Aquaporin-4 Protein Research Articles

Aquaporin-4 protein expression in normal canine brains

BackgroundAquaporin-4 (AQP4) is in growing recognition as potential marker for cancer progression, differentiation and therapeutic intervention. No information is available about AQP4 expression in the normal canine brain. The aim of this histopathological study is to confirm the presence of AQP4 by immunohistochemistry technique in a group of non-pathological canine brains and to describe its expression and distribution across the brain.ResultsTwelve non-pathological canine brains of various ages (ranging from 21 days to 17 years) and breeds were included in the study. Immunohistochemical expression of AQP4 was analyzed using formalin-fixed paraffin-embedded brain tissue sections. The findings were correlated between AQP4 expressing cells and astrocytes using glial fibrillary acidic protein (GFAP). AQP4 expression was more marked in the astrocyte foot processes of subpial, perivascular and periventricular surfaces in all specimens. The majority of the canine brain sections (9/12) presented with an AQP4 predilection for white matter tracts. Interestingly, the two youngest dogs (21 days and 3 months old) were characterized by diffuse AQP4 labelling in both grey and white matter tracts. This result may suggest that brain development and ageing may play a role in the AQP4 distribution throughout the canine brain.ConclusionsThis is the first study to describe immunohistochemical distribution of AQP4 in normal canine brains. The AQP4 expression and distribution in non-pathological canine brains was comparable to other species. Larger studies are needed to substantiate the influence of breed and ageing on AQP4 expression in the normal canine brain.

Activation of TGR5 restores AQP2 expression via the HIF pathway in renal ischemia-reperfusion injury.

Renal ischemia-reperfusion (I/R) injury is associated with markedly reduced protein expression of aquaporins (AQPs). Membrane G protein-coupled bile acid receptor-1 (TGR5) has shown protective roles in some kidney diseases. The purpose of the current study was to investigate whether activation of TGR5 prevented the decreased protein expression of AQPs in rodents with renal I/R injury and potential mechanisms. TGR5 agonist lithocholic acid (LCA) treatment reduced polyuria after renal I/R injury in rats. LCA prevented the decreased abundance of AQP2 protein and upregulated hypoxia-inducible factor (HIF)-1α protein expression, which were associated with decreased protein abundance of NF-κB p65 and IL-1β. After renal I/R, mice with tgr5 gene deficiency exhibited further decreases in AQP2 and HIF-1α protein abundance and increases of IL-1β and NF-κB p65 protein expression compared with wild-type mice. In primary cultured inner medullary collecting duct cells with hypoxia/reoxygenation, LCA induced markedly increased protein expression of AQP2 and HIF-1α, which were partially prevented by the PKA inhibitor H89. FG4592, a prolyl-4-hydroxylase domain-containing protein inhibitor, increased HIF-1α and AQP2 protein abundance in association with decreased NF-κB p65 protein expression in inner medullary collecting duct cells with hypoxia/reoxygenation. In conclusion, TGR5 stimulation by LCA prevented downregulation of renal AQPs in kidney with I/R injury, likely through activating HIF-1α signaling and suppressing inflammatory responses.NEW & NOTEWORTHY Stimulation of the membrane G protein-coupled bile acid receptor TGR5 by lithocholic acid (LCA) reduced polyuria in rats with renal ischemia-reperfusion (I/R) injury. LCA increased abundance of aquaporin-2 (AQP2) protein and upregulated hypoxia-inducible factor (HIF)-1α protein expression in association with decreased NF-κB p65 and IL-1β. After I/R, mice with tgr5 gene deficiency exhibited more severe decreases in AQP2 and HIF-1α protein abundance and inflammatory responses. TGR5 activation exhibits a protective role in acute renal injury induced by I/R.

Study of the rejection of contaminants of emerging concern by a biomimetic aquaporin hollow fiber forward osmosis membrane

Forward osmosis (FO) plays an increasingly important role in membrane processes because of its advantages compared to traditional pressure-driven membrane processes. There are different types of water-selective FO membranes. In this study, a biomimetic hollow fiber module comprising an active layer of polyamide thin film composite (TFC) with integrated aquaporin proteins and an effective area of 0.6 m2 is used to study the rejection of 24 Contaminants of Emerging Concern (CECs). The rejections obtained for all the contaminants studied were higher than 93 % and for 19 of them rejections of up to 99 % were reached. It was observed that although all the tested compounds showed rejections very close to 100 %, they were not completely recovered in the feed solution which makes the retention within the membrane an important factor to be considered. Hence, two membrane rinses were necessary after each membrane operation to completely recover each contaminant. The results were analyzed considering the physicochemical properties (molecular weight, charge and hydrophobicity) of the contaminants.

Satralizumab: an interleukin-6 (IL-6) receptor antagonist for the treatment of neuromyelitis optica spectrum disorders.

Neuromyelitis optica spectrum disorders (NMOSD) consist of a rare autoimmune disorder in which patients suffer from relapses that affect the optic nerve, spinal cord or brainstem. Few have a full recovery. NMOSD is more common in women, the age of onset being around 30-40 years of age depending on race. The prevalence of the condition varies from 0.5-4.4 per 100,000 population. About 80% of patients have antibodies directed against the aquaporin-4 (AQP4) protein that form membrane-bound water transporters in the central nervous system (CNS). This protein is highly expressed in those areas of the CNS often targeted in NMOSD relapses. Satralizumab is a humanized monoclonal antibody that binds to the interleukin-6 (IL-6) receptor and thus inhibits IL-6 signaling. Two recent phase III studies have demonstrated that satralizumab significantly reduced the relapse rate in NMOSD by 76-79%. This beneficial effect was apparently confined to patients who have anti-AQP4 antibodies and satralizumab did not reduce the rate of pain or fatigue in NMOSD patients.

Comparison of content-toxicity-activity of six ingenane-type diterpenoids between Euphorbia kansui before and after stir-fried with vinegar by using UFLC-MS/MS, zebrafish embryos and HT-29 cells

The dried roots of Euphorbia kansui (EK) are especially beneficial for the treatment of edema, but the severe toxicity limits their clinical applications. Euphorbia kansui stir-fried with vinegar (VEK) is traditionally employed to reduce the toxicity of EK. However, the material basis for the toxicity reduction with effectivity conservation is still unclear. Therefore, in this study, a rapid, sensitive, and reliable ultra-fast liquid chromatography tandem mass spectrometry (UFLC-MS/MS) method was firstly established to simultaneously determine six ingenane-type diterpenoids, i.e. kansuiphorin C (1), 5-O-benzoyl-20-deoxyingenol (2), 20-deoxyingenol (3), 3-O-(2’E,4’E-decadienoyl)-20-O-acetylingenol (4), 20-O-(2’E,4’Z-decadienoyl)ingenol (5), and ingenol (6), in EK and VEK based on the processing conversion. Then, the toxicity evaluation on zebrafish embryos and modulation of the expression of aquaporin-3 (AQP3) proteins in HT-29 cells were employed to investigate the toxicity-activity of six compounds. Chromatographic separation was obtained on Waters BEH RP18 column (2.1 mm × 100 mm, 2.5 μm) with the mobile phase composed of 0.1 % formic acid in acetonitrile and water, respectively. The column temperature was 35 ℃ at a flow rate of 0.4 mL min−1. Multiple reaction monitoring was conducted in both positive and negative modes for quantitative analysis. The method was then successfully used for the determination of six compounds in EK and VEK. In addition, 1, 2, 4, and 5 had evident cardiotoxicity, intestinal irritation and nutrient absorption disorders on zebrafish larvae, while no in-vivo toxicity was seen for groups given 3 and 6 (LC50 > 200 μM). Meanwhile, 1, 2, 4, 5, and 6 significantly increased the expression of AQP3 protein (p < 0.05) to promote the excretion of water in the colon. This study demonstrated that toxic ingenane-type diterpenoids converted into the less toxic compounds with the same core structure through the breakage of multiple ester bonds in the side chain. At the same time, the laxative effect was retained, providing useful information for the optimization of the process of EK and quality evaluation of other similar toxic Chinese herbal medicines.

Could Galectin-3 be a key player in the etiology of neuromyelitis optica spectrum disorder?

Neuromyelitis Optica Spectrum Disorder (NMOSD) is a chronic, inflammatory, demyelinating disorder of the central nervous system (CNS) characterized primarily by transverse myelitis (TM) and optic neuritis (ON). Serum antibodies of the IgG class to the water channel protein aquaporin-4 (AQP4) are associated with NMOSD in most cases. These antibodies are thought to cause functional abnormality or changed expressional pattern of AQP4 channel proteins in the CNS lesions.Activation of microglia is one of the chief antibody-mediated effects in NMOSD and it has opposing detrimental and protective effects in NMOSD. On the one hand, it promotes neuroinflammation, demyelination and BBB breakdown. On the other, it aids in remyelination. What controls the switch between these effects is unknown.Recently, Galectin- 3, a lectin, has been identified as a key player in several neurodegenerative diseases. In transient focal brain ischemia, alzheimer’s disease (AD), huntington disease (HD), and experimental autoimmune encephalitis (EAE), Galectin-3 promotes microglia-mediated inflammation.Conversely, in amyotrophic lateral sclerosis (ALS), Galectin-3 reduces inflammation. It also suppresses Th17 cytokines, which play a crucial role in NMOSD pathogenesis. Being devoid of a leader signal, Gal-3 localizes in different cellular compartments and is subject to various post-translational modifications. These reasons explain why Galectin-3 expression has opposing effects under different physiological conditions. Microglia-mediated inflammation in NMOSD has not been extensively studied. The factors that regulate microglia-mediated inflammation in NMOSD are unknown. Here, I hypothesize that Galectin-3 might be an etiological factor in NMOSD that regulates microglia-mediated inflammation. Analysing the role of Gal-3 in NMOSD could help in the development of novel therapies to treat NMOSD.

Human Pluripotent Stem Cell-Derived Kidney Organoids with Improved Collecting Duct Maturation and Injury Modeling

Maximizing the potential of human kidney organoids for drug testing and regenerative medicine and to model development and disease requires addressing cell immaturity, the lack of a mature collecting system, and off-target cell types. By independently generating two kidney progenitor cell populations-metanephric mesenchyme and ureteric bud (UB)-like cells-we could generate kidney organoids with a collecting system. We also identify the hormones aldosterone and arginine vasopressin (AVP) as critical to promote differentiation of collecting duct cell types including both principal cells (PCs) and intercalated cells (ICs). The resulting PCs express aquaporin-2 (AQP2) protein, which undergoes translocation to the apical membrane after vasopressin or forskolin stimulation. By single-cell RNA sequencing (scRNA-seq), we demonstrate improved proximal tubule maturation and reduced off-target cell populations. We also show appropriate downregulation of progenitor cell types, improved modeling of tubular injury, the presence of urothelium (Uro), and the ability of Notch pathway modulation to regulate PC:IC ratios during organoid development.

Controversial Regulation of Gene Expression and Protein Transduction of Aquaporins under Drought and Salinity Stress.

Enhancement of the passage of water through membranes is one of the main mechanisms via which cells can maintain their homeostasis under stress conditions, and aquaporins are the main participants in this process. However, in the last few years, a number of studies have reported discrepancies between aquaporin messenger RNA (mRNA) expression and the number of aquaporin proteins synthesised in response to abiotic stress. These observations suggest the existence of post-transcriptional mechanisms which regulate plasma membrane intrinsic protein (PIP) trafficking to the plasma membrane. This indicates that the mRNA synthesis of some aquaporins could be modulated by the accumulation of the corresponding encoded protein, in relation to the turnover of the membranes. This aspect is discussed in terms of the results obtained: on the one hand, with isolated vesicles, in which the level of proteins present provides the membranes with important characteristics such as resistance and stability and, on the other, with isolated proteins reconstituted in artificial liposomes as an in vitro method to address the in vivo physiology of the entire plant.

Aquaporins and cation transporters are differentially regulated by two arbuscular mycorrhizal fungi strains in lettuce cultivars growing under salinity conditions

The aim was to identify the effects of AM symbiosis on the expression patterns of genes associated with K+ and Na+ compartmentalization and translocation and on K+/Na+ homeostasis in some lettuce (Lactuca sativa) cultivars as well as the effects of the relative abundance of plant AQPs on plant water status. Two AM fungi species (Funneliformis mosseae and Claroideoglomus lamellosum) isolated from the hyper-arid Atacama Desert (northern Chile) were inoculated to two lettuce cultivars (Grand Rapids and Lollo Bionda), and watered with 0 and 60 mM NaCl. At 60 days of plant growth, the AM symbiotic development, biomass production, nutrient content (Pi, Na+, K+), physiological parameters, gene expressions of ion channels and transporters (NHX and HKT1), and aquaporins proteins abundance (phosphorylated and non-phosphorylated) were evaluated. Salinity increased the AM root colonization by both inocula. AM lettuce plants showed an improved growth, increased relative water content and improved of K/Na ratio in root. In Grand Rapids cultivar, the high efficiency of photosystem II was higher than Lollo Bionda cultivar; on the contrary, stomatal conductance was higher in Lollo Bionda. Nevertheless, both parameters were increased by AM colonization. In the same way, LsaHKT1;1, LsaHKT1;6, LsaNHX2, LsaNHX4, LsaNHX6 and LsaNHX8 genes and aquaporins PIP2 were up-regulated differentially by both AM fungi. The improved plant growth was closely related to a higher water status due to increased PIP2 abundance, as well as to the upregulation of LsaNHX gene expression, which concomitantly improved plant nutrition and K+/Na+ homeostasis maintenance.

Abstract 14736: Aquaporin 1 Modulates H 2 o 2 -mediated Endothelial Inflammaging

Background: Aquaporin 1 (AQP1), a key regulator of endothelial cell function, transports hydrogen peroxide (H 2 O 2 ) into the cells. H 2 O 2 -mediated inflammation has been implicated in endothelial dysfunction, yet specific roles of AQP1-mediated signaling pathways underlying age-dependent endothelial dysfunction remains incompletely understood. Methods: In these studies, we dissected AQP1-regulated inflammation modulation of eNOS signaling pathways in human aortic endothelial cells (HAEC) from passages 5 to 15 by exploiting siRNA approaches, live cell fluorescence imaging with genetically-encoded H 2 O 2 biosensor HyPer, biochemical and in vitro endothelial function assays. Results: We discovered that AQP1 expression remarkably increases in senescent HAEC (P.15) in association with significant increased SA-βgal activity compared to young cells (P.5). H 2 O 2 levels were increased in senescent cell cytosol revealed by fluorescence HyPer imaging. We found that senescence-associated increase in AQP1-mediated H 2 O 2 led to enhanced TNF-α, unlike HO-1, transcription (p&lt;0.01). Moreover, immunofluorescence assay documented that AQP1 gene knock-down significantly ameliorated senescence-associated elevation of adhesion molecule ICAM-1. Immunoblot analyses demonstrated that increased AQP1 protein levels in senescent HAEC leads to significant increase in caveolin-1 phosphorylation (2-fold) and decreases in phosphorylation of AMPK (Thr172; 4-fold) and eNOS (Ser1177; 2-fold) compared to young ECs (for each, p&lt;0.05, n&gt;6). We discovered that AQP1 knock-down improved the reduced angiogenesis and wound healing capacity in association with eNOS down-regulation in senescent endothelial cells. Conclusion: These results establish that AQP1 plays a crucial role in the regulation of H 2 O 2 -mediated inflammation-associated endothelial senescence, and AQP1 deletion improves endothelial dysfunction by ameliorating ROS-modulated inflammaging.

Identification of aquaporin markers in Solanum melongena for water stress response

Aim: The study focused on identifying markers linked to aquaporin genes from the expressed regions of S. melongena using bioinformatics applications. Methodology: The EST collections were explored for identification of aquaporin markers for water stress response using comparative analysis and in-house developed repeat motif detection program. An algorithm was developed to generate repeat motifs which can be effectively used for collecting EST of S. melongena to filter the sequences having repeat motifs for further analysis. Results: From the results generated, the 22 potential sequences with the markers were found to be associated with aquaporin proteins. The detected repeat motifs are inherent part of markers and these markers are found to be evolutionarily conserved and associated with aquaporin proteins. Hence, identifying markers for the presence of aquaporin proteins play an important role in water diffusion across cell membranes in plants. Interpretation: Identifying aquaporin markers are useful for plant breeders for developing water stress tolerant crops during elevated temperatures. These markers are linked to water channel proteins that belong to superfamily Major Intrinsic Protein, that primarily plays an important role in conduction of water in plants.

Toxicity reduction and water expelling effect preservation of Shizaotang after its toxic members processing with vinegar on rats with malignant pleural effusions

Ethnopharmacological relevanceShizaotang (SZT), consisted of Euphorbia kansui S.L.Liou ex S.B.Ho (EK), Euphorbia pekinensis Rupr. (EP), Daphne genkwa Sieb. et Zucc. (DG，fried) and Ziziphus jujuba Mill. (ZJ), is usually used for treating malignant pleural effusions (MPE), but the toxicity of EK and EP limits its clinical safe application. It was reported that vinegar processing can reduce the toxicity of EK and EP. Whether EK and EP processing with vinegar can cause the reduced toxicity and retained pharmacological effects of SZT, it still remains unknown.Aim of the study: We aimed to evaluate whether using vinegar processed EK and EP would reduce toxicity and preserve water expelling effect of SZT. Materials and methodsNetwork pharmacology and qualitative analysis of SZT/VSZT were used to construct compound-target-pathway network of their effects and toxicity. Pleural fluid weight, urine volume, uric electrolyte, pH, pro-inflammatory cytokines in pleural fluid, serum Renin-Angiotensin-Aldosterone System (RAAS), anti-diuretic hormone (ADH) and intestinal aquaporin 8 (AQP8) protein were used to evaluate the effect mechanisms involved in rats experiments. And liver damage, oxidative damage and HE staining (liver, stomach, and intestine) were used to determine the toxicity. ResultsNetwork pharmacology analysis reviewed inflammation-related pathways of the effect and toxicity of SZT/VSZT: VEGF-PI3K-AKT pathway inhibited MPE by changing the vasopermeability; PI3K-Akt/Mitogen-activated protein kinase (MAPK)/TNF-NF-κB signaling pathway inhibited MPE by up-regulating expression of AQP8 protein. In vivo experiments displayed that SZT/VSZT could reduce pleural fluid, increase urine volume, lower pro-inflammatory cytokines levels and up-regulate AQP8 protein expression significantly (P < 0.05, P < 0.01). In addition, disorders on electrolyte (Na+, K+ and Cl−) and pH were ameliorated (P < 0.05, P < 0.01). The levels of RAAS and ADH were significantly dose-dependently called back (P < 0.01). These findings were partly consistent with the results of network pharmacology analysis. Results of toxicity experiments demonstrated that SZT and VSZT exhibited certain toxicity on normal rats, and VSZT had lower toxicity than that of SZT. Interestingly, SZT and VSZT exerted alleviation effect to the liver damage and oxidative damage on model rats. ConclusionSZT/VSZT improved MPE by regulating associated inflammation pathways. Besides, compared to SZT, VSZT showed lower toxicity and equivalent expelling MPE effect. This study may provide scientific basis for guiding the clinical application of SZT.

Isolation, Cloning and Characterization of a Constitutive Plant from Potato Aquaporin Gene

Plasma membrane intrinsic proteins (PIP1) are the most common integral membrane proteins belong to a larger family of intrinsic aquaporin proteins. They are member of aquaporin gene family and have gained importance as highly expressed genes in plants. In this study, the promoter of aquaporin PIP1 gene was identified, analyzed and retrieved from high throughput genomic sequence (HTGS) database. The cis-acting regulatory elements, transcription start sites and transcription factor binding sites of selected promoter were identified through different bio-informatics tools. Many light responsive, phytohormone, stress and defense related cis-regulatory elements were detected in PIP1 promoter region indicating its role as a constitutive promoter. The PIP1 promoter was isolated from Solanum tuberosum. It was initially cloned in TA vector (pTZ57R/T) and later transferred to plant expression binary vectors, pGR1 and pGA482 for transient and stable expression studies in tobacco. The GUS expression results of PIP1 promoter in different tobacco tissues showed its functional importance in regulating gene expression in a constitutive manner. Further, it was concluded that the PIP1 aquaporin promoter is constitutively expressed with a strength equivalent to CaMV 2x35S promoter. These findings indicated the significance of isolated promoter for genetic engineering of plants for crop improvement.&#x0D;

Mechanisms Activating Latent Functions of PIP Aquaporin Water Channels via the Interaction between PIP1 and PIP2 Proteins.

Plant plasma membrane-type plasma membrane intrinsic protein (PIP) aquaporins are classified into two groups, PIP1s and PIP2s. In this study, we focused on HvPIP1;2, a PIP1 in barley (Hordeum vulgare), to dissect the molecular mechanisms that evoke HvPIP1-mediated water transport. No HvPIP1;2 protein was localized to the plasma membrane when expressed alone in Xenopus laevis oocytes. By contrast, a chimeric HvPIP1;2 protein (HvPIP1;2_24NC), in which the N- and C-terminal regions were replaced with the corresponding regions from HvPIP2;4, was found to localize to the plasma membrane of oocytes. However, HvPIP1;2_24NC showed no water transport activity in swelling assays. These results suggested that the terminal regions of PIP2 proteins direct PIP proteins to the plasma membrane, but the relocalization of PIP1 proteins was not sufficient to PIP1s functionality as a water channel in a membrane. A single amino acid replacement of threonine by methionine in HvPIP2;4 (HvPIP2;4T229M) abolished water transport activity. Co-expression of HvPIP1;2_24NC either with HvPIP2;4_12NC or with HvPIP2;4TM_12NC, in which the N- and C-terminal regions were replaced with the corresponding regions of HvPIP1;2, increased the water transport activity in oocytes. These data provided evidence that the HvPIP1;2 molecule has own water transport activity and an interaction with the middle part of the HvPIP2;4 protein (except for the N- and C-termini) is required for HvPIP1;2 functionality as a water channel. This molecular mechanism could be applied to other PIP1s and PIP2s in addition to the known mechanism that the terminal regions of some PIP2s lead some PIP1s to the plasma membrane.

Industrial wastewater volume reduction through osmotic concentration: Membrane module selection and process modeling

Osmotic concentration (OC), a form of forward osmosis (FO) but without draw solution recovery, can be applied for reducing wastewater disposal volumes in the oil & gas industry. Within this industry, wastewater is often disposed of by injection through disposal wells into deep underground reservoirs. By reducing wastewater disposal volumes, the sustainability of the disposal reservoir is improved. In this application of OC, seawater or brine from a desalination plant serves as the draw solution and the diluted seawater is discharged to the sea. This study compared 3 commercial hollow-fiber FO membranes (CTA, TFC, aquaporin proteins) for reducing the volume of low salinity wastewater generated during liquified natural gas (LNG) production. Additionally, a model was developed to predict the performance of commercial full-scale membranes by identifying optimum operating conditions, taking into consideration the trade-off between feed concentration factor and water flux. Bench-scale tests were conducted using synthetic and actual wastewater from an LNG facility to evaluate OC technology performance and validate model predictions.Based on model results with a feed mimicking the salinity of actual wastewater, a 4x concentration factor produced a reasonable compromise between feed recovery and draw solution dilution and was considered the optimum for future tests. At higher concentration factors, the increased dilution of the draw solution negatively impacted flux. In bench tests with real wastewater, the TFC chemistry had a ≈5x higher water flux (9.7 vs. 1.9 L/m2-h) and a ≈3x lower specific reverse solute flux (192 vs. 551 mg/L) compared to the CTA chemistry. However, both membranes showed less than 5% fouling and a specific forward organic solute flux of less than 0.5 mg/L of total organic carbon (TOC). Pilot testing for >50 h showed stable performance, comparable to bench scale data and model predictions.

Clostridium bolteae is elevated in neuromyelitis optica spectrum disorder in India and shares sequence similarity with AQP4.

ObjectiveTo understand the role of gut microbiome in influencing the pathogenesis of neuromyelitis optica spectrum disorders (NMOSDs) among patients of south Indian origin.MethodsIn this case-control study, stool and blood samples were collected from 39 patients with NMOSD, including 17 with aquaporin 4 IgG antibodies (AQP4+) and 36 matched controls. 16S ribosomal RNA (rRNA) sequencing was used to investigate the gut microbiome. Peripheral CD4+ T cells were sorted in 12 healthy controls, and in 12 patients with AQP4+ NMOSD, RNA was extracted and immune gene expression was analyzed using the NanoString nCounter human immunology kit code set.ResultsMicrobiota community structure (beta diversity) differed between patients with AQP4+ NMOSD and healthy controls (p < 0.001, pairwise PERMANOVA test). Linear discriminatory analysis effect size identified several members of the microbiota that were altered in patients with NMOSD, including an increase in Clostridium bolteae (effect size 4.23, p 0.00007). C bolteae was significantly more prevalent (p = 0.02) among patients with AQP4-IgG+ NMOSD (n = 8/17 subjects) compared with seronegative patients (n = 3/22) and was absent among healthy stool samples. C bolteae has a highly conserved glycerol uptake facilitator and related aquaporin protein (p59-71) that shares sequence homology with AQP4 peptide (p92-104), positioned within an immunodominant (AQP4 specific) T-cell epitope (p91-110). Presence of C bolteae correlated with expression of inflammatory genes associated with both innate and adaptive immunities and particularly involved in plasma cell differentiation, B cell chemotaxis, and Th17 activation.ConclusionOur study described elevated levels of C bolteae associated with AQP4+ NMOSD among Indian patients. It is possible that this organism may be causally related to the immunopathogenesis of this disease in susceptible individuals.

Aquaporin-1: a major modulator of aging-mediated platelet/endothelial dysfunction in atherothrombosis

Abstract Background Aging is associated with development of cardiovascular diseases, including atherothrombosis. Aquaporin-1 (AQP1) is a water channel that also transports hydrogen peroxide (H2O2). Regarding to the role of oxidative stress in atherothrombosis, we hypothesized that AQP1 modulates aging-associated platelet/endothelial dysfunction. Methods Human aortic endothelial cells (HAEC) from passages 5 (young) to 15 (senescent/old) were subjected to fluorescent immunocytochemistry to detect AQP1 protein expression and subcellular localization. The cells were also probed for AQP1 and the phospho/total proteins (AMPK, acetyl-coA-carboxylase (ACC), caveolin-1 and eNOS) for studying the signal transduction by immunoblotting. The endothelial cells were transfected with constructs containing H2O2 biosensor HyPer targeted to cell nucleus or cytosol followed by fluorescence imaging. The transcriptional levels of pro-inflammatory/pro-atherogenic vs. anti-inflammatory/atheroprotective genes in the cells were assessed by qRT-PCR. Human blood samples were taken and treated with or without AQP1 inhibitor (Bacopaside II, 10 μM) to examine the platelet adhesion and rolling velocity on vWF under high shear flow (100 dyn/cm2). Also, platelet aggregation in response to collagen (2 μM), ADP (1 μM) and TRAP (1 μM) were recorded. Results First, the senescence of HAEC was adjusted by a significant increase in β-galactosidase activity from passage 5 to 15. AQP1 immunofluorescence showed a remarkable increase in the young (P.5) compared to senescent (P.15) cells. Immunoblot analyses showed that aging leads to significant increases in AQP1 intensity and phosphorylation of caveolin-1 (Tyr14) and ACC (Ser79), along with decreases in phosphorylation of eNOS (Ser1177) and AMPK (Thr172) (p&amp;lt;0.01, n&amp;gt;6). Fluorescence imaging documented a robust H2O2 production in the senescent endothelial cell cytosol, but not nucleus, and activated TNF-α gene, whereas the transcription of hemoxygenase-1 gene enhanced in the young cells (p&amp;lt;0.01). AQP1 inhibition reduced platelet adhesion and thrombus formation, and elevated platelet rolling velocity on vWF under shear flow (p&amp;lt;0.01). Also, a decrease was found in platelet aggregation in response to AQP1 inhibition (p&amp;lt;0.05). Conclusion These studies, for the first time, demonstrate that aging induces AQP1 expression in endothelial cells and platelets, and modulates the dephosphorylation of AMPK/eNOS. These may lead to platelet/endothelial dysfunction and production of pro-coagulant/pro-inflammatory factors via ACC activation. Therefore, AQP1 inhibition could potentially be exploited as a therapeutic strategy for improving age-related atherothrombosis. Figure 1. Aging increases AQP1 expression in HAEC that leads to dephosphorylation of AMPK and eNOS. AQP inhibition also improves platelet function. *p&amp;lt;0.05, **p&amp;lt;0.01 compared to control. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): Swiss National Science Foundation (SNSF)

Immunohistological Study of Palpebral Lobe of the Lacrimal Gland in Severe Dry Eyes Secondary to Stevens–Johnson Syndrome

ABSTRACT Purpose To detect the immunohistochemical changes in the main lacrimal glands of patients suffering from chronic ocular sequelae of Stevens–Johnson syndrome (SJS). Methods Histological sections of biopsies from the lacrimal gland of three chronic SJS patients (mean age, 33 years; 2 males) with severe dry eye disease (Schirmer = 0 mm) were assessed using double immunofluorescence techniques. Changes in the expression of secretory proteins lactoferrin (Lf), lysozyme (Ly), aquaporin 5 (AQP5), S-100, and early apoptosis marker (Annexin V) were studied. Results Different morphological expressions of secretory proteins were present in the three samples. One sample had maintained the immunoreactivity for Lf, Ly, S-100, similar to healthy controls. Two samples had significantly reduced immunoreactivity for anti-Lf, anti-Ly, and S-100, the weakest being in the sample with distorted lobular architecture and mild interlobular fibrosis. AQP5 had a distinct vesicular intracytoplasmic immunoreactivity suggesting defective trafficking and integration of the protein to the apical membrane. There was no S-100 immunostaining in the acinar or ductal epithelium, whereas interstitial nerve fibers scattered in the periacinar region showed reduced immunoreactivity for S-100. There was strong Annexin V immunoreactivity in the nuclei of epithelial cells in the majority of acinar and ductal epithelia of all the samples, with distorted nuclear morphology in one sample. Conclusion Defective trafficking of AQP5 and variable expression of Ly, Lf, S-100 are the notable findings in the lacrimal glands of chronic SJS patients along with signs of early apoptosis. It suggests that the palpebral lobe of the lacrimal gland is involved in the pathological processes occurring in the conjunctiva of SJS patients.

Expression of transmembrane protein aquaporin-3 in oral epithelial dysplasia and oral squamous cell carcinoma

The objective of this study was to evaluate aquaporin-3 (AQP3) expression in patient samples of oral epithelial dysplasia (OED) and oral squamous cell carcinoma (OSCC), thereby assessing the potential of AQP3 as a molecular marker for tumor progression. An in vitro comparative study was done to determine the AQP3 expression on 20 surgical biopsy specimens each of OED and OSCC using immunohistochemistry. Twenty specimens of normal oral mucosa were kept as controls. The results were statistically analyzed using one-way analysis of variance and post hoc analysis. The expression of AQP3 was analyzed and further semiquantified using H-scores. The mean H-score showed a statistically significant difference between OSCC, OED, and normal oral mucosa (P < .05). There was a significant increase in the expression of AQP3 in OSCC and OED compared to normal oral mucosa. The highest expression was observed in OSCC (P < .01). The observations of the study indicate that staining intensity of AQP3 increased from dysplastic noninvasive lesion to invasive OSCC, suggesting a possible role of AQP3 as a biomarker for tumor progression.

Gradient washout and secondary nephrogenic diabetes insipidus after brain injury in an infant: a case report

BackgroundDisorders of water and sodium balance can occur after brain injury. Prolonged polyuria resulting from central diabetes insipidus and cerebral salt wasting complicated by gradient washout and a type of secondary nephrogenic diabetes insipidus, however, has not been described previously, to the best of our knowledge. We report an unusual case of an infant with glioblastoma who, after tumor resection, was treated for concurrent central diabetes insipidus and cerebral salt wasting complicated by secondary nephrogenic diabetes insipidus.Case presentationA 5-month-old Hispanic girl was found to have a large, hemorrhagic, suprasellar glioblastoma causing obstructive hydrocephalus. Prior to mass resection, she developed central diabetes insipidus. Postoperatively, she continued to have central diabetes insipidus and concurrent cerebral salt wasting soon after. She was managed with a vasopressin infusion, sodium supplementation, fludrocortisone, and urine output replacements. Despite resolution of her other major medical issues, she remained in the pediatric intensive care unit for continual and aggressive management of water and sodium derangements. Starting on postoperative day 18, her polyuria began increasing dramatically and did not abate with increasing vasopressin. Nephrology was consulted. Her blood urea nitrogen was undetectable during this time, and it was thought that she may have developed a depletion of inner medullary urea and osmotic gradient: a “gradient washout.” Supplemental dietary protein was added to her enteral nutrition, and her fluid intake was decreased. Within 4 days, her blood urea nitrogen increased, and her vasopressin and fluid replacement requirements significantly decreased. She was transitioned soon thereafter to subcutaneous desmopressin and transferred out of the pediatric intensive care unit.ConclusionsGradient washout has not been widely reported in humans, although it has been observed in the mammalian kidneys after prolonged polyuria. Although not a problem with aquaporin protein expression or production, gradient washout causes a different type of secondary nephrogenic diabetes insipidus because the absence of a medullary gradient impairs water reabsorption. We report a case of an infant who developed complex water and sodium imbalances after brain injury. Prolonged polyuria resulting from both water and solute diuresis with low enteral protein intake was thought to cause a urea gradient washout and secondary nephrogenic diabetes insipidus. The restriction of fluid replacements and supplementation of enteral protein appeared adequate to restore the renal osmotic gradient and efficacy of vasopressin.