Membrane water transport is critically involved in brain volume homeostasis and in the pathogenesis of brain edema. The cDNA encoding aquaporin-4 (AQP4) water channel protein was recently isolated from rat brain. We used immunocytochemistry and high-resolution immunogold electron microscopy to identify the cells and membrane domains that mediate water flux through AQP4. The AQP4 protein is abundant in glial cells bordering the subarachnoidal space, ventricles, and blood vessels. AQP4 is also abundant in osmosensory areas, including the supraoptic nucleus and subfornical organ. Immunogold analysis demonstrated that AQP4 is restricted to glial membranes and to subpopulations of ependymal cells. AQP4 is particularly strongly expressed in glial membranes that are in direct contact with capillaries and pia. The highly polarized AQP4 expression indicates that these cells are equipped with specific membrane domains that are specialized for water transport, thereby mediating the flow of water between glial cells and the cavities filled with CSF and the intravascular space. Key words: aquaporin-4 water channel; brain water permeability; glial cells; ependymal cells; immunogold electron microscopy; CSFWater metabolism is of major importance in a number of physiological processes in the CNS including CSF production and absorption, fluid transport across neuropil and vascular endothelium, and cell volume regulation (Fitzsimons, 1992;Robertson, 1992). In addition, water transport may serve to compensate for local changes in osmolality associated with potassium siphoning, which is essential for synaptic transmission. Alterations in water distribution in brain and CSF compartments is a common occurence in multiple neuropathological conditions including brain edema, brain tumors, stroke, hyponatremia, head injuries, and hydrocephalus. Despite its importance, little is known about the cellular and molecular mechanisms involved in transmembrane water movements in brain.Discovery of aquaporin-1 (Preston et al., 1992) answered the long-standing biophysical question of how water crosses plasma membranes (for review, see Agre et al., 1993;Knepper, 1994). Characterization of aquaporins provided molecular insight into fundamental processes of normal water balance and disorders of water balance outside brain (for review, see Nielsen et al., 1996). A cDNA for aquaporin-4 (AQP4) water channel protein was isolated recently from rat brain (Hasegawa et al., 1994;Jung et al., 1994), and abundant AQP4 was noted in brain including in cerebellum, hypothalamus, spinal cord, and ependymal cells lining the ventricles (Jung et al., 1994;Frigeri et al., 1995). Nevertheless, the cellular and subcellular distributions of AQP4 in brain remain unknown, and definition of the sites of AQP4 expression will be essential for understanding its physiological and pathophysiological roles.Immunocytochemistry and high-resolution immunogold electron microscopy were used to define the sites of AQP4 in brain. AQP4 expression is restricted to ependymal cell lining of ...

The discovery of aquaporin-1 (AQP1) answered the long-standing biophysical question of how water specifically crosses biological membranes. In the kidney, at least seven aquaporins are expressed at distinct sites. AQP1 is extremely abundant in the proximal tubule and descending thin limb and is essential for urinary concentration. AQP2 is exclusively expressed in the principal cells of the connecting tubule and collecting duct and is the predominant vasopressin-regulated water channel. AQP3 and AQP4 are both present in the basolateral plasma membrane of collecting duct principal cells and represent exit pathways for water reabsorbed apically via AQP2. Studies in patients and transgenic mice have demonstrated that both AQP2 and AQP3 are essential for urinary concentration. Three additional aquaporins are present in the kidney. AQP6 is present in intracellular vesicles in collecting duct intercalated cells, and AQP8 is present intracellularly at low abundance in proximal tubules and collecting duct principal cells, but the physiological function of these two channels remains undefined. AQP7 is abundant in the brush border of proximal tubule cells and is likely to be involved in proximal tubule water reabsorption. Body water balance is tightly regulated by vasopressin, and multiple studies now have underscored the essential roles of AQP2 in this. Vasopressin regulates acutely the water permeability of the kidney collecting duct by trafficking of AQP2 from intracellular vesicles to the apical plasma membrane. The long-term adaptational changes in body water balance are controlled in part by regulated changes in AQP2 and AQP3 expression levels. Lack of functional AQP2 is seen in primary forms of diabetes insipidus, and reduced expression and targeting are seen in several diseases associated with urinary concentrating defects such as acquired nephrogenic diabetes insipidus, postobstructive polyuria, as well as acute and chronic renal failure. In contrast, in conditions with water retention such as severe congestive heart failure, pregnancy, and syndrome of inappropriate antidiuretic hormone secretion, both AQP2 expression levels and apical plasma membrane targetting are increased, suggesting a role for AQP2 in the development of water retention. Continued analysis of the aquaporins is providing detailed molecular insight into the fundamental physiology and pathophysiology of water balance and water balance disorders.

Water excretion by the kidney is regulated by the peptide hormone vasopressin. Vasopressin increases the water permeability of the renal collecting duct cells, allowing more water to be reabsorbed from collecting duct urine to blood. Despite long-standing interest in this process, the mechanism of the water permeability increase has remained undetermined. Recently, a molecular water channel (AQP-CD) has been cloned whose expression appears to be limited to the collecting duct. Previously, we immunolocalized this water channel to the apical plasma membrane (APM) and to intracellular vesicles (IVs) of collecting duct cells. Here, we test the hypothesis that vasopressin increases cellular water permeability by inducing exocytosis of AQP-CD-laden vesicles, transferring water channels from IVs to APM. Rat collecting ducts were perfused in vitro to determine water permeability and subcellular distribution of AQP-CD in the same tubules. The collecting ducts were fixed for immunoelectron microscopy before, during, and after exposure to vasopressin. Vasopressin exposure induced increases in water permeability and the absolute labeling density of AQP-CD in the APM. In parallel, the APM:IV labeling ratio increased. Furthermore, in response to vasopressin withdrawal, AQP-CD labeling density in the APM and the APM:IV labeling ratio decreased in parallel to a measured decrease in osmotic water permeability. We conclude that vasopressin increases the water permeability of collecting duct cells by inducing a reversible translocation of AQP-CD water channels from IVs to the APM.Vasopressin (the antidiuretic hormone) is a 9-amino acid peptide hormone, secreted by the neurohypophysis, which acts on the kidney via the adenylyl cyclase-coupled vasopressin receptor (V2 receptor) to regulate water excretion. Vasopressin reduces urinary water excretion in part by increasing the water permeability of the renal collecting duct, thereby accelerating the osmotically driven absorption of water from the collecting duct lumen to the blood. Although the mechanism by which the water permeability increases is controversial, it presumably involves an increase in the number or unit conductance of water channels in the apical plasma membrane (APM), the rate-limiting barrier for net transepithelial water transport (1, 2). Several molecular water channels of the aquaporin (AQP) family are expressed in the kidney (3-5). One of these, AQP-CD (also called WCH-CD or AQP-2), appears to be expressed exclusively in the renal collecting duct (6, 7) and has been documented to be the major pathway responsible for vasopressin-regulated water transport across collecting duct cells (8,9). Several years ago, it was hypothesized (10) that vasopressin induces translocation of water channels from intracellular vesicles (IVs) to the APM by exocytosis (the "shuttle hypothesis"). However, direct experimental evidence for translocation ofwater channels is lacking. We recently demonstrated that the AQP-CD water channel is present in the APM and IVs of collecting d...

The high water permeability characteristic of mammalian red cell membranes is now known to be caused by the protein AQP1. This channel freely permits movement of water across the cell membrane, but it is not permeated by other small, uncharged molecules or charged solutes. AQP1 is a tetramer with each subunit containing an aqueous pore likened to an hourglass formed by obversely arranged tandem repeats. Cryoelectron microscopy of reconstituted AQP1 membrane crystals has revealed the three-dimensional structure at 3-6 A. AQP1 is distributed in apical and basolateral membranes of renal proximal tubules and descending thin limbs as well as capillary endothelia. Ten mammalian aquaporins have been identified in water-permeable tissues and fall into two groupings. Orthodox aquaporins are water-selective and include AQP2, a vasopressin-regulated water channel in renal collecting duct, in addition to AQP0, AQP4, and AQP5. Multifunctional aquaglyceroporins AQP3, AQP7, and AQP9 are permeated by water, glycerol, and some other solutes. Aquaporins are being defined in numerous other species including amphibia, insects, plants, and microbials. Members of the aquaporin family are implicated in numerous physiological processes as well as the pathophysiology of a wide range of clinical disorders.

The existence of water-selective channels has been postulated to explain the high water permeability of erythrocytes and certain epithelial cells. The aquaporin CHIP (channel-forming integral membrane protein of 28 kDa), a molecular water channel, is abundant in erythrocytes and water-permeable segments of the nephron. To determine whether CHIP may mediate transmembrane water movement in other water-permeable epithelia, membranes of multiple organs were studied by immunoblotting, immunohistochemistry, and immunoelectron microscopy using afmity-purified anti-CHIP IgG. The apical membrane of the choroid plexus epithelium was densely stained, implying a role for CHIP in the secretion ofcerebrospinal fluid. In the eye, CHIP was abundant in apical and basolateral domains of ciliary epithelium, the site of aqueous humor secretion, and also in lens epitheilum and corneal endothelium. CHIP was detected in membranes of hepatic bile ducts and water-resorptive epithelium of gall bladder, suggesting a role in bile secretion and concentration. CHIP was not detected in glandular epithelium of mammary, salivary, or lacrimal glands, suggesting the existence of other water-channel isoforms. CHIP was also not detected within the epithelium of the gastrointestinal mucosa. CHIP was abundant in membranes of intestinal lacteals and continuous capillaries in diverse tissues, including cardiac and skeletal muscle, thus providing a molecular explanation for the known water permeability of certain lymphatics and capillary beds. These studies underscore the hypothesis that CHIP plays a major role in transcellular water movement throughout the body.Much progress has been made in the quest for carriers ofions and other small molecules, but the molecular mechanism of transmembrane water movement has remained poorly understood (1). Northern analyses revealed CHIP mRNA in several organs (6,14,15). CHIP transcripts were demonstrated in diverse epithelia in fetal rats where three distinct patterns of expression were identified (16). CHIP has been proposed to be the major mechanism by which transmembrane water movement occurs in mammals (3), and this study was undertaken to document the tissue and cellular distributions of CHIP throughout the body. MATERIALS AND METHODSAntibodies to CHIP. Immunization of rabbits with highly purified CHIP and affinity purification of antibodies was described (5, 10). Antibodies to human erythrocyte spectrin and band 3 were a gift from Vann Bennett (Duke University Medical Center).Tissue Preparation for Immunoblots. Four mature SpragueDawley rats were injected with 100 units of heparin and killed by ether inhalation, and the carcasses were perfused through the right and left ventricles with 500 ml of0.15 M NaCl/7.5 mM sodium phosphate/i mM sodium EDTA, pH 7.5. Selected organs were removed and homogenized in buffer containing 0.25 M sucrose, 1 mM phenylmethylsulfonyl fluoride, diisopropylfluorophosphate at 0.5 mg/ml, and leupeptin at 4 ,ug/ml at 0°C as described (4). Membranes were solubilized in SDS, electr...

Aquaporin (AQP) water channels are abundant in the brain and spinal cord, where AQP1 and AQP4 are believed to play major roles in water metabolism and osmoregulation. Immunocytochemical analysis of the brain recently revealed that AQP4 has a highly polarized distribution, with marked expression in astrocyte end-feet that surround capillaries and form the glia limitans; however, the structural organization of AQP4 has remained unknown. In freezefracture replicas, astrocyte end-feet contain abundant square arrays of intramembrane particles that parallel the distribution of AQP4. To determine whether astrocyte and ependymocyte square arrays contain AQP4, we employed immunogold labeling of SDS-washed freeze-fracture replicas and stereoscopic confirmation of tissue binding. Antibodies to AQP4 directly labeled Ϸ33% of square arrays in astrocyte and ependymocyte plasma membranes in rat brain and spinal cord. Overall, 84% of labels were present beneath square arrays; 11% were beneath particle clusters that resembled square arrays that had been altered during fixation or cleaving; and 5% were beneath the much larger areas of glial plasma membrane that were devoid of square arrays. Based on this evidence that AQP4 is concentrated in glial square arrays, freeze-fracture methods may now provide biophysical insights regarding neuropathological states in which abnormal f luid shifts are accompanied by alterations in the aggregation state or the molecular architecture of square arrays.

Despite longstanding interest by nephrologists and physiologists, the molecular identities of membrane water channels remained elusive until recognition of CHIP, a 28-kDa channel-forming integral membrane protein from human red blood cells originally referred to as "CHIP28." CHIP functions as an osmotically driven, water-selective pore; 1) expression of CHIP conferred Xenopus oocytes with markedly increased osmotic water permeability but did not allow transmembrane passage of ions or other small molecules; 2) reconstitution of highly purified CHIP into proteoliposomes permitted determination of the unit water permeability, i.e., 3.9 x 10(9) water molecules.channel subunit-1 x s-1. Although CHIP exists as a homotetramer in the native red blood cell membrane, site-directed mutagenesis studies suggested that each subunit contains an individually functional pore that may be reversibly occluded by mercurial inhibitors reacting with cysteine-189. CHIP is a major component of both apical and basolateral membranes of water-permeable segments of the nephron, where it facilitates transcellular water flow during reabsorption of glomerular filtrate. CHIP is also abundant in certain other absorptive or secretory epithelia, including choroid plexus, ciliary body of the eye, hepatobiliary ductules, gall bladder, and capillary endothelia. Distinct patterns of CHIP expression occur at these sites during fetal development and maturity. Similar proteins from other mammalian tissues and plants were later shown to transport water, and the group is now referred to as the "aquaporins." Recognition of CHIP has provided molecular insight into the biological phenomenon of osmotic water movement, and it is hoped that pharmacological modulation of CHIP function may provide novel treatments of renal failure and other clinical problems.

The aquaporins are molecular water channels expressed in the kidney and other organs. To investigate long-term regulation of renal expression of these water channels, we carried out immunoblotting studies using membrane fractions from rat renal cortex and medulla. Both 48-h water restriction in Sprague-Dawley rats and 5-day arginine vasopressin (AVP) infusion in Brattleboro rats caused significant increases in the expression levels of two aquaporins, aquaporin-2 and aquaporin-3, while the levels of aquaporin-1 and aquaporin-4 were unchanged. The increases in aquaporin-2 and aquaporin-3 expression were seen in inner and outer medulla as well as cortex. Ablation of the corticomedullary interstitial osmotic gradient with an infusion of furosemide did not eliminate the upregulatory response to AVP infusion in Brattleboro rats. Furthermore, 5-day furosemide infusion to Sprague-Dawley rats did not decrease expression levels of the collecting duct aquaporins, but rather increased them. We conclude that the expression of aquaporin-2 and aquaporin-3, but not aquaporin-1 or aquaporin-4, is increased in response to elevated circulating AVP. Because regulation of aquaporin-2 and aquaporin-3 levels was observed in the cortex and because osmotic gradient ablation did not abrogate the increase, we conclude that changes in interstitial osmolality are not necessary for the AVP-induced upregulation of aquaporin-2 and aquaporin-3 expression.