Aquaporin Water Channels in Mammalian Kidney

Hypernatremia can occur with normal, increased or decreased total body sodium content. In healthy individuals and in normal conditions, the plasma concentration of sodium ranges between 136 and 143 mEq/l of plasma, despite large individual variations in the intake of salt and water. The concentration is maintained at constant levels because of the homeostatic mechanism in the body. Claude Bernard was the first to appreciate that higher animals: “have really two environments: a milieu exterieur in which the organism is situated, and a milieu interieur in which the tissue elements live.” The latter is the extracellular fluid (ECF) that bathes the cells of the body. ^1,2,3 Maintenance of this consistency of plasma sodium and solute activity is the function of the thirst–neurohypophyseal–renal axis. ^4,5 Thirst and urinary concentration are the main defenses against hyperosmolality, and hence hypernatremia. Hypernatremia is a relatively common problem, with prevalence in hospitalized patients of 0.5 to 2%. It is defined as plasma Na ⁺ concentration ([Na ⁺ ]) greater than 145 mEq/l. It can be produced by the administration of hypertonic sodium solutions or in almost all cases, by the loss of free water. Since [Na ⁺ ] is an effective osmole, the increase in the plasma osmolality (P _osm ) induced by hypernatremia creates an osmotic gradient that results in water movement out of the cells into the ECF. It is this cellular dehydration, particularly in the brain, that is primarily responsible for the neurologic symptoms associated with hypernatremia. A similar syndrome can be produced when the plasma osmolality is elevated by hyperglycemia. However, when hyperosmolality is due to the accumulation of cell-permeable solute, such as urea or ethanol, there is no water shift in the steady-state because osmotic equilibrium is reached by solute entering the cell.

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 ). 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 energy of this process is equivalent to the diffusion of water in solution, E _a <5 kcal/mol. In addition, reversible inhibition by HgCl ₂ and a subset of organomercurials suggested that the water transporter is a membrane protein (see review ). 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 well-characterized in the mammalian kidney (see review ). 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 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. 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 ). 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, HgCl ₂ 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. 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, a 28 kDa polypeptide was partially co-purified. 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 identified which permitted cDNA cloning. Also of note, radiation inactivation studies of water permeability by renal vesicles yielded a target size of 30 kDa. Because the 28 kDa polypeptide was found to be abundant in red cells, renal proximal tubules, and descending thin limbs, 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. Thus, CHIP28 was designated aquaporin-1 (symbol AQP1). The Human Genome Nomenclature Committee has embraced this nomenclature for all related proteins, 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. Oocytes injected with cRNA encoding AQP1 exhibit remarkably high osmotic water permeability (P _f ~200×10 ⁻¹⁷ cm/s), causing the cells to swell rapidly and eventually rupture in hyposmotic buffer. 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. Osmotically-induced swelling of oocytes expressing AQP1 occurs with a low activation energy, and is reversibly inhibited by HgCl ₂ . 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.

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. These studies permitted determination of the unit water permeability, which had an astonishingly high value (P _f ~3×10 ⁹ water molecules subunit ⁻¹¹¹ sec ⁻¹¹¹ ). Moreover, the water permeability is reversibly inhibited by HgCl ₂ , and exhibits low activation energy. Several of these studies have been confirmed by using red cell membranes partially depleted of other proteins, and attempts to demonstrate permeation by other small solutes or even protons showed that AQP1 is water selective. 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 long-sought-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, a feature in agreement with initial studies of red cell AQP1. 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), suggesting ancestral gene duplication. When evaluated by hydropathy analysis, six bilayer-spanning domains, five connecting loops (A–E), and intracellular N-and C-termini are predicted. Attenuated total reflection-FTIR (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.

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. 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 confirmed ( 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. 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 Cys ¹⁸⁹ in loop E is the site of mercurial inhibition. Site-directed mutagenesis experiments in oocytes revealed that substitution of Cys ¹⁸⁹ with a serine residue eliminates HgCl ₂ sensitivity and increases osmotic water permeability, while substitution with larger amino acids residues prevents facilitated water transport. These results suggest that water transport and selectivity in AQP1 is somehow dependent on the steric properties of Cys ¹⁸⁹ . In accordance with the internal repeat theory, Ala ⁷³ located in loop B, the equivalent to Cys ¹⁸⁹ in loop E, was investigated. By creating double mutants expressing Cys ¹⁸⁹ as a serine and Ala ⁷³ as a cysteine, the HgCl ₂ sensitivity and osmotic water permeability were restored. 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, in which these domains overlap midway between the leaflets of the bilayer, creating a constitutively open, narrow aqueous pathway ( Figure 41.2 ).

Structural Analyses and Molecular Dynamics of Aquaporins

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. 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. Image projections revealed the presence of multiple bilayer-spanning domains, and atomic force microscopy further defined the orientation and extramembranous dimensions of AQP1. 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 resolutions, down to 3.8 Å. X-ray crystallographic 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 2.2 Å. In contrast to the previous studies, the high 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 Å down to 3.5 Å. The residues surrounding the gap are hydrophobic, suggesting an interaction with a hydrophobic molecule. 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.

The dimensions of each monomer are 40 Å across and 60 Å 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 membrane ( Figure 41.3 ). This organization is strikingly similar to the proposed hourglass arrangement of the B and E loops. 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 Å at its widest. Following the extracellular vestibule towards the lipid bilayer, a decrease in diameter occurs and the pore becomes exceedingly small. The constriction site (also referred to as the aromatic/arginine constriction) is located 20 Å from the beginning of the extracellular vestibule, and is composed of several highly-conserved residues in conventional aquaporins. The constriction site is approximately 2.8 Å wide, about the diameter of a water molecule. After the constriction site the pore continues for 20 Å, a region termed the “selectivity filter”. The selectivity filter is slightly wider and part of the region is formed by the helical loops B and E. This locates the asparagine residues in the NPA motifs within the selectivity filter. Additionally, Cys ¹⁸⁹ in loop E protrudes into the pore, confirming earlier studies, suggesting that HgCl ₂ sensitivity was due to steric hindrance of water movement through the pore. Following the pore towards the intracellular vestibule, the diameter increases again, reaching 15 Å ( Figure 41.3 ).

Using molecular dynamic simulations, de Groot and Grubmüller obtained time-resolved, atomic resolution models of water transport through AQP1. From the extracellular vestibule, water molecules enter the constriction site, composed of side chains from the aromatic Phe ⁵⁶ and His ¹⁸⁰ , and the charged Arg ¹⁹⁵ . 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 Arg ¹⁹⁵ suggests that the constriction site is the main site for exclusion of protons (hydronium ions) and other ions. 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 Arg ¹⁹⁵ with a valine residue appears to facilitate proton transport. 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, 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. Evaluation of differences between AQP1 and GlpF using molecular dynamic simulations revealed a larger selectivity filter in GlpF. Moreover, the preference for glycerol could be explained by what de Groot and Grubmüller describe as a glycerol-mediated “induced fit” gating motion (i.e., glycerol transport serves as the prime mechanism for water exclusion).

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 4.5 Å in the membrane plane. As with AQP1, AQP2 is found in a tetrameric assembly in the lipid bilayer. The AQP2 monomer shows structural features similar to those earlier reported for AQP1, while structural variation is found around the tetramer’s axis.

The structure of AQP0 has also been determined down to a resolution of 2.2 Å. The water conductance of AQP0 is much lower than that reported for AQP1, possibly due to highly-conserved tyrosine residues in AQP0 imposing further constriction on the channel. The constriction site is smaller than that in AQP1, and the selectivity filter is also narrower. 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. 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. Copper and nickel also significantly block aquaporins. Over the past five years the overall structural concepts of aquaporins have been confirmed.

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. This was confirmed with polyclonal rabbit antiserum, and was defined in rat and human kidney with affinity-purified immunoglobulin specific for the N- and C-terminal domains of AQP1. 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. Enzyme-Linked Immunosorbent Assay (ELISA) measurements of microdissected tubules revealed that proximal tubules contain approximately 20 million copies of AQP1 per cell. Additionally, AQP1 is found in the plasma membrane of glomerular mesangial cells in humans, although not in rat. Other immunohistochemical and immunogold electron microscopic studies have demonstrated AQP1 in multiple capillary endothelia throughout the body, including the renal vasa recta. AQP1 is also abundant in peribronchiolar capillary endothelium, where expression is induced by glucocorticoids, apparently acting through the classic glucocorticoid response elements in the AQP1 gene. In addition, AQP1 has been defined in multiple water-permeable epithelia including choroid plexus, peritoneal mesothelial cells, fetal membranes, at multiple locations in eye, including ciliary epithelium, lens epithelium, and corneal endothelium, and in hepatobiliary epithelium, pancreatic interlobular ducts, heart and skeletal muscle, gall bladder, salivary glands, inner ear, and several other organs including the nervous system (see review ). AQP1 has also been localized in tumor cells and their vasculature. 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.

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 AQP1 ( 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. 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. Moreover, Co-null individuals display reduced pulmonary vascular permeability.

The development of AQP1 gene knockout mice has provided further insights into the role of AQP1 in renal water homeostasis. AQP1-null (AQP1 ^−/− ) mice appeared moderately polyuric under basal conditions. However, AQP1 ^−/− animals exhibited an extreme degree of polyuria and polydipsia when undergoing water deprivation, including rapid hyperosmolar extracellular fluid volume depletion. Additionally, AQP1 ^−/− mice failed to respond appropriately to vasopressin, suggesting that renal water conservation and urinary concentration is highly dependent on AQP1 protein. Detailed classic in vivo and in vitro physiological evaluation of proximal tubules from the AQP1 ^−/− mice established that transepithelial water permeability was reduced by 80%, and led to an approximate 50% decrease in proximal tubule fluid reabsorption. 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 AQP1 ^−/− mice. Despite these observations, distal fluid delivery in AQP1 ^−/− remained unchanged, due to a compensatory reduction in single nephron glomerular filtration. When blunting the TGF response, and thereby the compensatory reduction in glomerular filtration observed in AQP1 ^−/− mice, ambient urine osmolalities and urinary flow rates appeared no different from normal AQP1 ^−/− mice, probably due to distal tubular compensatory mechanisms. The impaired proximal fluid reabsorption observed in AQP1 ^−/− 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. 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. In the thin descending limb of AQP1 ^−/− mice, the abundance and size of these intramembrane particles was markedly reduced. 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 AQP1 ^−/− mice, leading to a predicted reduction in medullary interstitial osmolality and likely an impairment of countercurrent multiplication. Additional studies using adenoviral gene delivery restored AQP1 protein expression in the proximal tubule epithelia and renal microvessels of AQP1 ^−/− knockout mice, albeit not in the descending thin limbs. The adenovirus-treated 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. In conclusion, the severe concentrating defect seen in the AQP1 ^−/− mice primarily results from impaired water 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, and the biologic significance remains unclear. Forskolin was reported to induce a cation current in AQP1-expressing oocytes, but multiple other scientific groups have failed to reproduce this effect. 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. Likewise, secretin-induced membrane trafficking has been noted in isolated cholangiocytes ; however, this awaits confirmation by immunoelectron microscopy. Permeation of AQP1 by CO ₂ has also been evaluated. Rates of pH change are about 40% higher in oocytes expressing AQP1 than in control oocytes. Although the background permeation of lipid bilayers by CO ₂ , as well as oxygen, ammonia, nitric oxide, and other gases, may be high, the potential physiological relevance of AQP1 permeation by gases warrants more study (see review ). Additionally, AQP1 appears to play a role in cell migration. AQP1 ^−/− 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 AQP1 ^−/− mice showed marked impairment in cell migration. In primary cultures of proximal tubule cells from AQP1 ^−/− mice, in vitro cell migration was impaired compared to AQP1 ^+/+ mice. Furthermore, in an ischemia–reperfusion model of acute renal tubular injury, AQP1 ^−/− mice showed more severe pathological changes, including more prominent tubule degeneration. 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. Thirteen mammalian aquaporins are now known, and they form at least two subgroups: water-selective 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, 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, 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. AQP4, a HgCl ₂ -insensitive 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. AQP6 was identified at the cDNA level and found to be localized in intracellular vesicles in the collecting duct intercalated cells in the kidney. AQP7 is permeated by water and glycerol. First cloned from testis, AQP7 is present in segment 3 proximal tubule brush border membranes, where it facilitates glycerol and water transport. AQP8 is a HgCl ₂ -sensitive water channel found in intracellular domains of proximal tubule and collecting duct cells ; however, its function remains unclear. AQP11 is found in the cytoplasm of renal proximal tubule cells. The exact function is not established, although deletion of the AQP11 gene produces a severe phenotype with renal vacuolization and cyst formation.