postobstructive uropathy in rats. AJP-Renal Physiol 2010;298: F634
42. Jensen AM, Li C, Praetorius HA, Norregaard R, Frische S, Knepper MA, et al. Angiotensin II mediates downregulation of aquaporin water channels and key renal sodium transporters in response to urinary tract obstruction. AJP-Renal Physiol 2006;291(5):F1021
32. Frokiaer J, Christensen BM, Marples D, Djurhuus JC, Jensen UB, Knepper MA, et al. Downregulation of aquaporin-2 parallels changes in renal water excretion in unilateral ureteral obstruction. Am J Physiol 1997;273:F213
23. Hanley MJ. Isolated nephron segments in a rabbit model of ischemic acute renal failure. Am J Physiol 1980;239: F17
23. Tanner GA, Sloan KL, Sophasan S. Effects of renal artery occlusion on kidney function in the rat. Kidney Int 1973;4: 377
89. Venkatachalam MA, Bernard DB, Donohoe JF, Levinsky NG. Ischemic damage and repair in the rat proximal tubule: differences among the S1, S2, and S3 segments. Kidney Int 1978;14: 31
49. Anderson RJ, Gordon JA, Kim J, Peterson LM, Gross PA. Renal concentration defect following nonoliguric acute renal failure in the rat. Kidney Int 1982;21:583
91. Fernandez-Llama P, Andrews P, Turner R, Saggi S, Di Mari J, Kwon T-H, et al. Role of collecting duct aquaporins in polyuria of post-ischemic acute renal failure in rats. J Am Soc Nephrol 1999;10:1658
68. Kwon T-H, Frokiaer J, Fernandez-Llama P, Knepper MA, Nielsen S. Reduced abundance of aquaporins in rats with bilateral ischemia-induced acute renal failure: prevention by alphaMSH. Am J Physiol 1999;277:F413
27. Gong H, Wang W, Kwon TH, Jonassen T, Frokiaer J, Nielsen S. Reduced renal expression of AQP2, p-AQP2 and AQP3 in haemorrhagic shock-induced acute renal failure. Nephrol Dialysis Transplant 2003;18:2551
9. Sharples EJ, Patel N, Brown P, Stewart K, Mota-Philipe H, Sheaff M, et al. Erythropoietin protects the kidney against the injury and dysfunction caused by ischemia-reperfusion. J Am Soc Nephrol 2004;15:2115
24. Hocherl K, Schmidt C, Kurt B, Bucher M. Inhibition of NFkappaB ameliorates sepsis-induced downregulation of aquaporin-2/V2 receptor expression and acute renal failure in vivo. AJP-Renal Physiol 2010;298:F196
204. Tannen RL, Regal EM, Dunn MJ, Schrier RW. Vasopressinresistant hyposthenuria in advanced chronic renal disease. N Engl J Med 1969;280:1135
41. Teitelbaum I, McGuinness S. Vasopressin resistance in chronic renal failure. Evidence for the role of decreased V2 receptor mRNA. J Clin Invest 1995;96:378
85. Kwon T-H, Frokiaer J, Knepper MA, Nielsen S. Reduced AQP1, -2, and -3 levels in kidneys of rats with CRF induced by surgical reduction in renal mass. Am J Physiol 1998;275: F724
41. Nielsen S, Terris J, Andersen D, Ecelbarger C, Frokiaer J, Jonassen T, et al. Congestive heart failure in rats is associated with increased expression and targeting of aquaporin-2 water channel in collecting duct. Proc Natl Acad Sci USA 1997;94: 5450
5. Xu DL, Martin PY, Ohara M, St.John J, Pattison T, Meng X, et al. Upregulation of aquaporin-2 water channel expression in chronic heart failure rat. J Clin Invest 1997;99: 1500
5. Lutken SC, Kim SW, Jonassen T, Marples D, Knepper MA, Kwon TH, et al. Changes of renal AQP2, ENaC, and NHE3 in

[350]

[351]

[352]

[353]

[354]

[355]

[356]

[357]

[358]

[359]

[360]

[361]

[362]

[363]

[364]

experimentally induced heart failure: response to angiotensin II AT1 receptor blockade. Am J Physiol-Renal Physiol 2009;297: F1678
88. Martin PY, Abraham WT, Lieming X, Olson BR, Oren RM, Ohara M, et al. Selective V2-receptor vasopressin antagonism decreases urinary aquaporin-2 excretion in patients with chronic heart failure. J Am Soc Nephrol 1999;10: 2165
70. Starklint J, Bech JN, Nyvad O, Jensen P, Pedersen EB. Increased urinary aquaporin 2 excretion in response to furosemide in patients with chronic heart failure. Scandinavian J Clinical Lab Invest 2006;66:55
66. Portincasa P, Palasciano G, Svelto M, Calamita G. Aquaporins in the hepatobiliary tract. Which, where and what they do in health and disease? European J Clin Invest 2008;38:1
10. Jonassen TE, Nielsen S, Christensen S, Petersen JS. Decreased vasopressin-mediated renal water reabsorption in rats with compensated liver cirrhosis. Am J Physiol 1998;275: F216
25. Jonassen TE, Promeneur D, Christensen S, Petersen JS, Nielsen S. Decreased vasopressin-mediated renal water reabsorption in rats with chronic aldosterone-receptor blockade. Am J Physiol Renal Physiol 2000;278:F246
56. Asahina Y, Izumi N, Enomoto N, Sasaki S, Fushimi K, Marumo F, et al. Increased gene expression of water channel in cirrhotic rat kidneys. Hepatology 1995;21: 169
73. Fernandez-Llama P, Jimenez W, Bosch-Marce M, Arroyo V, Nielsen S, Knepper MA. Dysregulation of renal aquaporins and Na-Cl co-transporter in CCl4-induced cirrhosis. Kidney Int 2000;58:216
28. Esteva-Font C, Baccaro ME, Ferna´ndez-Llama P, Sans L, Guevara M, Ars E, et al. Aquaporin-1 and aquaporin-2 urinary excretion in cirrhosis: relationship with ascites and hepatorenal syndrome. Hepatology 2006;44:1555
63. Gines P, Berl T, Bernardi M, Bichet DG, Hamon G, Jimenez W, et al. Hyponatremia in cirrhosis: from pathogenesis to treatment. Hepatology 1998;28:851
64. Kim SW, Schou UK, Peters CD, de Seigneux S, Kwon TH, Knepper MA, et al. Increased apical targeting of renal epithelial sodium channel subunits and decreased expression of type 2 11beta-hydroxysteroid dehydrogenase in rats with CCl4induced decompensated liver cirrhosis. J Am Soc Nephrol 2005;16:3196
210. Wood LJ, Massie D, McLean AJ, Dudley FJ. Renal sodium retention in cirrhosis: tubular site and relation to hepatic dysfunction. Hepatology 1988;8:831
6. Levy M, Wexler MJ. Hepatic denervation alters first-phase urinary sodium excretion in dogs with cirrhosis. Am J Physiol 1987;253:F664
71. Huebert RC, Jagavelu K, Hendrickson HI, Vasdev MM, Arab JP, Splinter PL, et al. Aquaporin-1 promotes angiogenesis, fibrosis, and portal hypertension through mechanisms dependent on osmotically sensitive microRNAs. Am J Pathol 2011;179:1851
60. Apostol E, Ecelbarger CA, Terris J, Bradford AD, Andrews P, Knepper MA. Reduced renal medullary water channel expression in puromycin aminonucleoside: induced nephrotic syndrome. J Am Soc Nephrol 1997;8:15
24. Fernandez-Llama P, Andrews P, Ecelbarger CA, Nielsen S, Knepper M. Concentrating defect in experimental nephrotic syndrome: altered expression of aquaporins and thick ascending limb Na1 transporters. Kidney Int 1998;54: 170
9.

III. FLUID AND ELECTROLYTE REGULATION AND DYSREGULATION

1439

REFERENCES

[365] Fernandez-Llama P, Andrews P, Nielsen S, Ecelbarger CA, Knepper MA. Impaired aquaporin and urea transporter expression in rats with adriamycin-induced nephrotic syndrome. Kidney Int 1998;53:1244
53. [366] Flear CT, Gill GV, Burn J. Hyponatraemia: mechanisms and management. Lancet 1981;2:26
31. [367] Bartter FC, Schwartz WB. The syndrome of inappropriate secretion of antidiuretic hormone. Am J Med 1967;42: 790
806. [368] Ishikawa S, Saito T, Saito T, Kasono K, Funayama H. Pathophysiological role of aquaporin-2 in impaired water excretion. In: Neumann ID, editor. Progress in brain research

[369] [370] [371] [372]

advances in vasopressin and oxytocin
from genes to behaviour to disease. Elsevier; 2008. p. 581
8. Verbalis JG. Whole-body volume regulation and escape from antidiuresis. Am J Med 2006;119:S21
9. Heymann JB, Agre P, Engel A. Progress on the structure and function of aquaporin 1. J Struct Biol 1998;121:191
206. de Groot BL, Engel A, Grubmuller H. A refined structure of human aquaporin-1. FEBS Lett 2001;504:206
11. de Groot BL, Engel A, Grubmuller H. The structure of the aquaporin-1 water channel. A comparison between cryoelectron microscopy and X-ray crystallography. J Mol Biol 2003;325:485
93.

III. FLUID AND ELECTROLYTE REGULATION AND DYSREGULATION