Regulation and dysregulation of aquaporins in water balance disorders.

The discovery of aquaporin-1 (AQP1) explained the long-standing biophysical question of how water specifically crosses biological membranes. These studies led to the identification of a whole new family of membrane proteins, the aquaporin water channels. At present, at least eight aquaporins are expressed at distinct sites in the kidney and four members of this family (AQP1-4) have been demonstrated to play pivotal roles in the physiology and pathophysiology for renal regulation of body water balance. In the present review, a number of inherited and acquired conditions characterized by urinary concentration defects as well as common diseases associated with severe water retention are discussed with relation to the role of aquaporins in regulation and dysregulation of renal water transport.

Expression of aquaporin isoforms during human and mouse tooth development.

Previously, we described the development of hyaluronan (HA) deposition in human tooth germ tissues that are consistent with water transport in different stages of tooth development. The aquaporins (AQP) constitute a family of membrane water channels that are expressed in many organs. However, there are no data available about the expression pattern of aquaporin water channels in dental structures. In the present study we have characterised the expression of six different aquaporin isoforms (AQP1-5, AQP-9) in developing human and mouse tooth germs by immunohistochemistry using isoform specific antibodies. In the "bell stage" AQP1 was expressed in endothelial cells of small vessels whereas no other structures of the tooth primordial were labeled. AQP2, AQP3 and AQP9 immunoreactivity was not observed in tooth germs, whereas strong AQP4 and AQP5 expression was observed in dental lamina, inner enamel epithelium, stratum intermedium, stellate reticulum and the outer enamel epithelium. Oral epithelium also exhibited AQP4 and AQP5 immunolabeling. During development of the matrices of the dental hard tissues AQP4 and AQP5 immunostaining was observed in the odontoblasts and their processes, as well as in the secretory ameloblast and their apical processes. Immunolabeling controls were negative. In conclusion, AQP4 and AQP5 are expressed in tooth germ tissues in early development in cells that previously have been shown to express HA and/or CD44, indicating that AQP water channels may play a role for ECM hydration during tooth development.

Aquaporin in Candida: characterization of a functional water channel protein.

The Candida albicans genome database contains one ORF with homology to aquaporins, AQY1. Xenopus oocytes injected with cRNA encoding C. albicans Aqy1p displayed a coefficient of water permeability (P(f)) that was equivalent to the P(f) for oocytes injected with the cRNA of S. cerevisiae Aqy1p. In addition, as seen in Saccharomyces for Aqy1p and Aqy2p, deletion of AQY1 in C. albicans resulted in cells that were less sensitive than wild-type to osmotic shock. In Saccharomyces, aquaporin null cells also have a cell surface that is more hydrophobic. However, unlike Saccharomyces, there was no effect on the cell surface hydrophobicity, flocculation or cell aggregation in aqy1 null C. albicans cells. Perhaps as a result, there was no difference between the virulence of C. albicans wild-type and aqy1 null strains in a murine model for systemic candidiasis.

Syntrophin-dependent expression and localization of Aquaporin-4 water channel protein.

The Aquaporin-4 (AQP4) water channel contributes to brain water homeostasis in perivascular astrocyte endfeet where it is concentrated. We postulated that AQP4 is tethered at this site by binding of the AQP4 C terminus to the PSD95-Discs large-ZO1 (PDZ) domain of syntrophin, a component of the dystrophin protein complex. Chemical cross-linking and coimmunoprecipitations from brain demonstrated AQP4 in association with the complex, including dystrophin, beta-dystroglycan, and syntrophin. AQP4 expression was studied in brain and skeletal muscle of mice lacking alpha-syntrophin (alpha-Syn(-/-)). The total level of AQP4 expression appears normal in brains of alpha-Syn(-/-) mice, but the polarized subcellular localization is reversed. High-resolution immunogold analyses revealed that AQP4 expression is markedly reduced in astrocyte endfeet membranes adjacent to blood vessels in cerebellum and cerebral cortex of alpha-Syn(-/-) mice, but is present at higher than normal levels in membranes facing neuropil. In contrast, AQP4 is virtually absent from skeletal muscle in alpha-Syn(-/-) mice. Deletion of the PDZ-binding consensus (Ser-Ser-Val) at the AQP4 C terminus similarly reduced expression in transfected cell lines, and pulse-chase labeling demonstrated an increased degradation rate. These results demonstrate that perivascular localization of AQP4 in brain requires alpha-Syn, and stability of AQP4 in the membrane is increased by the C-terminal PDZ-binding motif.

Cancer-related patient education: an overview of the last decade of evaluation and research.

PURPOSE/OBJECTIVES: To provide an overview of cancer-related patient-education research to determine future research needs. DATA SOURCES: A literature search of peer-reviewed articles from 1989-1999. Databases that were searched included Medline, CINAHL, HealthStar, ERIC, CancerLit, and PubMed. DATA SYNTHESIS: 176 articles were analyzed and synthesized into narrative form. CONCLUSIONS: Patients with cancer want and benefit from information, especially when making treatment decisions. Education helps patients manage side effects and improves adherence. Literacy is an important factor in materials development. The efficacy of computer-assisted learning, audio and video programs, and telephone interventions is supported in a variety of patient groups. Pain education can improve pain control, but the impact on fatigue has not been well researched. IMPLICATIONS FOR NURSING PRACTICE: Patient education is an important component of nursing care. Research has confirmed its impact in many areas but questions still remain.

Highly selective water channel activity measured by voltage clamp: analysis of planar lipid bilayers reconstituted with purified AqpZ.

Aquaporins are membrane channels selectively permeated by water or water plus glycerol. Conflicting reports have described ion conductance associated with some water channels, raising the question of whether ion conductance is a general property of the aquaporin family. To clarify this question, a defined system was developed to simultaneously measure water permeability and ion conductance. The Escherichia coli water channel aquaporin-Z (AqpZ) was studied, because it is a highly stable tetramer. Planar lipid bilayers were formed from unilamellar vesicles containing purified AqpZ. The hydraulic conductivity of bilayers made from the total extract of E. coli lipids increased 3-fold if reconstituted with AqpZ, but electric conductance was unchanged. No channel activity was detected under voltage-clamp conditions, indicating that less than one in 10(9) transport events is electrogenic. Microelectrode measurements were simultaneously undertaken adjacent to the membrane. Changes in sodium concentration profiles accompanying transmembrane water flow permitted calculation of the activation energies: 14 kcal/mol for protein-free lipid bilayers and 4 kcal/mol for lipid bilayers containing AqpZ. Neither the water permeability nor the electric conductivity exhibited voltage dependence. This sensitive system demonstrated that AqpZ is permeated by water but not charged ions and should permit direct analyses of putative electrogenic properties of other aquaporins.

Water and ion permeation of aquaporin-1 in planar lipid bilayers. Major differences in structural determinants and stoichiometry.

The aquaporin-1 (AQP1) water channel protein is known to facilitate the rapid movement of water across cell membranes, but a proposed secondary role as an ion channel is still unsettled. Here we describe a method to simultaneously measure water permeability and ion conductance of purified human AQP1 after reconstitution into planar lipid bilayers. Water permeability was determined by measuring Na(+) concentrations adjacent to the membrane. Comparisons with the known single channel water permeability of AQP1 indicate that the planar lipid bilayers contain from 10(6) to 10(7) water channels. Addition of cGMP induced ion conductance in planar bilayers containing AQP1, whereas cAMP was without effect. The number of water channels exceeded the number of active ion channels by approximately 1 million-fold, yet p-chloromethylbenzenesulfonate inhibited the water permeability but not ion conductance. Identical ion channel parameters were achieved with AQP1 purified from human red blood cells or AQP1 heterologously expressed in Saccharomyces cerevisae and affinity purified with either N- or C-terminal poly-histidine tags. Rp-8-Br-cGMP inhibited all of the observed conductance levels of the cation selective channel (2, 6, and 10 pS in 100 mm Na(+) or K(+)). Deletion of the putative cGMP binding motif at the C terminus by introduction of a stop codon at position 237 yielded a truncated AQP1 protein that was still permeated by water but not by ions. Our studies demonstrate a method for simultaneously measuring water permeability and ion conductance of AQP1 reconstituted into planar lipid bilayers. The ion conductance occurs (i) through a pathway distinct from the aqueous pathway, (ii) when stimulated directly by cGMP, and (iii) in only an exceedingly small fraction of AQP1 molecules.

Abnormal distribution of aquaporin-5 water channel protein in salivary glands from Sjögren's syndrome patients.

Patients with Sjögren's syndrome (SS) suffer from deficient secretion of saliva due to an autoimmune destruction of salivary glands, however, glandular dysfunction also occurs without destruction. Based upon its abnormal distribution in SS salivary glands, a potential role for the water channel protein aquaporin-5 (AQP5) is proposed in the pathogenesis of SS. The immunohistochemical distribution of AQP5 was compared in minor salivary gland biopsies obtained from women after informed consent: primary SS (53.2 +/- 14 years old, n = 10), healthy volunteers (46.2 +/- 17 years old, n = 10), patients with sarcoidosis (37 and 48 years old), and patients with non-specific sialoadenitis (54 and 61 years old). Biopsies from normal subjects revealed AQP5 primarily at the apical membrane of the salivary gland acinus. In contrast, biopsies from SS patients revealed AQP5 primarily at the basal membranes of the acinus. The AQP5 distribution in biopsies from patients with other dry mouth disorders, such as non-specific sialoadenitis or sarcoidosis, was similar to biopsies from control subjects. Computer-assisted microscopy was performed to quantitatively evaluate AQP5 distribution in the immunoreactive acini of both SS and control subjects. Biopsies from SS patients had higher labeling indices (percentage of acinus area immunoreactive for AQP5) at the basal membrane when compared with biopsies from control subjects. In contrast, biopsies of SS patients exhibited lower labeling indices at the apical membrane when compared with biopsies from control subjects. To verify the specificity of the AQP5 antibody, Western blot analysis was performed on membranes from Xenopus oocytes injected with AQP5 cRNA or on membranes from minor salivary glands of control subjects and SS patients. In each case, the immunoblots had a 27 kd band, corresponding to the expected molecular weight of AQP5. Abnormal distribution of AQP5 in salivary gland acini is likely to contribute to the deficiency of fluid secretion, which is a defining feature of Sjögren's syndrome.

Defective cellular trafficking of lacrimal gland aquaporin-5 in Sjögren's syndrome.

Dry eyes and dry mouth are clinical hallmarks of Sjögren's syndrome. We assessed the distribution of aquaporin-5 (AQP5) in lacrimal gland biopsy samples. Healthy controls and patients with Mikulicz's disease or non-Sjögren's syndrome dry eye had the expected apical distribution of AQP5 in lacrimal acinar cells. By contrast, cytoplasmic AQP5 was seen in patients with Sjögren's syndrome. Sodium channel and sodium-potassium ATPase distributions were normal in all groups. These findings show a selective defect in lacrimal gland AQP5 trafficking in Sjögren's syndrome that might contribute to decreased lacrimation and dry eye in these patients.

Reconstitution and functional comparison of purified GlpF and AqpZ, the glycerol and water channels from Escherichia coli.

A large family of membrane channel proteins selective for transport of water (aquaporins) or water plus glycerol (aquaglyceroporins) has been found in diverse life forms. Escherichia coli has two members of this family-a water channel, AqpZ, and a glycerol facilitator, GlpF. Despite having similar primary amino acid sequences and predicted structures, the oligomeric state and solute selectivity of AqpZ and GlpF are disputed. Here we report biochemical and functional characterizations of affinity-purified GlpF and compare it to AqpZ. Histidine-tagged (His-GlpF) and hemagglutinin-tagged (HA-GlpF) polypeptides encoded by a bicistronic construct were expressed in bacteria. HA-GlpF and His-GlpF appear to form oligomers during Ni-nitrilotriacetate affinity purification. Sucrose gradient sedimentation analyses showed that the oligomeric state of octyl glucoside-solubilized GlpF varies: low ionic strength favors subunit dissociation, whereas Mg(2+) stabilizes tetrameric assembly. Reconstitution of affinity-purified GlpF into proteoliposomes increases glycerol permeability more than 100-fold and water permeability up to 10-fold compared with control liposomes. Glycerol and water permeability of GlpF both occur with low Arrhenius activation energies and are reversibly inhibited by HgCl(2). Our studies demonstrate that, unlike AqpZ, a water-selective stable tetramer, purified GlpF exists in multiple oligomeric forms under nondenaturing conditions and is highly permeable to glycerol but less well permeated by water.

Altered ubiquitination and stability of aquaporin-1 in hypertonic stress.

Aquaporin-1 (AQP1) water channel protein expression is increased by hypertonic stress. The contribution of changes in protein stability to hypertonic induction of AQP1 have not been described. Incubation of BALB/c fibroblasts spontaneously expressing AQP1 with proteasome inhibitors increased AQP1 expression, suggesting basal proteasome-dependent degradation of the protein. Degradation by the proteasome is thought to be triggered by polyubiquitination of a target protein. To determine whether AQP1 is ubiquitinated, immunoprecipitation with anti-AQP1 antibodies was performed, and the resultant samples were probed by protein immunoblot for the presence of ubiquitin. Immunoblots demonstrated ubiquitination of AQP1 under control conditions that increased after treatment with proteasome inhibitors (MG132, lactacystin). Exposure of cells to hypertonic medium for as little as 4 h decreased ubiquitination of AQP1, an effect that persisted through 24 h in hypertonic medium. Using metabolic labeling with [(35)S]methionine, the half-life of AQP1 protein under isotonic conditions was found to be <4 h. AQP1 protein half-life was markedly increased by exposure of cells to hypertonic medium. These observations provide evidence that aquaporins are a target for ubiquitination and proteasome-dependent degradation. Additionally, these studies demonstrate that reduced protein ubiquitination and increased protein stability lead to increased levels of AQP1 expression during hypertonic stress.

Aquaporins in Saccharomyces: Characterization of a second functional water channel protein.

The Saccharomyces cerevisiae genome database contains two ORFs with homology to aquaporins, AQY1 and AQY2. Aqy1p has been shown to be a functional aquaporin in some strains, such as Sigma1278b. AQY2 is disrupted by a stop codon in most strains; however, Sigma1278b has an intact ORF. Because Sigma1278b Aqy2p has an intracellular localization in Xenopus oocytes and in yeast, other strains of yeast were examined. Aqy2p from Saccharomyces chevalieri has a single amino acid in the third transmembrane domain (Ser-141) that differs from Sigma1278b Aqy2p (Pro-141). S. chevalieri Aqy2p is a functional water channel in oocytes and traffics to the plasma membrane of yeast. The Sigma1278b parental strain, the aqy1-aqy2 double null yeast, and null yeast expressing S. chevalieri Aqy2p were examined under various conditions. Comparison of these strains revealed that the aquaporin null cells were more aggregated and their surface was more hydrophobic. As a result, the aquaporin null cells were more flocculent and more efficient at haploid invasive growth. Despite its primary intracellular localization, Sigma1278b Aqy2p plays a role in yeast similar to Aqy1p and S. chevalieri Aqy2p. In addition, Aqy1p and Aqy2p can affect cell surface properties and may provide an advantage by dispersing the cells during starvation or during sexual reproduction.

Regulation of AQP6 mRNA and protein expression in rats in response to altered acid-base or water balance.

In the rat, aquaporin-6 (AQP6) is mainly localized in intercalated cells (ICs) in collecting ducts, where it is exclusively associated with intracellular vesicles. In this study, we examined whether AQP6 protein and mRNA expression were regulated in the inner medulla or inner stripe of the outer medulla. Rats treated with dietary alkali or acid load for 7 days with a fixed daily water intake revealed appropriate changes in urine pH but unchanged urine output. AQP6 protein and mRNA abundance were increased in alkali-loaded rats (187 +/- 18 and 151 +/- 17% of control, respectively), whereas no changes were observed in acid-loaded rats. Immunohistochemistry revealed increased IC AQP6 labeling in alkali-loaded rats but not in acid-loaded rats. In contrast, administration of NH(4)Cl in the drinking water for 2 wk (free access to water) revealed a significant increase in AQP6 protein abundance (194 +/- 9% of control), but this was associated with increased water intake. Combined, this suggests that AQP6 expression was not affected by acid loading per se but rather was in response to changes in water intake. Consistent with this, water loading for 48 h was associated with increased AQP6 protein abundance, compared with thirsted rats. Moreover, rats with lithium-induced nephrogenic diabetes insipidus had a threefold increase in both AQP6 protein and mRNA expression. Overall, these results suggest that AQP6 expression in collecting duct ICs is regulated by altered acid/alkali load or water balance. Thus AQP6 may contribute to maintenance of acid-base homeostasis and water balance.

Structural determinants of water permeation through aquaporin-1.

Human red cell AQP1 is the first functionally defined member of the aquaporin family of membrane water channels. Here we describe an atomic model of AQP1 at 3.8A resolution from electron crystallographic data. Multiple highly conserved amino-acid residues stabilize the novel fold of AQP1. The aqueous pathway is lined with conserved hydrophobic residues that permit rapid water transport, whereas the water selectivity is due to a constriction of the pore diameter to about 3 A over a span of one residue. The atomic model provides a possible molecular explanation to a longstanding puzzle in physiology-how membranes can be freely permeable to water but impermeable to protons.

Functional impairment of lens aquaporin in two families with dominantly inherited cataracts.

Opacities in the crystalline lens of eye appear with high frequency in the general population. Dominantly inherited cataracts with differing clinical features were found in two families carrying different point mutations in the gene encoding lens water channel protein AQP0 (major intrinsic protein, MIP). Families with E134G have a uni-lamellar cataract which is stable after birth, whereas families with T138R have multi-focal opacities which increase throughout life. To establish pathophysiological relevance of cataract formation, the Xenopus laevis oocyte expression system was employed to evaluate functional defects in the mutant proteins, E134G and T138R. Both substitutions cause loss of membrane water channel activity due to impaired trafficking of the mutant proteins to the oocyte plasma membrane. Although missense mutations in AQP1 and AQP2 proteins are known to result in recessive traits in vivo and in vitro, when E134G or T138R are co-expressed with wild-type AQP0 protein, the mutant proteins exhibit dominant negative behaviour. To our knowledge, these studies represent the first in vitro demonstration of functionally defective AQP0 protein from humans with congenital cataracts. Moreover, these observations predict that less severe defects in the AQP0 protein may contribute to lens opacity in patients with common, less fulminant forms of cataracts.

The aquaporin sidedness revisited.

Aquaporins are transmembrane water channel proteins, which play important functions in the osmoregulation and water balance of micro-organisms, plants, and animal tissues. All aquaporins studied to date are thought to be tetrameric assemblies of four subunits each containing its own aqueous pore. Moreover, the subunits contain an internal sequence repeat forming two obversely symmetric hemichannels predicted to resemble an hour-glass. This unique arrangement of two highly related protein domains oriented at 180 degrees to each other poses a significant challenge in the determination of sidedness. Aquaporin Z (AqpZ) from Escherichia coli was reconstituted into highly ordered two-dimensional crystals. They were freeze-dried and metal-shadowed to establish the relationship between surface structure and underlying protein density by electron microscopy. The shadowing of some surfaces was prevented by protruding aggregates. Thus, images collected from freeze-dried crystals that exhibited both metal-coated and uncoated regions allowed surface relief reconstructions and projection maps to be obtained from the same crystal. Cross-correlation peak searches along lattices crossing metal-coated and uncoated regions allowed an unambiguous alignment of the surface reliefs to the underlying density maps. AqpZ topographs previously determined by AFM could then be aligned with projection maps of AqpZ, and finally with human erythrocyte aquaporin-1 (AQP1). Thereby features of the AqpZ topography could be interpreted by direct comparison to the 6 A three-dimensional structure of AQP1. We conclude that the sidedness we originally proposed for aquaporin density maps was inverted.