Water rapidly crosses the plasma membrane of red blood cells (RBCs) and renal tubules through specialized channels. Although selective for water, the molecular structure of these channels is unknown. The CHIP28 protein is an abundant integral membrane protein in mammalian RBCs and renal proximal tubules and belongs to a family of membrane proteins with unknown functions. Oocytes from Xenopus laevis microinjected with in vitro-transcribed CHIP28 RNA exhibited increased osmotic water permeability; this was reversibly inhibited by mercuric chloride, a known inhibitor of water channels. Therefore it is likely that CHIP28 is a functional unit of membrane water channels.

The aquaporins transport water through membranes of numerous tissues, but the molecular mechanisms for sensing changes in extracellular osmolality and regulating water balance in brain are unknown. We have isolated a brain aquaporin by homology cloning. Like aquaporin 1 (AQP1, also known as CHIP, channel-forming integral membrane protein of 28 kDa), the deduced polypeptide has six putative transmembrane domains but lacks cysteines at the known mercury-sensitive sites. Two initiation sites were identified encoding polypeptides of 301 and 323 amino acids; expression of each in Xenopus oocytes conferred a 20-fold increase in osmotic water permeability not blocked by 1 mM HgCI2, even after substitution of cysteine at the predicted mercury-sensitive site. Northern analysis and RNase protection demonstrated the mRNA to be abundant in mature rat brain but only weakly detectable in eye, kidney, intestine, and lung. In situ hybridization of brain localized the mRNA to ependymal cells lining the aqueduct, glial cells forming the edge of the cerebral cortex and brainstem, vasopressin-secretory neurons in supraoptic and paraventricular nuclei of hypothalamus, and Purkinje cells of cerebellum. Its distinctive expression pattern implicates this fourth mammalian member of the aquaporin water channel family (designated gene symbol, AQP4) as the osmoreceptor which regulates body water balance and mediates water flow within the central nervous system. The aquaporins are a family of water-selective membrane channels found in animals, plants, and microorganisms (reviewed in refs. 1 and 2). Aquaporin 1 (AQP1, also known as CHIP, channel-forming integral membrane protein of 28 kDa) was the first protein shown to function as a molecular water channel (3) and is naturally expressed in mammalian red cells, renal proximal tubules (4-6), and other waterpermeable epithelia (7). AQP2 is the vasopressin-regulated water channel in renal collecting ducts (8, 9) and is the site of mutations in some forms of nephrogenic diabetes insipidus (10). AQP3 is the water channel in basolateral membranes of renal medullary collecting duct (11).Because of restricted space within the cranium, regulation of salt and water balance is essential for normal functions of the mammalian brain (reviewed in ref. 12). Moreover, vasopressin is released by the neurohypophysis in response to small changes in osmolality around the supraoptic and paraventricular nuclei (13). AQP1 (CHIP) is abundant in the choroid plexus, but not elsewhere in brain where molecular mechanisms responsible for transmembrane water movements and osmoreception are still unknown (7). By homology cloning we have isolated a cDNA from a rat brain library and have established its function and distribution. As this work was nearing completion, a highly related cDNA was cloned from rat lung (14); however, discrepancies with our studies were observed in the initiation site, in the coding and 3' untranslated sequences, and in the sites of expression and relative abundance. Thus, additional studie...

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.

CHIP28 is a 28-kDa integral membrane protein with similarities to membrane chanch and Is found in erythrocytes and renal tubules. A cDNA for CHIP28 was isolated from human fetal liver cDNA template by a the-step polymerase chain reaction (PCR) Analysis of the deduced amino acid sequence sugss that CHEIP28 protein contains six bilayer-sannin domains, two exofacial potential N-glycosylation sites, and intraceflular N and C termini. Search of the DNA sequence data bas revealed a strong homology with the major intrinsic protein of bovine lens, which is the prototype of an ancient but recently recognized family of membrane channels. These proteins are believed to form channels permeable to water and possibly other small molecules. CHIP28 shares homology with all known members of this channel family, and it is speculated that CHIP28 has a similar function.The erythrocyte membrane has proven to be an accessible source of new proteins with structural or metabolic functions and a useful model with general relevance to plasma membranes (for reviews, see refs. 1 and 2). During isolation of the 32-kDa Rh polypeptides from human erythrocytes, a 28-kDa integral membrane protein copurified and was considered a breakdown product (3, 4). The 28-kDa protein was later shown to be a unique molecule that is abundant in erythrocytes and renal tubules, and a subpopulation is N-glycosy-The function of the 28-kDa protein is not yet known, but several observations suggest that it is a membrane channel. The 28-kDa protein behaves as a tetramer when solubilized in Triton X-100, and the N-terminal amino acid sequence of the purified 28-kDa protein (6) is related to that of MIP26, the 26-kDa major intrinsic protein of bovine lens fiber cells (7). When reconstituted into planar lipid bilayers, MIP26 forms tetrameric channels with voltage-regulated conductance (8,9), and MIP26 appears to function as a channel through which lens fiber cells absorb interstitial fluid (8). MMP26 is the prototype of a family of membrane proteins recently identified in diverse species (10). This report describes isolation of the cDNA* for the 28-kDa protein, which has homology with all known members of the MIP channel family and is referred to as

The Aquaporin family of water channels plays a fundamental role in transmembrane water movements in numerous plant and animal tissues. Since the molecular pathway by which water is secreted by salivary glands is unknown, a cDNA was isolated from rat submandibular gland by homology cloning. Similar to other Aquaporins, the salivary cDNA encodes a 265-residue polypeptide with six putative transmembrane domains separated by five connecting loops (A-E); the NH2- and COOH-terminal halves of the polypeptide are sequence-related, and each contains the motif Asn-Pro-Ala. A mercurial-inhibition site is present in extracellular loop E, and cytoplasmic loop D contains a cAMP-protein kinase phosphorylation consensus. In vitro translation yielded a 27-kDa polypeptide, and expression of the cRNA in Xenopus oocytes conferred a 20-fold increase in osmotic water permeability (Pf) which was reversibly inhibited by 1 mM HgCl2. Northern analysis demonstrated a 1.6-kilobase mRNA in submandibular, parotid, and sublingual salivary glands, lacrimal gland, eye, trachea, and lung. In situ hybridization revealed a strong hybridization over the corneal epithelium in eye and over the secretory lobules in salivary glands. These studies have identified a new mammalian member of the Aquaporin water channel family (gene symbol AQP5) which is implicated in the generation of saliva, tears, and pulmonary secretions.

The gene aquaporin-1 encodes channel-forming integral protein (CHIP), a member of a large family of water transporters found throughout nature. Three rare individuals were identified who do not express CHIP-associated Colton blood group antigens and whose red cells exhibit low osmotic water permeabilities. Genomic DNA analyses demonstrated that two individuals were homozygous for different nonsense mutations (exon deletion or frameshift), and the third had a missense mutation encoding a nonfunctioning CHIP molecule. Surprisingly, none of the three suffers any apparent clinical consequence, which raises questions about the physiological importance of CHIP and implies that other mechanisms may compensate for its absence.

OBJECTIVECirculating levels of soluble receptor for advanced glycation end products (sRAGE) likely comprise both a secreted isoform (esRAGE) and wild-type RAGE cleaved from the cell membrane. Both sRAGE and esRAGE have been proposed as biomarkers of cardiovascular disease (CVD), but prospective data are limited. We examined the relationship of sRAGE and esRAGE to incident coronary heart disease (CHD) and stroke in type 2 diabetic patients followed for 3.9 years in a trial of atorvastatin: the Collaborative Atorvastatin Diabetes Study (CARDS).RESEARCH DESIGN AND METHODSWe used a nested case-control design sampling all incident cases of CVD with available plasma and randomly selecting three control subjects, who were free of CVD throughout follow-up, per case. Analysis was by Cox regression with adjustment for treatment allocation and relevant covariates.RESULTSsRAGE and esRAGE were strongly correlated (ρ = 0.88) and were both higher in those with lower BMI (P < 0.001), higher adiponectin (P < 0.001), lower estimated glomerular filtration rate (P = 0.009), and white ethnicity (P < 0.001). Both sRAGE and esRAGE were associated with incident CHD events, independently of treatment allocation and the above factors; hazard ratio (HR) = 1.74 (95% CI 1.25–2.41; P = 0.002) for a doubling of the sRAGE level; HR = 1.45 (1.11–1.89; P = 0.006) for a doubling of the esRAGE level. There was no significant association with stroke; HR for sRAGE = 0.66 (0.38–1.14). Atorvastatin, 10 mg daily, did not alter sRAGE.CONCLUSIONSHigher levels of sRAGE and esRAGE are associated with incident CHD but not stroke in type 2 diabetes.

Serum levels of TBARS were strongly predictive of cardiovascular events in patients with stable CAD, independently of traditional risk factors and inflammatory markers.