Aquaporins, anti-aquaporin-4 autoantibodies and neuromyelitis optica

Clinica Chimica Acta 415 (2013) 350–360

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Invited critical review

Aquaporins, anti-aquaporin-4 autoantibodies and neuromyelitis optica Concepción González a, José M. González-Buitrago b, c,⁎, Guillermo Izquierdo d a

Servicio de Bioquímica Clínica, Hospital Universitario Virgen Macarena, 41009 Sevilla, Spain Unidad de Investigación, Hospital Universitario, Salamanca, Spain c Departamento de Bioquímica y Biología Molecular, Universidad de Salamanca, 37007 Salamanca, Spain d Servicio de Neurología, Hospital Universitario Virgen Macarena, 41009 Sevilla, Spain b

a r t i c l e

i n f o

Article history: Received 21 March 2012 Received in revised form 25 April 2012 Accepted 27 April 2012 Available online 2 May 2012 Keywords: Anti-aquaporin antibodies Aquaporins Neuromyelitis optica

a b s t r a c t The classification, distribution and functions of the different molecules of aquaporins (AQPs), including aquaporins, aquaglyceroporins and superaquaporins are reviewed together with their potential diagnostic and therapeutic uses. We analyzed the pathogenic importance of anti-AQP4 autoantibodies in neuromyelitis optica and related syndromes, as well as their diagnostic and predictive potential, prognosis, and monitoring of the disease. Finally, the analytical methods and current recommendations for testing anti-AQP4 autoantibodies in clinical practice are described. © 2012 Elsevier B.V. All rights reserved.

Contents 1. 2. 3.

Introduction . . . . . . . . . . Membrane channels for water . The aquaporin family of proteins 3.1. Type I aquaporins . . . . 3.2. Aquaglyceroporins . . . . 3.3. Superaquaporins . . . . 4. Clinical applications of aquaporin 4.1. Diagnostic usefulness . . 4.2. Therapeutic usefulness . . 5. Neuromyelitis optica . . . . . . 5.1. Pathogenic mechanisms . 6. Anti-AQP4 antibodies . . . . . . 7. Anti-AQP4 assays . . . . . . . 8. Conclusion . . . . . . . . . . . References . . . . . . . . . . . . .

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1. Introduction The diffusion of water across biological membranes has long been known. It occurs across all lipid bilayers and is a slow and low-capacity bidirectional process. However, many physiological processes require rapid movement of water. More than 30 years ago, it was considered that in certain specialized epithelia (renal tubules, red cells, secretory glands) there should be an additional system for the rapid movement

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of large masses of water. This system would possess high capacity for water and be specific, not allowing the passage of other substances, such as hydronium ions (H3O +). Water would move according to the saline osmotic gradient and the system could be modified or inhibited by various substances. The evidence pointed to the notion that it might be a membrane channel for water.

2. Membrane channels for water ⁎ Corresponding author at: Unidad de Investigación, Hospital Universitario de Salamanca, Paseo de San Vicente 58-132, 37007 Salamanca, Spain. E-mail address: [email protected] (J.M. González-Buitrago). 0009-8981/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2012.04.027

In 1988, in studies addressing Rh cell antigens, a 28 kDa protein shared by red cells from different species that participated in the

C. González et al. / Clinica Chimica Acta 415 (2013) 350–360

movement of water across biological membranes was found unexpectedly [1]. Later, coding DNA was isolated and a membrane polypeptide with several transmembrane domains with repeated amino acid sequences in the amino and carboxyl ends were identified. Its structure suggested that it might be part of a channel and that it could correspond to a component of water channels. Likewise, it was observed that this protein had homologous molecules in bacteria (E. coli), plants, and other mammalian cells [2]. Later, the name aquaporin was used for this membrane channel for water [3]. In 2003, Peter Agree was awarded Nobel Prize in Chemistry for the discovery of aquaporins (AQPs). 3. The aquaporin family of proteins Aquaporin-1 (AQP1) was the first aquaporin described in humans. Currently, for aquaporins at least 13 physiological sequences have been described (Table 1), which can be divided into two broad groups. On one hand, there are the channels that transport water exclusively, and on the other there are the aquaglyceroporins, which transport water and glycerol. A third group of aquaporins called super-AQP, with less homology with the above, has been proposed. 3.1. Type I aquaporins Using specific antibodies, it was observed that AQP1 is expressed in the nephron proximal tubules and not in the collecting duct (which could perhaps express another AQP). It was located in the apical membrane, in the basal membrane and in the intercellular borders, and it was involved in water movement across the cell, in favour of a concentration gradient between the apical and basal ends [4]. Later, it was demonstrated that AQP1 is also expressed in the basal and luminal membranes of the capillary endothelium [5]. AQP1 also participates in the secretion of fluid by the choroid plexus where CSF is produced, and seems to be involved in the regulation of intracranial pressure [6]. The AQP1 gene is located on the short arm of chromosome 7 (7p14) [7]. AQP1-null individuals have a limited capacity to concentrate water when subjected to functional testing of renal concentration; however, AQP1-null individuals are infrequent [8]. In the plasma membrane, AQP1 is located as a homotetramer, with each subunit containing an individually functional water pore [9]. In the centre of the four monomers lies a fifth pore, composed mainly of hydrophobic amino acids, which might provide a path for non-polar molecules such as gas molecules. Early studies of aquaporin-1 pointed to an “hourglass” model in which repeated sequences were responsible for forming the pore [10]. The crystal structure revealed that pore conformation enabled the electrostatic repulsion of protonated water (H3O +) [11] (Fig. 1). Recent evidence has indicated that AQP1 is also involved in gas transport through cellular membranes. Thus, AQP1 transports CO2, NO, and NH3 across plasma membranes and AQP1-dependent CO2 and NO transport appears to play an important role in mammalian Table 1 Classification of aquaporins.

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physiology. A channel-dependent transport of gaseous molecules could match the tightly controlled intracellular environment and the rapid paracrine actions of gaseous molecules better and offers a means of controlling directional release [12]. Otto et al. have found that AQP1 tetramers exhibit higher rates of CO2 transport than monomers [13], consistent with the concept that CO2 crosses AQP1 via the central pore of the aquaporin tetramer. Also, NO molecules appear to cross the highly hydrophobic central pore formed by the AQP1 tetramer. Whether additional gas paths are created in between tetramers or not remains to be answered. In the proximal tubule of mammals, AQP1 may play an important role in acid/base balance by facilitating CO2 influx and therefore HCO3reabsorption. AQP1 could play an important role in regulating arterial pH during metabolic acidosis, possibly by acting as a CO2 transporter in the proximal tubule [14,15]. AQP1 carries NO across cell membranes by facilitated transport, which is three times faster than free diffusion. The studies of Herrera et al. have confirmed that endothelium-dependent relaxation requires AQP1-dependent transport of NO across cell membranes [16,17]. Thus, transport of NO by AQP1 is of physiological relevance because it mediates NO-dependent vasorelaxation. Also, the ability of aquaporins to transport NO may permit tight control of intracellular NO concentrations in target cells and directional release from cells where it is produced [12]. Moreover, AQP-1 is also involved in tumour angiogenesis, tumour cell proliferation and migration [18]. AQP2 is similar to AQP1, but it is expressed in renal collecting tubules. AQP2 forms a water channel regulated by vasopressin. It is expressed in the apical membrane of collecting tubules and it is also observed in intracellular vesicles. Its expression in the apical membrane is dependent on environmental conditions [19,20]. The main water channel in the brain is AQP4 (Fig. 2). This aquaporin is expressed in the endings of astrocytes surrounding blood vessels and, therefore, at the border of the blood–brain barrier. Similarly, it is located in the limiting glia (in the subarachnoid cerebrospinal fluid-brain interface) and in the ependyma (ventricular cerebrospinal fluid-brain interfaces). Through its expression in these interfaces it regulates water movement inside and outside the brain [21–23]. AQP4-deficient mice do not manifest significant baseline water balance abnormalities in the central nervous system (CNS), but show, under the appropriate stress, impairment in the brain and spinal cord [24]. Experiments in mice null for AQP4 or with alpha-syntropin, which down-regulates AQP4, have shown that AQP4 facilitates oedema formation in diseases that produce cytotoxic oedema (cell swelling), including cerebral ischemia,

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Aquaglyceroporins (type 2)

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AQP0 AQP1 AQP2 AQP4 AQP5 AQP8

AQP3 AQP6 AQP7 AQP9 AQP10

AQP11 AQP12

A short sequence of hydrophobic amino acids (NPA boxes) which form the pore and six transmembrane domains are common for all types of aquaporins. Type-2 AQPs have an aspartic residue that expands the pore and permit molecules with a higher molecular weight than H2O, such as glycerol, to pass through. Type 3 AQPs do not have the aspartic residue in the pore but they have a characteristic cysteine residue near the pore and the pore amino acid sequence is less preserved.

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Fig. 1. Aquaporin-1 structure. Fragments of the AQP1 included within the membrane and in the interior and exterior of the cell. As can be seen, two repeats in tandem next to the amino and carboxyl ends are responsible for the shape of the pore for the passage of H2O.

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Extracellular loops

Arg216

His201

Fig. 2. AQP4 structure. Side view of AQP4 with the M1 to M8 helices joined by intra and extracellular handles. The selectivity filter residues Arg216 ande His20 allow the passage of H2O (yellow areas) but not of glycerol (hexahedral in red).

hyponatremia or meningitis. By contrast, it facilitates the elimination of oedema in diseases producing vasogenic oedema (from blood vessels), such as tumours and brain abscesses [25]. AQP4 is also important in the swelling of the spinal cord, where it appears to reduce post-traumatic oedema by facilitating the clearing of excess water [26,27]. AQP-4 in gliomas and meningiomas is highly concentrated in both tumoural and peritumoural tissue, playing a role in vasogenic oedema formation. Chemotherapy and radiotherapy induce a down-regulation in AQP-4 expression, restoring its perivascular rearrangement [28–31]. AQP4 is expressed in supporting cells adjacent to electrically excitable cells, such as glia cells vs. brain neurons; Müller cells vs. retinal bipolar cells; hair vs. supporting cells in the inner ear, and supporting cells in the olfactory epithelium vs. olfactory neurons. In AQP4-null mice, electrophysiological measurements have shown a decrease in the excitability of these cells. Although the mechanisms remain speculative, alterations in water volume and K+ concentration in the extracellular medium would affect neurons or other electrically excitable cells from other locations (retina, inner ear, olfactory neurons) [32,33]. AQP4 is also expressed in cell plasma membranes in several peripheral tissues, including kidney collecting ducts, skeletal muscle, gastric parietal cells, tracheal epithelial cells, airway epithelium, and exocrine gland epithelium. However, AQP4-deficient mice do not manifest significant peripheral abnormalities, such as skeletal muscle dysfunction [34] or reduced gastric acid secretion [35], except for a very mild impairment in maximal urinary concentrating ability [36]. An AQP4 function related to its role in water transport is cell migration. A model in which the tip of a cell protrusion (lamellipodium) creates an osmotic gradient through the fibre breakage of actin and ion uptake has been proposed. This gradient directs water entry through AQP channels, facilitating protrusion, spreading and cell migration [37]. In the RGMI gastric epithelial cell line, AQP1 inhibitors reduce membrane protrusion formation and slowdown of closure of traumatic rupture caused in the confluent layer of the cells [38]. AQP-4 could also be involved in brain tumour migration and invasion, and may accelerate glioma migration by facilitating the rapid changes in cell volume that accompany changes in cell shape. In peripheral areas of tumours, glioma cells are strongly labelled by AQP-4, indicative of their migratory activity [30]. AQP4, like AQP1, could participate in CO2 and NO transport through cellular membranes [39]. Recently, a pro-inflammatory role of AQP-4 in experimental autoimmune encephalomyelitis has been documented [40,41]. AQP5 is expressed in the glandular epithelium and participates in the active transport of water across the epithelium. In mice, AQP5 gene deletion worsens fluid secretion by salivary gland and airway submucosa, leading to a reduced secretion of a hyperosmolar fluid [42], while the AQP5 salivary concentration correlates with its secretion in health and disease [43]. In Sjögreen syndrome, an autoimmune disorder characterized by a deficient secretion of tears and saliva, a defective cellular trafficking of AQP5 in salivary and lachrymal glands has been

detected [44,45]. AQP5 can also transport CO2, although its affinity varies with respect to AQP1 or AQP4 [39]. AQP8 is the only aquaporin that passes H2O2, a molecule very similar to water in terms of both size and dielectric properties, although to our knowledge in mammals its physiological significance has not been established [46]. AQP8 has been localized in canalicular membranes, and modulates membrane water permeability, providing a molecular mechanism for the osmotically-coupled transport of solute and water during bile formation. Moreover, AQP8 gene silencing in the human hepatocyte-derived cell line HepG2 inhibits canalicular water secretion. It is possible that AQP8 could contribute, to cholestasis [47]. AQP0 is expressed in lens cells. Classified as an aquaporin its function remains unclear. Recent studies have shown that AQP0 has a very low permeability to water and seems to influence cell–cell adhesion [48]. Individuals with genetic deficiencies of AQP0 develop congenital cataracts [49].

3.2. Aquaglyceroporins Aquaglyceroporins have a larger pore size than aquaporins, thus allowing the passage of other large counterparts such as glycerol, nitrate or arsenic [50]. They regulate the glycerol content in the epidermis, in the fatty tissue and in other tissues, and they are involved in skin hydration, cell proliferation, carcinogenesis and fat metabolism [51,52]. Among aquaglyceroporins, AQP3 is expressed in kidney, erythrocytes and skin; its cutaneous expression decreases with aging [51]. AQP3 is constitutively expressed in the basolateral membrane of the epithelium of collecting tubules. AQP3-facilitated glycerol transport in skin is an important determinant of epidermal and stratum corneum hydration [53]. Verkman has pointed out that AQP3-facilitated glycerol transport is a key determinant of cell proliferation through a mechanism involving reduced epidermal glycerol concentrations in AQP3 deficiency, an impaired lipid biosynthesis, a reduced glycerol and ATP metabolism, an impaired MAPK signalling cascade (particularly p38 kinase) and, finally, a reduced cell proliferation [24]. AQP7 and AQP9 are involved in glycerol metabolism. AQP7 is expressed in adipose tissue and AQP9 in liver. In fasting states, AQP7 releases glycerol from fat catabolism in adipose tissue. Aqp7-null mice manifest a remarkable progressive increase in fat mass and adipocyte hypertrophy, accumulating glycerol and triglycerides in their adipocytes as they age. This adipocyte hypertrophy appears to be the consequence of a reduced plasma membrane glycerol permeability, suggesting adipocyte glycerol permeability as a novel regulator of adipocyte size and whole-body fat mass and indicating that the modulation of adipocyte AQP7 expression and/or function can alter fat mass [24]. AQP9 facilitates glycerol uptake by the liver for gluconeogenesis. Both aquaglyceroporins are also involved in arsenic transport. Arsenic toxicity is greater in the AQP9-null model [54]. Finally, AQP3 and AQP9 can serve as channels for NH3 and are able to regulate pH through an enhanced NH3 influx across the cell membrane [55]. Among the aquaporins, AQP8-dependent NH3 transport has also been demonstrated [56].

3.3. Superaquaporins Within this group are AQP11 and AQP12. They have been implicated in the transport of water, and in mice AQP11 deficiency generates polycystic kidneys, but to date their function is only partially known [57].

4. Clinical applications of aquaporin measurements The current and potential usefulness of aquaporin measurements can be divided into two major diagnostic and therapeutic areas.

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4.1. Diagnostic usefulness Genetic studies have led to the detection of aquaporin mutations that lead to a loss of function. Mutations in the AQP2 gene cause nonX-linked nephrogenic diabetes insipidus, with recessive inheritance. However, its incidence is very low, less than 1/20 million newborns, representing less than 10% of congenital diabetes insipidus [58]. The incidence of mutations is ever lower in other AQPs. Few individuals with mutations in the AQP1 gene have been described. They are phenotypically normal but show a defect in the capacity to concentrate urine when they are water-deprived [8]. Few mutations of AQP0 (an intrinsic major lens protein) that cause congenital cataracts have been described [59], and few individuals with a lack of function of other AQPs, including AQP3 and AQP7, have been reported. AQP4 polymorphisms in patients with thrombosis of the middle cerebral artery have been detected, apparently with a polymorphism associated with increased cerebral oedema [60]. AQP4 polymorphisms associated with several diseases have been observed, and may act as predisposing factors to sudden death syndrome in childhood [61]. AQP1 polymorphisms are associated with diabetic nephropathy, to priapism in sickle cell disease, and to loss of body water in athletes [62–64]. Likewise, polymorphisms and changes in the expression of AQP7 and AQP9 genes are associated with obesity and type II diabetes [65–67]. Measurement of aquaporins in body fluids may be of diagnostic value; however, currently this is not used. A method that uses urinary AQP1 levels to distinguish between different causes of nephrogenic diabetes insipidus (absent in AQP2 deficiency) or enuresis nocturna has been published [68,69] but its clinical usefulness has not been demonstrated. The same applies to tissue expression in tumours and brain. 4.2. Therapeutic usefulness The function of aquaporins in the kidney suggests that AQP1 and AQP2 inhibitors should reduce urine concentrations, giving rise to a diuresis with more water than salt. AQP4 inhibitors would reduce the brain swelling in cerebral oedema that occurs after spinal cord injury, ischemia and meningitis, theoretically providing neuroprotection and reducing mortality. In tumours, AQPs inhibitors would reduce the migration and expansion of tumour as adjuncts to chemotherapy. Topical inhibitors of AQP1 in the eye would reduce intraocular pressure in glaucoma. Recently, using small-interference RNA technology and a pharmacological inhibitor to knock down the expression of AQP-4, a specific and massive impairment of glioblastoma cell migration and invasion in vitro and in vivo has been demonstrated [70]. In gliomas, corticosteroids help to reduce peritumoural brain oedema to a significant extent. Animal experiments have revealed a decrease in cerebral AQP-4 protein expression upon dexamethasone treatment, suggesting that AQP-4 may be considered one of the major molecular targets of steroid treatment for the formation of brain oedema [71]. To date, no inhibitors with clinical usefulness have been obtained. Several AQPs are inhibited by mercury and gold, but these are nonselective and highly toxic. While other candidates have been proposed as AQP inhibitors, they have not been proved useful. Conversely, components that increase the role of AQPs could reduce body mass in obesity. 5. Neuromyelitis optica Neuromyelitis optica (NMO), also known as Devic's disease, is a severe immune-mediated demyelinating and necrotizing disease that predominantly affects the optic nerves and spinal cord. It has been considered a form of multiple sclerosis in which inflammatory lesions are restricted to the optic nerve and spinal cord, but currently it is considered a distinct clinical entity. NMO causes acute eye pain

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with loss of function and myelitis with symmetric paraplegia, sensory loss, and sphincter dysfunction. In NMO, the lesions in the spinal cord extend over three or more vertebral segments, although they may be shorter in atrophic residual lesions, in relapse or early stages of the disease. They are mainly located in the cervical or thoracic spinal cord with affectation of central gray matter. Magnetic resonance imaging (MRI) shows hyperintensities in stage T2 and hypointensities in stage T1 [72]. These lesions are highly characteristic of NMO and play a central role in diagnosis. In the optic nerve, characteristic images are also seen on magnetic resonance imaging (enhancement with gadolinium in the T1 stage), and abnormal evoked potentials in the electrophysiological evaluation [73]. Recently, magnetic resonance imaging of the central nervous system has shown that injuries occur in up to 79% of patients with NMO and AQP4 antibodies. These injuries are of different types, less characteristic, and develop during the course of the disease after an average of 6 years [74]. In NMO, cerebrospinal fluid (CSF) pleocytosis can be found, with a predominance of monocytes and lymphocytes (14–79%), although it may be dominated by neutrophils and eosinophils. It is mainly mild (median, 19 cells/μl; range 6–380), and frequently includes neutrophils, eosinophils, activated lymphocytes, and/or plasma cells [75]. In 13–35% of patients the cell count exceeds 50 cells/mL, and in some patients 1000 cells/mL. In 46–75% of patients there is an increase in CSF protein with oligoclonal bands absent or not very prevalent (0–37%). If present, intrathecal IgG (and, more rarely, IgM) synthesis is low, transient, and significantly restricted to acute relapses. The albumin CSF/serum ratio, total protein and L-lactate levels are significantly correlated with disease activity [75]. Pleocytosis and blood CSF barrier dysfunction are associated with activity, but in some patients are also present during remission, possibly indicating sustained subclinical disease activity [75,76]. The CSF levels of soluble intercellular adhesion molecule 1 and soluble vascular cell adhesion molecule 1 increased in patients with NMO compared with patients with MS [77], indicating a severe blood–brain barrier breakdown in patients with NMO. Measuring adhesion molecules is useful to evaluate this barrier disruption [77]. As a result of injuries, neurofilament heavy-chain and glial fibrillary acidic protein are released into the CSF, with levels apparently higher than those observed in multiple sclerosis [78]. CSF should be obtained during or shortly after an acute attack. The findings are useful, but not very sensitive or specific. NMO can occur in partial or limited clinical forms that are considered to be part of the clinical spectrum of NMO. Among these limited forms are longitudinally extensive transverse myelitis (LETM) and optic nerve neuritis in its recurrent isolated form (recurrent isolated optic neuritis, RION) or bilateral form (bilateral optic neuritis, BON). Also included within the clinical spectrum of NMO are conditions that develop in the context of a systemic or organ-specific autoimmune disease, including systemic lupus erythematosus, Sjögren syndrome, celiac disease and myasthenia gravis (MG). When NMO and MG coexists, the latter usually precedes NMO (or NMO-limited forms), often by more than a decade. It occurs almost exclusively in Caucasian and non-Caucasian females with juvenile or early-onset MG and frequently after thymectomy [79]. MG usually follows an unusually mild course in these patients. The coexisting of NMO and MG seemed to also be often seen in Asian, especially Japanese along with Caucasian [80]. Testing for AQP4 antibodies should be considered in MG patients presenting with atypical motor or optic symptoms [79]. Finally, in the spectrum of NMO, atypical cases with subclinical or clinically manifest brain lesions could be included. Brain abnormalities are being recognized more frequently in patients with an NMO spectrum disorder, and most brain lesions are accompanied by pre-existing NMO [81]. Anti-aquaporin-4 antibody was measured in the sera of 257 patients with inflammatory diseases of the central nervous system who attended a multiple sclerosis clinic. Eighty-three were seropositive, and

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15 of these presented brain symptoms. The initial manifestations were classified into two groups according to clinical characteristics: encephalopathy mimicking acute disseminated encephalomyelitis or posterior reversible encephalopathy syndrome and characteristic brainstem symptoms such as intractable hiccup and vomiting [81]. Recently, an asymptomatic radiological longitudinally extensive transverse myelitis without symptoms or signs referable to the spinal cord or optic nerves has been associated with a clinical course dominated by severe anorexia and weight loss in a 14-year-old female with positive anti-AQP4 antibody [82]. An implication of brainstem with diplopia, poor appetite and/or prolonged hiccup has been described in 37% of NMO patients with most of these events occurring during the first demyelinating attack [83]. The prevalence of NMO is lower than that of multiple sclerosis and is higher in non-Caucasians. Within demyelizing disorders, in East Asia NMO can affect up to 48% of patients, its prevalence decreasing in African-Brazilians (15%) and Europeans (1.5%) [75]. In Europeans the MS:NMO ratio is 42.7 [84]. The prevalence of NMO in a north European Caucasian population in South East Wales was fourteen Caucasian patients (11 patients with NMO and three with NMO-limited forms) identified in a population of 712,572 (19.6/million; 95% CIs: 12.2–29.7) [85]. The yearly incidence rate of NMO in a Caucasian population from Denmark was estimated to be 0.4 per 10 5 personyears (95% confidence interval [CI] 0.30–0.54) and the prevalence was 4.4 per 10 5 (95% CI 3.1–5.7) [86]. Neuromyelitis optica is more common in women than in men, with a ratio of 3 to 10 women for every male. It usually appears in the third decade of life, but may occur at any age, including childhood and old age. The most usual initial inflammatory event is myelitis in 44–50% (longitudinally extensive transverse myelitis [LETM] in 14–17%), followed by optic neuritis in 28–42%, and concurrent myelitis and optic neuritis in 9-18% of patients [86–88]. The median interval between NMO onset and the time until fulfilment of the 2006 NMO criteria is about 28 months [87]. Recurrent clinical courses are the most frequent (80–90% of patients), with a rapid development of repeated events, with partial recoveries that lead to a progressive deterioration of motor, sensory, visual, intestinal and urinary bladder functions and ultimately to death from respiratory failure, often neurogenic. Single-phase (10–20%) or progressive (rare) forms of NMO are less frequent. Due to the severity of NMO attacks and the high risk for disability, treatment should be implemented as soon as diagnosis is confirmed. Acute NMO attacks are usually treated with high-dose intravenous corticosteroid pulses and plasmapheresis. Maintenance therapy is also required to avoid further attacks and this is based on low-dose oral corticosteroids and non-specific immunosuppressant drugs such as azathioprine and mycophenolate mofetil. New therapeutic strategies using monoclonal antibodies, such as rituximab, have been tested in NMO, with positive results in open-label studies [89].

5.1. Pathogenic mechanisms Cases of anti-AQP4 antibody-positive familial NMO in mothers and daughters have been described [90] and these cases may help in our understanding of the genetic contribution to NMO. The HLA background in Caucasian patients (Spanish cohort) shows an increased frequency of the DRB1*03 allele (OR = 2.27; 95% CI = 1.44–3.58; p b 0.0008) in NMO, which has been related to AQP4 antibody seropositivity (OR = 2.74; 95% CI = 1.58–4.77; p b 0.0008) [91]. Regarding multiple sclerosis, NMO has been associated with an increased frequency of the DRB1*10 allele (odds ratio, OR= 15.1; 95% confidence interval, 95% CI= 3.26–69.84; p = 0.012), having a different HLA-DRB1 allelic distribution [91]. In Asian populations different HLA alleles associated with NMO, such as DRB1*1602 and DPB1*0501, have been described [92]. Genetic analysis of AQP4 single-nucleotide polymorphisms in NMO

provides no support for the hypothesis that genetic variation in AQP4 accounts for the overall susceptibility to NMO [93]. In mice, in vivo intravenous administration of a recombinant monoclonal human IgG anti-AQP4 has shown that it localizes to AQP4-expressing cell membranes in kidney (collecting duct), skeletal muscle, trachea (epithelial cells) and stomach (parietal cells), and astrocytes in the brain (area postrema). However, NMO pathology has CNS specificity. Following intracerebral injection of anti-AQP4, this targets astrocyte foot-processes [94]. In NMO, it has been shown that in spinal cord lesions there is a marked loss of immunoreactivity against AQP4 in astrocytes, whereas this reactivity is preserved in multiple sclerosis [95,96]. Along with AQP4, there is a parallel loss of fibrillary acidic protein of glia, which is a marker of astrocytes. In CSF, this protein, as well as other proteins such as S100B, indicates a severe injury to astrocytes in the phases of activity of NMO; this injury is more pronounced than in multiple sclerosis. Although markers of neuronal destruction and demyelination, such as neurofilament protein and myelin basic protein, are also detected, damage to astrocytes appears to be greater than neuronal demyelination [97]. In NMO lesions, there is also a perivascular deposition of immunoglobulins (IgG and IgM) and complement. Immunoglobulins are arranged around blood vessels and activate complement, suggesting their pathogenic involvement in NMO [95,96]. In the inflammatory infiltrates there is a predominance of neutrophils and eosinophils, which usually accompanies a type-2 helper (Th2) cellular immune response. Cytokines that promote B cell activation and antibody production, such as B cell activation factor and interleukin 6, are elevated in the serum and CSF of patients with NMO [98–100], while the Th1-type cytokines (interleukin 2, interferon gamma or tumour necrosis factor) are not increased in CSF [98,99]. Uzawa et al. have measured 27 cytokines/chemokines in the CSF of NMO and MS patients [101]. The CSF levels of IL-1 receptor antagonist, IL-6, IL-8, IL-13 and granulocyte colony-stimulating factor were significantly increased in NMO, while IL-9, fibroblast growth factor-basic, granulocyte macrophage colony-stimulating factor, macrophage inflammatory protein-1-beta and tumour necrosis factor-alpha were increased in MS [101]. In serum analyses, only the IL-6 level showed significant elevation [101]. This findings support the view that different immunology and pathophysiologies exist between them. There is also growing evidence that IgG removal by plasmapheresis exerts a beneficial effect on the disease [102,103]. Passive transfer of anti-AQP4 to animal models with experimental autoimmune encephalomyelitis exacerbates the disease and replicates the histopathological lesions of NMO [104]. Consistent with the pathogenic role of AQP4 autoantibodies, an intrathecal humoral immune response against AQP4 at the onset of clinical disease has been detected. Furthermore, recombinant antibodies generated from clonally expanded plasma cells in NMO CSF are AQP4-specific and immunopathologic [105]. However, injection of AQP4 to animals without encephalitis does not seem dangerous. An inflammatory brain context such as that found in experimental encephalomyelitis might be required for the injection of AQP4 to become pathogenic [106,107]. In humans, a CNS infection or a general infection with neurotropic viruses could generate this context. A history of viral infections is detected in 15–35% of patients with NMO [108]. Also, in a patient with the disease and prostate adenocarcinoma after a pneumococcal polysaccharide vaccination NMO relapsed, suggesting that activation of the immune system by adenocarcinoma and complement activation induced by vaccination could be involved in the onset and relapse of NMO [109]. Recent evidence has suggested that what is really important is complement. Thus, intracerebral administration of immunoglobulin G from neuromyelitis optica patients and human complement produced neuromyelitis optica-like lesions in mice, including inflammation, a loss of AQP4 expression, perivascular deposition of activated complement components, extensive demyelination, loss of reactive astrocytes and neuronal cell death [110]. Probably, in this experimental encephalomyelitis model there is a blood–brain

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barrier breakdown and encephalomyelitis in itself is not a prerequisite for anti-AQP4 to exert its pathogenic effect. The role of T cells on NMO pathology remains to be elucidated, although recently it has been shown that AQP4-specific T cells react against astrocytes of the limiting glia, induce inflammation, disrupt the blood brain barrier, and allow the entry of pathogenic anti-AQP4 [111]. T cells are not required for the formation of NMO lesions in the mouse model of NMO produced by intracerebral injection of IgG antiAQP4 and complement [112]. T cells and other cell-mediated mechanisms could cause the antibody-independent AQP4 astrocytopathy that appears to occur in demyelinating conditions, including Baló's disease (a variant of multiple sclerosis), some patients with NMO, and opticospinal multiple sclerosis [113]. Anti-AQP4 antibodies initiate endocytosis of antigen (AQP4) and its incorporation into endosomes, with probable final degradation. Thus, they decrease the expression of AQP4 in astrocyte membranes, and possibly alter the homeostasis of water and cause oedema. However, it has recently been shown that anti-AQP4 binding impairs water flux directly, independently of antigen down-regulation [114]. In any case, NMO is an inflammatory demyelinating disease in which there is oedema in the spinal cord and optic nerves [115–117]. AQP4 forms a macromolecular complex with Na+-dependent excitatory amino acid transporter 2 (EAAT2) [118]. Thus, anti-AQP4 antibodies will reduce both astrocyte membrane proteins in a coupled manner. This is followed by a decrease of glutamate uptake, its extracellular concentration increasing. This increase results in neuron excitotoxicity, especially in oligodendrocytes that express glutamate receptors, some of which, when activated, sensitize oligodendrocytes to complement attack [119]. There is circumstantial evidence of a direct cytotoxic effect of antiAQP4 on astrocytes. Anti-AQP4 antibodies are of the IgG1 subclass, and also possibly IgG4, and can activate complement. In transfected cells expressing AQP4 in the membrane, antibody binding and complement deposition can be observed. In vivo, AQP4 tends to aggregate, offering the largest number of antigens, making it easier for the antibodies with higher affinity to join to the cell surface, forming oligomers and activating complement. The binding to extracellular epitopes of densely packed AQP4 aggregates activates complement more effectively than the binding to dispersed AQP4 tetramers. Larger size aggregates are induced by anti-AQP4 binding favouring more complement activation [114]. Complement activation induces lytic lesions in astrocyte cell membranes even though these cells are able to express complement regulatory proteins in their membranes to increase their resistance to it. Therefore, the binding of anti-NMO antibodies and complement active molecules appears to increase astrocyte apoptosis [119]. In sum, in astrocytes anti-QAP4 IgG binding to AQP4 is thought to initiate a cascade of inflammatory events, including antibody-dependent complement and cell-mediated damage, leukocyte recruitment, cytokine release and demyelination. Thus, anti-AQP4 antibodies produce CNS inflammation, demyelination, and oedema. They seem to be able to disrupt the blood brain barrier and to contribute to the damage of astrocytes and oligodendrocytes. 6. Anti-AQP4 antibodies In 2004, Lennon et al. found anti-AQP4 antibodies in the serum of individuals with NMO and related disorders, but not in the serum of subjects with multiple sclerosis and normal individuals. Therefore, these antibodies are also called anti-NMO [120]. Anti-AQP4 antibodies are directed against epitopes located in extracellular loops of AQP4 (Fig. 2) [121]. Specifically, anti-AQP4 antibodies recognize an epitope located in the E outer loop of AQP4, which is considered the major epitope of anti-AQP4, although intracellular and conformational epitopes (when AQP4 aggregates) have been described [122,123]. Like other AQPs, AQP4 is grouped; forming tetramers and even larger groups as orthogonal arrays (OAPs) of different size that are maintained by inter-tetrameric N-terminal interactions involving specific residues

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[124]. OAPs are an ultrastructural characteristic of membrane specializations, facing sites of rapidly fluxing potassium ions and water (Fig. 3). There are two isoforms of AQP4, M1-AQP4 and M23-AQP, which differ in size. In rats, but not in humans or mice, there is a longer Mz-AQP4 isoform with similar characteristics to M1-AQP4 [125]. M1AQP4 has 22 more amino acids at the cytoplasmic N terminus, while the M23-AQP4 isoform is slightly shorter and tends to form larger OAPs. The proportion of both isoforms varies among OAPs [126,127]. M1, without M23, does not form arrays but exists as individual tetramers [128]. In the absence of M1, M23 tetramers form high-order arrays in the plasma membrane. When coexpressed, interacting N termini of M1 and M23 form heterotetramers that limit the M23 array size to approximately the size of OAPs in astrocytic membranes [129]. The two isoforms have identical extracellular residues. AQP4 antibodies recognize both isoforms, although a study published in 2009 postulated that there are antibodies that recognize conformational epitopes that are formed exclusively on OAPs [130]. Recently, it has been observed that there are at least two conformational epitopes on OAPs and that anti-AQP4 antibodies vary in their form of union. Anti-AQP4 antibodies bind with greater affinity to M23-AQP4 than to M1-AQP4, apparently contributing to the bivalent binding of IgG resulting from the supply of antigen and structural changes in the major epitope occurring in the AQP4 of OAPs [123,130]. However, Crane et al. consider that the structural changes in the AQP4 epitope upon array assembly, and not bivalent cross-linking of whole IgG, are responsible for in the greater binding affinity to OAPs [129]. Despite these observations, the majority of diagnostic tests use M1-AQP4 as antigen, with acceptable sensitivity and specificity. Anti-AQP4 antibodies can be detected years before the clinical onset of NMO [131]. In spinal cord and optic nerve injury the presence of antiAQP4 predicts a later conversion to final NMO. Fifty to sixty percent of patients with a single episode of LETM, with positive anti-AQP4 antibodies, evolved into relapse or optic neuritis during the first year after presentation [132], whereas in patients with RION the positivity of the anti-NMO predicted the development of NMO and was also associated with a more severe visual impairment [133]. In one study, 50% of patients with acute monosymptomatic optic neuritis and the presence of anti-AQP4 evolved to NMO during the first year of evolution [134]. In a retrospective study of 273 patients with inflammatory CNS demyelination, anti-AQP4 was negative in persistent monophasic NMO patients, while in 74% of samples of recurrent cases it was positive, and remained positive during follow-up [135]. Despite our incomplete knowledge of anti-AQP4 in the pathogenesis of NMO, its detection has opened an important field in the diagnosis of the disease. The seropositivity of anti-AQP4 is reasonably sensitive (74%) and specific (> 90%) for NMO, allowing early diagnosis and permitting a distinction between NMO and MS [136]. In general, anti-AQP4 has been detected in 60–90% of patients with NMO and less frequently in limited forms such as LETM or RION (disorders of the spectrum of NMO), and other forms of optic neuritis, but not in multiple sclerosis [87,119,137]. Also, anti-AQP4 antibodies have been described less frequently in patients with brain stem encephalitis (affecting mainly the medulla oblongata), diencephalitis (mainly affecting the hypothalamus) and posterior reversible encephalopathy than the pathophysiological events they seem to share with NMO [138]. The high specificity of the anti-AQP4 for the syndrome is also seen in patients with connective tissue diseases or vasculitis that are only positive when NMO spectrum disorders coexist [79,139]. Anti-AQP4 antibodies are included in the revised criteria of the NMO [140] and the NMSS task force criteria for the differential diagnosis of multiple sclerosis [141]; in both cases as minor or supporting criteria (Table 2). The European Task Force has developed a diagnostic guide for disorders of the clinical spectrum of NMO (LETM, RION / BON) that also includes anti-AQP4 antibodies (Fig. 4) [75]. Evidence-based guidelines consider testing anti-AQP4 useful to determine the cause of transverse myelitis in patients presenting with clinical acute complete transverse

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a

membrane

b

*

Gas

Fig. 3. Aggregation of AQP4 in the cell membrane. a. Schematic representation of the AQP4 tetramer. Each AQP4 monomer transports water and via the central pore of the tetramer is thought to conduct gas. b. AQP4 monomers are shown as assembling into tetramers (top) or OAPs (bottom).* OAPs within the plasma membrane by electronic microscopy.

myelitis and are considered useful for the prediction of the risk of TM reoccurrence [142]. There is growing evidence correlating high levels of anti-NMO with greater NMO severity [143,144]. In a recent study of 36 NMO patients, AQP4 antibodies were absent in the monophasic NMO individuals, while were positive in 74% of recurrent NMO cases and remained positive during follow-up. It also appears that the concentration of anti-NMO increases in both phases of activity, although there is a considerably variability among individuals and no general threshold to predict relapse can be established [136]. In patients treated with drugs that cause B-cell depletion, an increase in CD19+ cells appears to increase the risk of relapse [136]. However, it remains to be determined whether anti-AQP4 antibodies are a reliable marker of disease activity or response to treatment. Recent studies have indicated that autoimmune responses to myelin oligodendrocyte glycoprotein can induce an NMO-like disease in experimental animal models, and an MOG-IgG-mediated immune response in a subset of AQP4-IgG seronegative patients with NMO and disorders of the spectrum of NMO has also been described [145]. Also, the paraneoplasic antibodies to CV2/CRMP5 have been described in a few AQP4-Ab-negative NMO patients [146]. 7. Anti-AQP4 assays At least 15 methods for assaying anti-AQP4 antibodies have been reported. The sensitivity varies widely between 33 and 91% (median 63%), while the overall specificity remains high (85% to 100% with a median of 99%) [119]. Methods depending on the substrate can be divided into indirect immunofluorescence or immunohistochemistry using tissue sections as substrate. Sensitivity is variable (38–87%); the methods are semiquantitative (titres) and depend on the observer's experience. These methods can use cells transfected with M1-AQP4 isoforms and/or M23-AQP4 and analyzed by indirect immunofluorescence (Fig. 5), immunocytochemistry, flow cytometry and quantitative cell-based assays [147]. Sensitivity varies between 42% and 91%, with specificities between 94% and 100%. It appears that M23 AQP4expressing cells are superior and a recent study of human serum samples reported an improvement from 70% to 97% in sensitivity for NMO-IgG upon using M23-expressing cells instead of M1-expressing cells [148]. HEK293 cells transfected with full-length recombinant human AQP4 are 78% sensitive for patients with clinically defined NMO and 71% for all patients with NMO syndrome [134]. When cell lysates are used, Western blot or immunoprecipitation (with or radiologic marker enzyme) are employed. Sensitivity ranges between 57 and 81% and specificity between 91% and 100%. Finally, there are ELISAs and on-line blots or dots using purified AQP4, usually from rodents, with a sensitivity around 67% and a specificity close to 90% (87%). Recently, recombinant antigens have been described with similar or better results than with purified antigens. ELISAs using recombinant M1 or M23 isoforms of human AQP4 have

the same sensitivity, although the M23-AQP4 ELISA resulted in an improved signal-to-noise ratio. The sensitivity of the M23-AQP4 assay was 72% (95% CI 64.9–75.5) in defined NMO, and 55% (95% CI 42.6–62.2) in the NMO spectrum. Specificity in both defined and high-risk NMO was 98% (95% CI 94.8–99.0, 95.2–98.9, respectively) [149]. Recently, 14 high-affinity linear and conformational peptides have been identified. These peptides represent epitopes of the AQP4 autoantigen. The linear peptides shared sequence homologies with the AQP4 autoantigen on the extracellular surface [150]. At present, there is no standard test, and for this reason it might be appropriate and reasonable to analyze each sample by means of two independent assays when there is a high clinical suspicion of NMO. The analysis could be repeated in the patients for whom the result is

Table 2 Diagnostic criteria of neuromyelitis optica. 1. The 2006 revised NMO diagnostic criteria [137] Absolute criteria 1. Optical neuritis 2. Myelitis Supportive criteria 1. Evidence of a contiguous spinal cord lesion that consists of three or more segments in length on magnetic resonance imaging (MRI). 2. Negativity for the diagnostic criteria for multiple sclerosis on brain MRI scans conducted at onset. 3. NMO Immunoglobulin G (or anti-AQP4 antibody) seropositivity. NMO diagnostic requires the presence of absolute criteria and at least two of the three supportive criteria. Sensitivity: 87.5% and specificity: 83.3%a 2. Miller's NMO criteria in the differential diagnosis of multiple sclerosis [138] Major criteria 1. Optical neuritis in one or two eyes. 2. Transverse myelitis, clinically complete or incomplete, but associated with radiological evidence of spinal cord lesion extending over three or more spinal segments on T2-weighted MRI images and hypointensities on T1-weighted images when obtained during acute episode of myelitis. 3. No evidence for sarcoidosis, vasculitis, clinically manifest systemic lupus erythematosus or Sjögren´s syndrome, or other explanation for the syndrome. Minor criteria 1. Most recent brain MRI scan of the head must be normal or may show abnormalities not fulfilling the Barkhof criteria used for McDonald diagnostic criteria including: a. Non-specific brain T2-signal abnormalities not satisfying the Barkhof criteria for dissemination in space used in the revised McDonald criteria. b. Lesions in the dorsal medulla, either in contiguity or not in contiguity with a spinal cord lesion. c. Hypothalamic and/or brainstem lesions d. Linear periventricular/corpus callosum signal abnormality, but not ovoid, not extending into the parenchyma of the cerebral hemispheres in Dawson finger configuration. 2. Positive test in serum or CSF for NMO-IgG/AQP4 antibodies. All major criteria are required (although they may be separated by an unspecified interval) and at least one of the minor criteria. a A. Saiz, L. Zuliani, Y. Blanco, B. Tavolato, B. Giometto, F. Graus; Spanish‐Italian NMO Study Group, Revised diagnostic criteria for neuromyelitis óptica (NMO). Application in a series of suspected patients, J. Neurol. 254 (2007) 1233–1237.

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LETM1 or RION/BON2

Serum anti-AQP4 IgG

Positive

Definitive LETM o RION/BON

Negative

MRI of brain/spinal cord espinal and CSF Criteria fullfilled3

Probable LETM o RION/BON4 Fig. 4. Algorithm recommended by the EFNS (European Federation of Neurological Societies) for the diagnosis of the limited forms of the NMO spectrum.1The diagnosis of LETM is based on a medullary syndrome in combination with lesions upon MRI in three or more vertebral segments.2The diagnosis of optic neuritis (RION/BON) is based on clinical, ophthalmologic examinations and visual evoked potentials.3Criteria: Negative brain MRI images or brain lesions typical of NMO and negative oligoclonal bands or no pathological IgG intrathecal synthesis.4Pathological evoked visual potentials (for LETM) or evoked somatosensorial potentials (for RION/BON) are further supportive of probable NMO spectrum disorder if criteria3 are fulfilled.

negative, especially when in remission or under immunosuppressive therapy. Monitoring can be useful in patients who are on immunosuppressive therapy since relapses are often preceded by increased levels of anti-AQP4 antibodies. Of 38 patients with NMO spectrum disorders who were initially seropositive, 21 became seronegative under effective immunosuppressive therapy, and 17 continued to be seropositive. During most relapses, the serum AQP4-Ab levels were either higher or increasing compared with previous levels. However, rising antibody levels did not always lead to acute exacerbation. AQP4-Ab titres correlated with B cell counts during therapy with anti-CD20 (B cell depletion) [151]. Repeated treatment with anti-CD20 based on the assessment of peripheral circulating memory B cells appeared to produce consistent and sustained efficacy in patients with relapsing NMO [151]. Currently, there is no indication to analyze the anti-AQP4 antibodies of the IgM isotype. They are present in almost 10% of patients with NMO but practically all AQP4-IgM-positive patients are also positive for AQP4-IgG, and none of the AQP4-IgG-negative samples were positive for AQP4-IgM [152]. Nor is it clear whether CSF anti-AQP4 antibodies should be analyzed. In general, the intrathecal synthesis

Fig. 5. Anti-AQP4 antibodies by indirect immunofluorescence using M1-AQP4-transfected HEK293 cells as substrate in a patient with NMO.

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of antibodies is low, but a few patients with anti-AQP4 exclusively in CSF have been detected [153–155]. Therefore, perhaps it could be analyzed in CSF, when seronegativity or low serum concentrations are detected, when clinical suspicion is high in negative serum and in clinical NMO spectrum disorders. Finally, we wish to point out that the lack of sensitivity of the analytical methods cannot exclude NMO in seronegative patients. 8. Conclusion AQPs are a family of protein channels that allow the movement of water, gas, glycerol and other molecules. AQP4 is the one that has gained the greatest clinical relevance; it is abundantly expressed in the brain, where it regulates the movement of water into and out of it, and it is the target of an autoimmune response in NMO and other related syndromes. NMO is a rare disease that is difficult to diagnose and has atypical partially clinical forms. Differential diagnostic problems with multiple sclerosis often cause a delay of more than two years after the onset of symptoms. Anti-AQP4 autoantibodies are responsible for this pathology. It has been shown that anti-AQP4 IgG binding to AQP4 has inflammatory effects, including antibody-dependent complementand cell-mediated damage, leukocyte recruitment, cytokine release and demyelination, leading to CNS inflammation, demyelination and oedema. Anti-AQP4 antibodies recognize the two AQP4 isoforms (M1-AQP4 and M23-AQP4) but seem to have a greater affinity for the M23-AQP4 to form larger groups such as orthogonal arrays of OAPs. Anti-AQP4 autoantibodies can be detected years before the clinical onset of NMO and have predictive value in the initial partial clinical forms of progression to the full clinical picture of NMO. Anti-AQP4 autoantibodies have diagnostic usefulness and reasonable sensitivity (~74%) and, especially, they have high specificity (>90%). They have been included in the latest diagnostic criteria of NMO and disorders of the clinical spectrum of NMO. Recent evidence points to a possible prognostics and monitoring of the disease. Finally, there are different methods for the analysis of antiAQP4, although there is no standard method. The ones used most commonly in clinical practice are indirect immunofluorescence and ELISA in serum, and occasionally in CSF. References [1] 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. [2] 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 U S A 1991;88:11110–4. [3] Agre P, Preston GM, Smith BL, et al. Aquaporin CHIP: the archetypal molecular water channel. Am J Physiol 1993;265:F463–76. [4] Nielsen S, Pallone T, Smith BL, Christensen EI, Agre P, Maunsbach AB. Aquaporin-1 water channels in short and long loop descending thin limbs and in descending vasa recta in rat kidney. Am J Physiol 1995;268:F1023–37. [5] King LS, Nielsen S, Agre P, Brown RH. Decreased pulmonary vascular permeability in aquaporin-1-null humans. Proc Natl Acad Sci U S A 2002;99:1059–63. [6] Oshio K, Watanabe H, Song Y, Verkman AS, Manley GT. Reduced cerebrospinal fluid production and intracranial pressure in mice lacking choroid plexus water channel Aquaporin-1. FASEB J 2005;19:76–8. [7] Deen PM, Weghuis DO, Geurs van Kessel A, Wieringa B, van Os CH. The human gene for water channel aquaporin 1 (AQP1) is localized on chromosome 7p15–>p14. Cytogenet Cell Genet 1994;65:243–6. [8] 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. [9] 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. [10] 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 U S A 1994;91:13052–6. [11] Kozono D, Yasui M, King LS, Agre P. Aquaporin water channels: atomic structure molecular dynamics meet clinical medicine. J Clin Invest 2002;109:1395–9. [12] Herrera M, Garvin JL. Aquaporins as gas channels. Pflugers Arch 2011;462:623–30. [13] Otto B, Uehlein N, Sdorra S, et al. Aquaporin tetramer composition modifies the function of tobacco aquaporins. J Biol Chem 2010;285:31253–60. [14] Skelton LA, Boron WF, Zhou Y. Acid–base transport by the renal proximal tubule. J Nephrol 2010;23:S4–S18.

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