RESUMO
OBJECTIVES: To examine the prevalence of temporomandibular disorders (TMD) in patients with juvenile fibromyalgia syndrome (JFS) and identify TMD characteristics specifically associated to JFS. METHODS: Signs and symptoms of TMD were assessed using a novel clinical tool specifically devised for children that consists of: 1. a self-report multiple-choice questionnaire; 2. a protocol for the clinical examination of the orofacial region. Multivariate logistic regression model was used to identify TMD features associated with JFS. RESULTS: Thirty JFS patients (median age 15.5 years) and 45 healthy controls (median age 15.0 years) were included in this cross-sectional study. Orofacial pain was reported by 26 of 30 JFS patients (86.7%) and by 3 of 45 controls (6.7%; p<0.001). Pain on TMJ palpation was present in 18 of 30 JFS patients (60%) and in 5 of 45 controls (11.1%; p<0.001). Median values of maximum spontaneous mouth opening, voluntary active opening and assisted passive opening were significantly higher in JFS patients than in controls. On multiple regression analysis spontaneous orofacial pain (OR: 21.0; p=0.005), diffuse tenderness on palpation of the masticatory muscles (OR: 14.9; p=0.026) and TMJ hypermobility (OR 1.42; p=0.008) were independently associated with JFS. CONCLUSIONS: The high prevalence of TMD in JFS highlights the need for a broader interdisciplinary evaluation of JFS patients. TMJ hypermobility, in addition to orofacial and masticatory muscle pain, is an important clue for the diagnosis of TMD in adolescents with JFS. Elucidating the link between these disorders will advance individualised management and improve treatment efficacy.
Assuntos
Dor Facial , Fibromialgia , Medição da Dor , Transtornos da Articulação Temporomandibular , Humanos , Fibromialgia/epidemiologia , Fibromialgia/diagnóstico , Fibromialgia/fisiopatologia , Adolescente , Dor Facial/epidemiologia , Dor Facial/diagnóstico , Dor Facial/fisiopatologia , Dor Facial/etiologia , Feminino , Transtornos da Articulação Temporomandibular/epidemiologia , Transtornos da Articulação Temporomandibular/diagnóstico , Transtornos da Articulação Temporomandibular/fisiopatologia , Prevalência , Masculino , Estudos Transversais , Criança , Estudos de Casos e Controles , Modelos Logísticos , Valor Preditivo dos Testes , Palpação , Análise Multivariada , Inquéritos e Questionários , Fatores Etários , Razão de Chances , Articulação Temporomandibular/fisiopatologia , Autorrelato , Fatores de RiscoRESUMO
Increasing evidence indicates that aquaporins (AQPs) exert an influence in cell signaling by the interplay with the transient receptor potential vanilloid 4 (TRPV4) channel. We previously found that TRPV4 physically and functionally interacts with AQP2 in cortical collecting ducts (CCD) cells, favoring cell volume regulation and cell migration. Because TRPV4 was implicated in ATP release in several tissues, we investigated the possibility that TRPV4/AQP2 interaction influences ATP release in CCD cells. Using two CCD cell lines expressing or not AQP2, we measured extracellular ATP (ATPe) under TRPV4 activation and intracellular Ca2+ under ATP addition. We found that AQP2 is critical for the release of ATP induced by TRPV4 activation. This ATP release occurs by an exocytic and a conductive route. ATPe, in turn, stimulates purinergic receptors leading to ATPe-induced ATP release by a Ca2+ -dependent mechanism. We propose that AQP2 by modulating Ca2+ and ATP differently could explain AQP2-increased cell migration.
Assuntos
Trifosfato de Adenosina/metabolismo , Aquaporina 2/metabolismo , Sinalização do Cálcio , Cálcio/metabolismo , Movimento Celular , Túbulos Renais Coletores/metabolismo , Canais de Cátion TRPV/metabolismo , Animais , Comunicação Autócrina , Sinalização do Cálcio/efeitos dos fármacos , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Exocitose , Túbulos Renais Coletores/efeitos dos fármacos , Leucina/análogos & derivados , Leucina/farmacologia , Comunicação Parácrina , Ratos , Receptores Purinérgicos P2/metabolismo , Sulfonamidas/farmacologia , Canais de Cátion TRPV/agonistasRESUMO
Aquaporin-2 (AQP2) promotes renal cell migration by the modulation of integrin ß1 trafficking and the turnover of focal adhesions. The aim of this study was to investigate whether AQP2 also works in cooperation with Na+ /H+ exchanger isoform 1 (NHE1), another well-known protein involved in the regulation of cell migration. Our results showed that the lamellipodia of AQP2-expressing cells exhibit significantly smaller volumes and areas of focal adhesions and more alkaline intracellular pH due to increased NHE1 activity than AQP2-null cells. The blockage of AQP2, or its physically-associated calcium channel TRPV4, significantly reduced lamellipodia NHE1 activity. NHE1 blockage significantly reduced the rate of cell migration, the number of lamellipodia, and the assembly of F-actin only in AQP2-expressing cells. Our data suggest that AQP2 modulates the activity of NHE1 through its calcium channel partner TRPV4, thereby determining pH-dependent actin polymerization, providing mechanical stability to delineate lamellipodia structure and defining the efficiency of cell migration.
Assuntos
Aquaporina 2/metabolismo , Rim/citologia , Trocador 1 de Sódio-Hidrogênio/metabolismo , Animais , Aquaporina 2/genética , Linhagem Celular , Tamanho Celular , Células Epiteliais , Adesões Focais , Regulação da Expressão Gênica/efeitos dos fármacos , Guanidinas/farmacologia , Concentração de Íons de Hidrogênio , Pseudópodes/fisiologia , Ratos , Trocador 1 de Sódio-Hidrogênio/genética , Sulfonas/farmacologiaRESUMO
BACKGROUND: Immediate changes in vascular mechanics during aortic cross-clamping remain widely unknown. By using a numerical model of the arterial network, vascular compliance and resistance can be estimated and the time constant of pressure waves can be calculated and compared with results from the classic arterial waveform analysis. METHODS: Experimental data were registered from continuous invasive radial artery pressure measurements from 11 patients undergoing vascular surgery. A stable set of beats were chosen immediately before and after each clamping event. Through the arterial waveform analysis, the time constant was calculated for each individual beat and for a mean beat of each condition as to compare with numerical simulations. Overall proportional changes in resistance and compliance during clamping and unclamping were calculated using the numerical model. RESULTS: Arterial waveform analysis of individual beats indicated a significant 10% median reduction in the time constant after clamping, and a significant 17% median increase in the time constant after unclamping. There was a positive correlation between waveform analysis and numerical values of the time constant, which was moderate (ρ = 0.51; P = 0.01486) during clamping and strong (ρ = 0.77; P ≤ 0.0001) during unclamping. After clamping, there was a significant 16% increase in the mean resistance and a significant 23% decrease in the mean compliance. After unclamping, there was a significant 19% decrease in the mean resistance and a significant 56% increase in the mean compliance. CONCLUSIONS: There are significant hemodynamic changes in vascular compliance and resistance during aortic clamping and unclamping. Numerical computer models can add information on the mechanisms of injury due to aortic clamping.
Assuntos
Pressão Arterial , Modelos Teóricos , Monitorização Intraoperatória/métodos , Artéria Radial/fisiologia , Resistência Vascular , Procedimentos Cirúrgicos Vasculares , Idoso , Idoso de 80 Anos ou mais , Constrição , Estudos Transversais , Feminino , Humanos , Complicações Intraoperatórias/prevenção & controle , Masculino , Pessoa de Meia-Idade , Artéria Radial/lesões , Lesões do Sistema Vascular/etiologia , Lesões do Sistema Vascular/prevenção & controleRESUMO
There is increasing evidence indicating that aquaporins (AQPs) exert an influence in cell signaling by the interplay with the TRPV4 Ca2+ channel. Ca2+ release from intracellular stores and plasma membrane hyperpolarization due to opening of Ca2+ -activated potassium channels (KCa) are events that have been proposed to take place downstream of TRPV4 activation. A major mechanism for Ca2+ entry, activated after depletion of intracellular Ca2+ stores and driven by electrochemical forces, is the store-operated Ca2+ entry (SOCE). The consequences of the interplay between TRPV4 and AQPs on SOCE have not been yet investigated. The aim of our study was to test the hypothesis that AQP2 can modulate SOCE by facilitating the interaction of TRPV4 with KCa channels in renal cells. Using fluorescent probe techniques, we studied intracellular Ca2+ concentration and membrane potential in response to activation of TRPV4 in two rat cortical collecting duct cell lines (RCCD1 ), one not expressing AQPs (WT-RCCD1 ) and the other transfected with AQP2 (AQP2-RCCD1 ). We found that AQP2 co-immunoprecipitates with TRPV4 and with the small-conductance potassium channel (SK3). We also showed that AQP2 is crucial for the activation of SK3 by TRPV4, leading to hyperpolarization of the plasma membrane. This seems to be relevant to modulate the magnitude of SOCE and is accompanied by TRPV4 translocation to the plasma membrane only in AQP2 expressing cells. These findings open the perspective to further investigate whether the interplay between different AQPs with TRPV4 and KCa channels can be an important mechanism to modulate SOCE with physiological relevance.
Assuntos
Aquaporina 2/metabolismo , Sinalização do Cálcio , Cálcio/metabolismo , Potenciais da Membrana , Canais de Cátion TRPV/metabolismo , Animais , Aquaporina 2/genética , Linhagem Celular , Ratos , Canais de Cátion TRPV/genéticaRESUMO
Neuronal activity in the retina generates osmotic gradients that lead to Müller cell swelling, followed by a regulatory volume decrease (RVD) response, partially due to the isoosmotic efflux of KCl and water. However, our previous studies in a human Müller cell line (MIO-M1) demonstrated that an important fraction of RVD may also involve the efflux of organic solutes. We also showed that RVD depends on the swelling-induced Ca2+ release from intracellular stores. Here we investigate the contribution of taurine (Tau) and glutamate (Glu), the most relevant amino acids in Müller cells, to RVD through the volume-regulated anion channel (VRAC), as well as their Ca2+ dependency in MIO-M1 cells. Swelling-induced [3H]Tau/[3H]Glu release was assessed by radiotracer assays and cell volume by fluorescence videomicroscopy. Results showed that cells exhibited an osmosensitive efflux of [3H]Tau and [3H]Glu (Tau > Glu) blunted by VRAC inhibitors 4-(2-butyl-6,7-dichloro-2-cyclopentylindan-1-on-5-yl)-oxybutyric acid and carbenoxolone reducing RVD. Only [3H]Tau efflux was mainly dependent on Ca2+ release from intracellular stores. RVD was unaffected in a Ca2+-free medium, probably due to Ca2+-independent Tau and Glu release, but was reduced by chelating intracellular Ca2+. The inhibition of phosphatidylinositol-3-kinase reduced [3H]Glu efflux but also the Ca2+-insensitive [3H]Tau fraction and decreased RVD, providing evidence of the relevance of this Ca2+-independent pathway. We propose that VRAC-mediated Tau and Glu release has a relevant role in RVD in Müller cells. The observed disparities in Ca2+ influence on amino acid release suggest the presence of VRAC isoforms that may differ in substrate selectivity and regulatory mechanisms, with important implications for retinal physiology. NEW & NOTEWORTHY The mechanisms for cell volume regulation in retinal Müller cells are still unknown. We show that swelling-induced taurine and glutamate release mediated by the volume-regulated anion channel (VRAC) largely contributes the to the regulatory volume decrease response in a human Müller cell line. Interestingly, the hypotonic-induced efflux of these amino acids exhibits disparities in Ca2+-dependent and -independent regulatory mechanisms, which strongly suggests that Müller cells may express different VRAC heteromers formed by the recently discovered leucine-rich repeat containing 8 (LRRC8) proteins.
Assuntos
Cálcio/metabolismo , Tamanho Celular , Células Ependimogliais/citologia , Células Ependimogliais/metabolismo , Ácido Glutâmico/metabolismo , Taurina/metabolismo , Análise de Variância , Ânions/metabolismo , Antiulcerosos/farmacologia , Carbenoxolona/farmacologia , Ciclopentanos/farmacologia , Humanos , Indanos/farmacologia , Canais Iônicos/antagonistas & inibidores , Microscopia de Vídeo , Osmorregulação/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/metabolismo , Retina/fisiologiaRESUMO
We have previously shown in renal cells that expression of the water channel Aquaporin 2 (AQP2) increases the rate of cell proliferation by shortening the transit time through the S and G2 /M phases of the cell cycle. This acceleration is due, at least in part, to a down-regulation of regulatory volume decrease (RVD) mechanisms when volume needs to be increased in order to proceed into the S phase. We hypothesize that in order to increase cell volume, RVD mechanisms may be overtaken by regulatory volume increase mechanisms (RVI). In this study, we investigated if the isoform 2 of the Na+ /H+ exchanger (NHE2), the main ion transporter involved in RVI responses, contributed to the AQP2-increased renal cell proliferation. Three cortical collecting duct cell lines were used: WT-RCCD1 (not expressing AQPs), AQP2-RCCD1 (transfected with AQP2), and mpkCCDc14 (with inducible AQP2 expression). We here demonstrate, for the first time, that both NHE2 protein activity and expression were increased in AQP2-expressing cells. NHE2 inhibition decreased cell proliferation and delayed cell cycle progression by slowing S and G2 /M phases only if AQP2 was expressed. Finally, we observed that only in AQP2-expressing cells a NHE2-dependent RVI response was activated in the S phase. These observations suggest that the AQP2-increased proliferation involves the activation of a regulatory volume increase mechanism dependent on NHE2. Therefore, we propose that the accelerated proliferation of AQP2-expressing cells requires a coordinated modulation of the RVD/RVI activity that contributes to cell volume changes during cell cycle progression. J. Cell. Biochem. 118: 967-978, 2017. © 2016 Wiley Periodicals, Inc.
Assuntos
Aquaporina 2/metabolismo , Córtex Renal/citologia , Trocadores de Sódio-Hidrogênio/genética , Trocadores de Sódio-Hidrogênio/metabolismo , Animais , Aquaporina 2/genética , Ciclo Celular , Linhagem Celular , Proliferação de Células , Tamanho Celular , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Córtex Renal/metabolismo , RatosRESUMO
Neural activity alters osmotic gradients favoring cell swelling in retinal Müller cells. This swelling is followed by a regulatory volume decrease (RVD), partially mediated by an efflux of KCl and water. The transient receptor potential channel 4 (TRPV4), a nonselective calcium channel, has been proposed as a candidate for mediating intracellular Ca2+ elevation induced by swelling. We previously demonstrated in a human Müller cell line (MIO-M1) that RVD strongly depends on ion channel activation and, consequently, on membrane potential (Vm ). The aim of this study was to investigate if Ca2+ influx via TRPV4 contributes to RVD by modifying intracellular Ca2+ concentration and/or modulating Vm in MIO-M1 cells. Cell volume, intracellular Ca2+ levels, and Vm changes were evaluated using fluorescent probes. Results showed that MIO-M1 cells express functional TRPV4 which determines the resting Vm associated with K+ channels. Swelling-induced increases in Ca2+ levels was due to both Ca2+ release from intracellular stores and Ca2+ influx by a pathway alternative to TRPV4. TRPV4 blockage affected swelling-induced biphasic response (depolarization-repolarization), suggesting its participation in modulating Vm changes during RVD. Agonist stimulation of Ca2+ influx via TRPV4 activated K+ channels hyperpolarizing Vm and accelerating RVD. We propose that TRPV4 forms a signaling complex with Ca2+ and/or voltage-dependent K+ channels to define resting Vm and Vm changes during RVD. TRPV4 involvement in RVD depends on the type of stimuli and/or degree of channel activation, leading to a maximum RVD response when Ca2+ influx overcomes a threshold and activates further signaling pathways in cell volume regulation. J. Cell. Biochem. 118: 2302-2313, 2017. © 2017 Wiley Periodicals, Inc.
Assuntos
Cálcio/metabolismo , Células Ependimogliais/metabolismo , Canais de Cátion TRPV/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Tamanho Celular/efeitos dos fármacos , Células Ependimogliais/efeitos dos fármacos , Imunofluorescência , Humanos , Leucina/análogos & derivados , Leucina/farmacologia , Potenciais da Membrana/efeitos dos fármacos , Morfolinas/farmacologia , Pirróis/farmacologia , Sulfonamidas/farmacologia , Canais de Cátion TRPV/agonistas , Canais de Cátion TRPV/antagonistas & inibidoresRESUMO
Introduction: Cardiovascular surgery risk prediction models are widely applied in medical practice. However, they have been criticized for their low methodological quality and scarce external validation. An additional limitation added in Latin America is that most of these models have been developed in the United States or Europe, which present marked geographical differences. The objective of this study is to characterize the postoperative clinical events of cardiovascular surgeries with the use of cardiopulmonary bypass pump in a local setting and to evaluate the prediction of postoperative mortality using the EuroSCORE II predictive model. Methods: Cross-sectional study in an urban university hospital in Buenos Aires. Patients ≥21 years of age were included, with a clinical indication for on-pump cardiovascular surgery. Patients with incomplete clinical data regarding EuroSCORE II variables or in-hospital survival, ≥95 years of age, or undergoing heart transplantation were excluded. Results: 195 patients were enrolled. Postoperative mortality estimated by EuroSCORE II presented a clear underestimation of risk (3.0% vs 7.7%). Discrimination (AUC = 0.82; 95% CI 0.74-0.92) and goodness of fit of the model were adequate (χ2 = 7.91; p = 0.4418). The most frequent postoperative complications were postoperative heart failure (35.9%), vasoplegic shock (13.3%), and cardiogenic shock (10.26%). Conclusion: The EuroSCORE II is an appropriate tool to discriminate between different risk categories in patients undergoing on-pump cardiovascular surgery, although it underestimates the risk.
Introducción: Los modelos de predicción de riesgo de cirugías cardiovasculares se aplican ampliamente a la práctica médica. Sin embargo, han sido criticados por su baja calidad metodológica y escasa validación externa. En América Latina se agrega la limitación de que la mayoría de estos modelos fueron desarrollados en Estados Unidos o Europa, existiendo diferencias geográficas marcadas. Objetivo: El objetivo de este estudio es caracterizar los eventos clínicos postoperatorios de cirugías cardiovasculares con uso de bomba de circulación extracorpórea en un escenario local y evaluar la predicción de mortalidad postoperatoria del modelo predictivo EuroSCORE II. Métodos: Corte transversal en un hospital universitario urbano de Buenos Aires. Se incluyeron a pacientes ≥21 años de edad, con indicación de cirugía cardiovascular con uso de bomba. Se excluyeron a pacientes con datos clínicos incompletos respecto a las variables del EuroSCORE II o respecto a la sobrevida intrahospitalaria, con ≥95 años de edad o sometidos a trasplante cardíaco. Resultados: Se enrolaron 195 pacientes. La mortalidad postoperatoria estimada por el EuroSCORE II presentó una clara subestimación del riesgo (3,0% vs 7,7%). La discriminación (AUC = 0,82; IC95% 0,74-0,92) y la bondad del ajuste del modelo fueron adecuadas (χ2 = 7,91; p = 0,4418). Las complicaciones postoperatorias más frecuentes fueron insuficiencia cardíaca postoperatoria (35,9%), shock vasopléjico (13,3%) y shock cardiogénico (10,26%). Conclusión: El EuroSCORE II es una herramienta apropiada para discriminar entre diferentes categorías de riesgo en pacientes sometidos a cirugías cardiovasculares con uso de bomba, si bien subestima el riesgo.
Assuntos
Circulação Extracorpórea , Complicações Pós-Operatórias , Humanos , Feminino , Masculino , Pessoa de Meia-Idade , Medição de Risco/métodos , Circulação Extracorpórea/efeitos adversos , Idoso , Estudos Transversais , Fatores de Risco , Procedimentos Cirúrgicos Cardiovasculares/efeitos adversos , Argentina , Mortalidade Hospitalar , AdultoRESUMO
We have previously demonstrated that renal cortical collecting duct cells (RCCD(1)), responded to hypotonic stress with a rapid activation of regulatory volume decrease (RVD) mechanisms. This process requires the presence of the water channel AQP2 and calcium influx, opening the question about the molecular identity of this calcium entry path. Since the calcium permeable nonselective cation channel TRPV4 plays a crucial role in the response to mechanical and osmotic perturbations in a wide range of cell types, the aim of this work was to test the hypothesis that the increase in intracellular calcium concentration and the subsequent rapid RVD, only observed in the presence of AQP2, could be due to a specific activation of TRPV4. We evaluated the expression and function of TRPV4 channels and their contribution to RVD in WT-RCCD(1) (not expressing aquaporins) and in AQP2-RCCD(1) (transfected with AQP2) cells. Our results demonstrated that both cell lines endogenously express functional TRPV4, however, a large activation of the channel by hypotonicity only occurs in cells that express AQP2. Blocking of TRPV4 by ruthenium red abolished calcium influx as well as RVD, identifying TRPV4 as a necessary component in volume regulation. Even more, this process is dependent on the translocation of TRPV4 to the plasma membrane. Our data provide evidence of a novel association between TRPV4 and AQP2 that is involved in the activation of TRPV4 by hypotonicity and regulation of cellular response to the osmotic stress, suggesting that both proteins are assembled in a signaling complex that responds to anisosmotic conditions.
Assuntos
Aquaporina 2/metabolismo , Rim/citologia , Canais de Cátion TRPV/metabolismo , Animais , Bloqueadores dos Canais de Cálcio/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Capsaicina/farmacologia , Tamanho Celular , Células Cultivadas , Citocalasina D/farmacologia , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Expressão Gênica , Pressão Osmótica , Forbóis/farmacologia , Ligação Proteica , Transporte Proteico , Ratos , Rutênio Vermelho/farmacologia , Estresse Fisiológico , Canais de Cátion TRPV/agonistas , Canais de Cátion TRPV/genética , Moduladores de Tubulina/farmacologiaRESUMO
We have previously demonstrated that in renal cortical collecting duct cells (RCCD(1)) the expression of the water channel Aquaporin 2 (AQP2) raises the rate of cell proliferation. In this study, we investigated the mechanisms involved in this process, focusing on the putative link between AQP2 expression, cell volume changes, and regulatory volume decrease activity (RVD). Two renal cell lines were used: WT-RCCD(1) (not expressing aquaporins) and AQP2-RCCD(1) (transfected with AQP2). Our results showed that when most RCCD(1) cells are in the G(1)-phase (unsynchronized), the blockage of barium-sensitive K(+) channels implicated in rapid RVD inhibits cell proliferation only in AQP2-RCCD(1) cells. Though cells in the S-phase (synchronized) had a remarkable increase in size, this enhancement was higher and was accompanied by a significant down-regulation in the rapid RVD response only in AQP2-RCCD(1) cells. This decrease in the RVD activity did not correlate with changes in AQP2 function or expression, demonstrating that AQP2-besides increasing water permeability-would play some other role. These observations together with evidence implying a cell-sizing mechanism that shortens the cell cycle of large cells, let us to propose that during nutrient uptake, in early G(1), volume tends to increase but it may be efficiently regulated by an AQP2-dependent mechanism, inducing the rapid activation of RVD channels. This mechanism would be down-regulated when volume needs to be increased in order to proceed into the S-phase. Therefore, during cell cycle, a coordinated modulation of the RVD activity may contribute to accelerate proliferation of cells expressing AQP2.
Assuntos
Aquaporina 2/metabolismo , Proliferação de Células/efeitos dos fármacos , Tamanho Celular/efeitos dos fármacos , Rim/citologia , Animais , Aquaporina 2/genética , Compostos de Bário/farmacologia , Sinalização do Cálcio , Linhagem Celular , Membrana Celular/metabolismo , Permeabilidade da Membrana Celular , Cloretos/farmacologia , Citometria de Fluxo , Pontos de Checagem da Fase G1 do Ciclo Celular , Microscopia de Vídeo , Pressão Osmótica , Canais de Potássio/efeitos dos fármacos , Canais de Potássio/metabolismo , Transporte Proteico , Ratos , Rutênio Vermelho/farmacologia , Fase S , Canais de Cátion TRPV/genética , Canais de Cátion TRPV/metabolismo , TransfecçãoRESUMO
NMO-IgG autoantibody selectively binds to aquaporin-4 (AQP4), the most abundant water channel in the central nervous system and is now considered a useful serum biomarker of neuromyelitis optica (NMO). A series of clinical and pathological observations suggests that NMO-IgG may play a central role in NMO physiopathology. The current study evaluated, in well-differentiated astrocytes cultures, the consequences of NMO-IgG binding on the expression pattern of AQP4 and on plasma membrane water permeability. To avoid or to facilitate AQP4 down-regulation, cells were exposed to inactivated sera in two different situations (1 hr at 4°C or 12 hr at 37°C). AQP4 expression was detected by immunofluorescence studies using a polyclonal anti-AQP4 or a human anti-IgG antibody, and the water permeability coefficient was evaluated by a videomicroscopy technique. Our results showed that, at low temperatures, cell exposure to either control or NMO-IgG sera does not affect either AQP4 expression or plasma membrane water permeability, indicating that the simple binding of NMO-IgG does not affect the water channel's activity. However, at 37°C, long-term exposure to NMO-IgG induced a loss of human IgG signal from the plasma membrane along with M1-AQP4 isoform removal and a significant reduction of water permeability. These results suggest that binding of NMO-IgG to cell membranes expressing AQP4 is a specific mechanism that may account for at least part of the pathogenic process.
Assuntos
Aquaporina 4/metabolismo , Astrócitos/efeitos dos fármacos , Imunoglobulina G/farmacologia , Neuromielite Óptica/imunologia , Água/metabolismo , Adulto , Animais , Animais Recém-Nascidos , Antígenos de Neoplasias/metabolismo , Astrócitos/fisiologia , Biotinilação , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Células Cultivadas , Córtex Cerebral/citologia , Feminino , Proteína Glial Fibrilar Ácida/metabolismo , Humanos , Imunoglobulina G/sangue , Glicoproteínas de Membrana/metabolismo , Pessoa de Meia-Idade , Neuromielite Óptica/patologia , Permeabilidade/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/fisiologia , Ratos , Adulto JovemRESUMO
Uroguanylin (UGN) has been proposed as a key regulator of salt and water intestinal transport. Uroguanylin activates cell-surface guanylate cyclase C receptor (GC-C) and modulates cellular function via cyclic GMP (cGMP), thus increasing electrolyte and net water secretion. It has been suggested that the action of UGN could involve the Na(+)/H(+) exchanger, but the actual contribution of this transporter still remains unclear. The objective of our study was to investigate the putative effects of UGN on some members of the Na(+)/H(+) exchanger family (NHEs), as well as to clarify its consequences on transepithelial fluid flow in T84 cells. In order to do so, transepithelial fluid flow (J(v)) was studied by optic techniques and intracellular pH (pH(i)) was measured with a fluorescence method. Results showed that NHE2 is found at the apical membrane and has a major role in Na(+) absorption; NHE1 and NHE4 are localized at the basolateral membrane with a house-keeping role in steady state pH(i). In the assayed conditions, cell exposure to apical UGN increases net secretory J(v), without changing short-circuit currents nor transepithelial resistance, and reduces NHE2 activity. Therefore, at physiological pH, the effect on net J(v) was produced mainly by a reduction in normal Na(+) absorption through NHE2, rather than by the stimulation of electrolyte secretion. Our study shows that the effect of UGN on pH(i) is GC-C/cGMP-mediated and enhanced by sildenafil, thus involving PDE5 enzyme. Additionally, cell exposure to apical UGN results in intracellular alkalinization, probably due to indirect effects on basolateral NHE1 and NHE4, which have a major role in pH(i) regulation.
Assuntos
Peptídeos Natriuréticos/farmacologia , Trocadores de Sódio-Hidrogênio/metabolismo , Transporte Biológico , Linhagem Celular Tumoral , Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/enzimologia , Humanos , Concentração de Íons de Hidrogênio , Intestinos/citologia , Intestinos/enzimologia , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Sódio/metabolismo , Trocadores de Sódio-Hidrogênio/genéticaRESUMO
Retinal Müller glial cells (MGs) are among the first to demonstrate metabolic changes during retinal disease and are a potential source of regenerative cells. In response to a harmful stimulus, they can dedifferentiate acquiring neural stem cells properties, proliferate and migrate to the damaged retinal layer and differentiate into lost neurons. However, it is not yet known how this reprogramming process is regulated in mammals. Since glucose and oxygen are important regulatory elements that may help directing stem cell fate, we aimed to study the effect of glucose variations and oxidative stress in Müller cells reprogramming capacity and analyze the participation the histone deacetylase SIRT6, as an epigenetic modulator of this process. We found that the combination of high glucose and oxidative stress induced a decrease in the levels of the marker glutamine synthetase, and an increase in the migration capacity of the cells suggesting that these experimental conditions could induce some degree of dedifferentiation and favor the migration ability. High glucose induced an increase in the levels of the pluripotent factor SOX9 and a decrease in SIRT6 levels accompanied by the increase in the acetylation levels of H3K9. Inhibiting SIRT6 expression by siRNA rendered an increase in SOX9 levels. We also determined SOX9 levels in retinas from mice with a conditional deletion of SIRT6 in the CNS. To further understand the mechanisms that regulate MGs response under metabolic impaired conditions, we evaluated the gene expression profile and performed Gene Ontology enrichment analysis of Müller cells from a murine model of Diabetes. We found several differentially expressed genes and observed that the transcriptomic change involved the enrichment of genes associated with glucose metabolism, cell migration, development and pluripotency. We found that many functional categories affected in cells of diabetic animals were directly related to SIRT6 function. Transcription factors enrichment analysis allowed us to predict several factors, including SOX9, that may be involved in the modulation of the differential expression program observed in diabetic MGs. Our results underline the heterogeneity of Müller cells response and the challenge that the study of metabolic impairment in vivo represents.
RESUMO
Aquaporin-4 (AQP4) is the target of the specific immunoglobulin G autoantibody (AQP4-IgG) produced in patients with neuromyelitis optica spectrum disorders (NMOSD). Previous studies demonstrated that AQP4-IgG binding to astrocytic AQP4 leads to cell-destructive lesions. However, the early physiopathological events in Müller cells in the retina are poorly understood. Here, we investigated the consequences of AQP4-IgG binding to AQP4 of Müller cells, previous to the inflammatory response, on two of AQP4's key functions, cell volume regulation response (RVD) and cell proliferation, a process closely associated with changes in cell volume. Experiments were performed in a human retinal Müller cell line (MIO-M1) exposed to complement-inactivated sera from healthy volunteers or AQP4-IgG positive NMOSD patients. We evaluated AQP4 expression (immunofluorescence and western blot), water permeability coefficient, RVD, intracellular calcium levels and membrane potential changes during hypotonic shock (fluorescence videomicroscopy) and cell proliferation (cell count and BrdU incorporation). Our results showed that AQP4-IgG binding to AQP4 induces its partial internalization, leading to the decrease of the plasma membrane water permeability, a reduction of swelling-induced increase of intracellular calcium levels and the impairment of RVD in Müller cells. The loss of AQP4 from the plasma membrane induced by AQP4-IgG positive sera delayed Müller cells' proliferation rate. We propose that Müller cell dysfunction after AQP4 removal from the plasma membrane by AQP4-IgG binding could be a non-inflammatory mechanism of retinal injury in vivo, altering cell volume homeostasis and cell proliferation and consequently, contributing to the physiopathology of NMOSD.
Assuntos
Aquaporina 4/sangue , Membrana Celular/metabolismo , Células Ependimogliais/metabolismo , Imunoglobulina G/metabolismo , Neuromielite Óptica/sangue , Retina/metabolismo , Aquaporina 4/administração & dosagem , Biomarcadores/sangue , Linhagem Celular Transformada , Membrana Celular/patologia , Proliferação de Células/fisiologia , Tamanho Celular , Células Ependimogliais/patologia , Homeostase/fisiologia , Humanos , Imunoglobulina G/administração & dosagem , Neuromielite Óptica/patologia , Retina/lesões , Retina/patologiaRESUMO
Aortic cross-clamping is a common strategy during vascular surgery, however, its instantaneous impact on hemodynamics is unknown. We, therefore, developed two numerical models to estimate the immediate impact of aortic clamping on the vascular properties. To assess the validity of the models, we recorded continuous invasive pressure signals during abdominal aneurysm repair surgery, immediately before and after clamping. The first model is a zero-dimensional (0D) three-element Windkessel model, which we coupled to a gradient-based parameter estimation algorithm to identify patient-specific parameters such as vascular resistance and compliance. We found a 10% increase in the total resistance and a 20% decrease in the total compliance after clamping. The second model is a nine-artery network corresponding to an average human body in which we solved the one-dimensional (1D) blood flow equations. With a similar parameter estimation method and using the results from the 0D model, we identified the resistance boundary conditions of the 1D network. Determining the patient-specific total resistance and the distribution of peripheral resistances through the parameter estimation process was sufficient for the 1D model to accurately reproduce the impact of clamping on the pressure waveform. Both models gave an accurate description of the pressure wave and had a high correlation (R2 > .95) with experimental blood pressure data.
Assuntos
Aorta , Hemodinâmica , Pressão Sanguínea , Constrição , Humanos , Resistência VascularRESUMO
Collecting ducts (CD) not only constitute the final site for regulating urine concentration by increasing apical membrane Aquaporin-2 (AQP2) expression, but are also essential for the control of acid-base status. The aim of this work was to examine, in renal cells, the effects of chronic alkalosis on cell growth/death as well as to define whether AQP2 expression plays any role during this adaptation. Two CD cell lines were used: WT- (not expressing AQPs) and AQP2-RCCD(1) (expressing apical AQP2). Our results showed that AQP2 expression per se accelerates cell proliferation by an increase in cell cycle progression. Chronic alkalosis induced, in both cells lines, a time-dependent reduction in cell growth. Even more, cell cycle movement, assessed by 5-bromodeoxyuridine pulse-chase and propidium iodide analyses, revealed a G2/M phase cell accumulation associated with longer S- and G2/M-transit times. This G2/M arrest is paralleled with changes consistent with apoptosis. All these effects appeared 24 h before and were always more pronounced in cells expressing AQP2. Moreover, in AQP2-expressing cells, part of the observed alkalosis cell growth decrease is explained by AQP2 protein down-regulation. We conclude that in CD cells alkalosis causes a reduction in cell growth by cell cycle delay that triggers apoptosis as an adaptive reaction to this environment stress. Since cell volume changes are prerequisite for the initiation of cell proliferation or apoptosis, we propose that AQP2 expression facilitates cell swelling or shrinkage leading to the activation of channels necessary to the control of these processes.
Assuntos
Adaptação Fisiológica , Alcalose/patologia , Alcalose/fisiopatologia , Apoptose , Aquaporina 2/metabolismo , Ciclo Celular , Túbulos Renais Coletores/patologia , Animais , Proliferação de Células , Córtex Renal/patologia , Ratos , Fatores de TempoRESUMO
BACKGROUND INFORMATION: A major hallmark of apoptosis is cell shrinkage, termed apoptotic volume decrease, due to the cellular outflow of potassium and chloride ions, followed by osmotically obliged water. In many cells, the ionic pathways triggered during the apoptotic volume decrease may be similar to that observed during a regulatory volume decrease response under hypotonic conditions. However, the pathways involved in water loss during apoptosis have been largely ignored. It was recently reported that in some systems this water movement is mediated via specific water channels (aquaporins). Nevertheless, it is important to identify whether this is a ubiquitous aspect of apoptosis as well as to define the mechanisms involved. The aim of the present work was to investigate the role of aquaporin-2 during apoptosis in renal-collecting duct cells. We evaluated the putative relationship between aquaporin-2 expression and the activation of the ionic pathways involved in the regulatory volume response. RESULTS: Apoptosis was induced by incubating cells with a hypertonic solution or with cycloheximide in two cortical collecting duct cell lines: one not expressing aquaporins and the other stably transfected with aquaporin-2. Typical features of apoptosis were evaluated with different approaches and the water permeability was measured by fluorescence videomicroscopy. Our results show that the rate of apoptosis is significantly increased in aquaporin-2 cells and it is linked to the rapid activation of volume-regulatory potassium and chloride channels. Furthermore, the water permeability of cells expressing aquaporin-2 was strongly reduced during the apoptotic process and it occurs before DNA degradation. CONCLUSIONS: These results let us propose that under apoptotic stimulation aquaporin-2 would act as a sensor leading to a co-ordinated activation of specific ionic channels for potassium and chloride efflux, resulting in both more rapid cell shrinkage and more rapid achievement of adequate levels of ions necessary to activate the enzymatic apoptotic cascade.
Assuntos
Apoptose/fisiologia , Aquaporina 2/metabolismo , Túbulos Renais Coletores , Animais , Aquaporina 2/genética , Compostos de Bário/metabolismo , Linhagem Celular , Permeabilidade da Membrana Celular , Tamanho Celular , Cloretos/metabolismo , Cicloeximida/metabolismo , Fragmentação do DNA , Glibureto/metabolismo , Humanos , Canais Iônicos/metabolismo , Túbulos Renais Coletores/citologia , Túbulos Renais Coletores/metabolismo , Concentração Osmolar , Ratos , Água/metabolismoRESUMO
We have previously shown in renal cells that expression of the water channel Aquaporin-2 increases cell proliferation by a regulatory volume mechanism involving Na+/H+ exchanger isoform 2. Here, we investigated if Aquaporin-2 (AQP2) also modulates Na+/H+ exchanger isoform 1-dependent cell proliferation. We use two AQP2-expressing cortical collecting duct models: one constitutive (WT or AQP2-transfected RCCD1 cell line) and one inducible (control or vasopressin-induced mpkCCDc14 cell line). We found that Aquaporin-2 modifies Na+/H+ exchanger isoform 1 (NHE1) contribution to cell proliferation. In Aquaporin-2-expressing cells, Na+/H+ exchanger isoform 1 is anti-proliferative at physiological pH. In acid media, Na+/H+ exchanger isoform 1 contribution turned from anti-proliferative to proliferative only in AQP2-expressing cells. We also found that, in AQP2-expressing cells, NHE1-dependent proliferation changes parallel changes in stress fiber levels: at pH 7.4, Na+/H+ exchanger isoform 1 would favor stress fiber disassembly and, under acidosis, NHE1 would favor stress fiber assembly. Moreover, we found that Na+/H+ exchanger-dependent effects on proliferation linked to Aquaporin-2 relied on Transient Receptor Potential Subfamily V calcium channel activity. In conclusion, our data show that, in collecting duct cells, the water channel Aquaporin-2 modulates NHE1-dependent cell proliferation. In AQP2-expressing cells, at physiological pH, the Na+/H+ exchanger isoform 1 function is anti-proliferative and, at acidic pH, Na+/H+ exchanger isoform 1 function is proliferative. We propose that Na+/H+ exchanger isoform 1 modulates proliferation through an interplay with stress fiber formation.
Assuntos
Aquaporina 2/fisiologia , Proliferação de Células , Células Epiteliais/citologia , Túbulos Renais Coletores/citologia , Trocador 1 de Sódio-Hidrogênio/fisiologia , Animais , Linhagem Celular , Concentração de Íons de Hidrogênio , Isoformas de Proteínas/fisiologia , RatosRESUMO
OBJECTIVES: Cardiopulmonary bypass (CPB) use is an essential strategy for many cardiovascular surgeries. However, its use and duration have been associated with a higher rate of postoperative complications, such as low cardiac output syndrome due to myocardial oedema and dysfunction. Though Aquaporin water channels have been implicated in myocardial water balance, their specific role in this clinical scenario has not been established. METHODS: In a consecutive study of 17 patients with severe aortic stenosis undergoing aortic valve replacement surgery, 2 myocardial biopsies of the left ventricle were taken: 1 before and 1 after CPB use. Sociodemographic, clinical and laboratory data were collected. Western blot and immunohistochemistry studies were performed. RESULTS: After CPB use, there was a mean increase of â¼62% in Aquaporin 1 protein levels (P = 0.001) and a mean reduction of â¼38% in Aquaporin 4 protein levels (P = 0.030). In immunohistochemistry assays, Aquaporin 1 was found lining small blood vessels, while Aquaporin 4 formed a circular label in cardiomyocytes. There were no changes in the localization of either protein following CPB use. During the observed on-pump time interval, there was a 1.7%/min mean increase in Aquaporin 1 (P = 0.021) and a 2.5%/min mean decrease in Aquaporin 4 (P = 0.018). Myocardial interstitial oedema increased by 42% (95% confidence interval 31-54%) after CPB use. Patients who developed low cardiac output syndrome were in the upper half of the median percentage change of Aquaporin expression. CONCLUSION: Time-dependent changes in cardiac Aquaporin expression may be associated with myocardial oedema and dysfunction related to CPB use.