P2X7 receptor-nitric oxide interaction mediates apoptosis in mouse immortalized mesangial cells exposed to high glucose / A interação do receptor P2X7 com o óxido nítrico medeia a apoptose em células mesangiais imortalizadas de camundongos expostas a níveis elevados de glicose

Abstract Introduction: Diabetes mellitus (DM) is a chronic disease characterized by hyperglycemia that leads to diabetic nephropathy (DN). We showed that P2X7, a purinergic receptor, was highly expressed in DM; however, when oxidative stress was controlled, renal NO recovered, and the activation of this receptor remained significantly reduced. The aim of this study was to assess the influence of NO on the P2X7 and apoptosis in mouse immortalized mesangial cells (MiMC) cultured in high glucose (HG) medium. Methods: MiMCs were cultured with DMEM and exposed to normal glucose (NG), mannitol (MA), or HG. Cell viability was assessed by an automated counter. Supernatants were collected for NO quantification, and proteins were extracted for analysis of NO synthases (iNOS and eNOS), caspase-3, and P2X7. Results: Cell viability remained above 90% in all groups. There was a significant increase in the proliferation of cells in HG compared to MA and NG. NO, iNOS, caspase-3, and P2X7 were significantly increased in HG compared to NG and MA, with no changes in eNOS. We observed that there was a strong and significant correlation between P2X7 and NO. Discussion: The main finding was that the production of NO by iNOS was positively correlated with the increase of P2X7 in MCs under HG conditions, showing that there is a common stimulus between them and that NO interacts with the P2X7 pathway, contributing to apoptosis in experimental DM. These findings could be relevant to studies of therapeutic targets for the prevention and/or treatment of hyperglycemia-induced kidney damage to delay DN progression.

Resumo Introdução: Diabetes mellitus (DM) é uma doença crônica caracterizada por hiperglicemia levando à nefropatia diabética (ND). Mostramos que P2X7, um receptor purinérgico, foi altamente expresso na DM; entretanto, quando o estresse oxidativo foi controlado, o NO renal recuperou-se, e a ativação deste receptor permaneceu significativamente reduzida. Este estudo objetivou avaliar a influência do NO no P2X7 e a apoptose em células mesangiais imortalizadas de camundongos (CMiC) cultivadas em meio de glicose elevada (GE). Métodos: CMiCs foram cultivadas em meio DMEM e expostas à glicose normal (GN), manitol (MA), ou GE. A viabilidade celular foi avaliada por contador automático. Sobrenadantes foram coletados para quantificação de NO, e foram extraídas proteínas para análise de NO sintases (iNOS e eNOS), caspase-3, e P2X7. Resultados: A viabilidade celular permaneceu acima de 90% em todos os grupos. Houve aumento significativo na proliferação de células na GE comparado com MA e GN. NO, iNOS, caspase-3 e P2X7 foram significativamente aumentados na GE comparados com GN e MA, sem alterações na eNOS. Observamos que houve correlação forte e significativa entre P2X7 e NO. Discussão: O principal achado foi que a produção de NO pela iNOS foi positivamente correlacionada com aumento de P2X7 em CMs sob condições de GE, mostrando que existe um estímulo comum entre eles e que o NO interage com a via do P2X7, contribuindo para apoptose na DM experimental. Estes achados podem ser relevantes para estudos de alvos terapêuticos para a prevenção e/ou tratamento de danos renais induzidos por hiperglicemia para retardar a progressão da ND.

Relationship between short and long-term glycemic variability and oxidative stress in type 1 diabetes mellitus under daily life insulin treatment

ABSTRACT Objectives: The purpose of this study was to investigate the heterogeneity of the association between glycemic variability and oxidative stress markers in T1DM patients under daily life insulin treatment. Subjects and methods: We studied, in a cross-sectional analysis, 76 T1DM patients without clinical chronic diabetes complications and 22 healthy individuals. Were evaluated the short-term glycemic variability (STGV), long-term glycemic variability (LTGV), oxidative stress markers [8-isoprostaglandin-F2α (Ur-8-iso-PGF2α), nitric oxide (NO), thiobarbituric acid reactive substances (TBARS) and erythrocytes reduced/oxidized glutathione (GSH/GSSG)] and biochemical dosages (glycaemia, HbA1c, lipidogram, albuminuria). Results: Plasmatic NO was positively associated with LTGV (last year average of HbA1c) (8.7 ± 1.6% or 71 ± 18 mmol) (rS: 0.278; p: 0.042). Plasmatic TBARS, erythrocytes GSH/GSSH and Ur-8-iso-PGF-2α didn't show correlation with glycemic variability. GSH/GSSG was inversely correlated with LDL-cholesterol (rS: - 0.417; p: 0.047) and triglycerides (rS: −0.521; p: 0.013). Albuminuria was positive correlated with age (rS: 0.340; p: 0.002), plasmatic NO (rS: 0.267; p 0.049) and TBARS (rS: 0.327; p: 0.015). Conclusion: In daily life insulin treatment, young T1DM patients have higher plasmatic NO than healthy subjects. However, the correlation between glycemic variability and oxidative stress markers is heterogeneous. Lipid profile and albuminuria are associated with different oxidative stress markers. These data collaborate to explain the controversial results in this issue.

Cbfa1 expression in vascular smooth muscle cells may be elevated by increased nitric oxide/iNOS.

INTRODUCTION: Vascular calcification is a common complication of chronic kidney disease. Osteoblast differentiation factor (Cbfa1) is present in histologic sections of arteries from patients with end-stage renal disease. Vascular smooth muscle cells (VSMC) can dedifferentiate to osteoblast-like cells, possibly by up-regulation of Cbfa1. There is evidence that the production of nitric oxide (NO) may have an important role in the regulation of osteoblast metabolism. The aim of this study is to evaluate whether increased NO/iNOS expression causes an increase in cbfa1 expression in VSMC. METHODS: VSMC were obtained from renal artery of Wistar male rats, treated for 72 hours with lipopolysaccharide (LPS), ß-glycerophosphate (BGF), a donor of phosphate and aminoguanidine (AG), an inhibitor of iNOS, in the following groups: CTL (control), LPS, BGF, LPS + BGF, and LPS + AG. NO synthesis was determined by chemiluminescence. Cbfa1 and iNOS mRNA expressions were analyzed by RT-PCR, Cbfa1 protein expression by immunohistochemistry and cellular viability by acridine orange. RESULTS: Cbfa1 and iNOS mRNA expressions were higher in LPS and LPS+ BGF vs CTL (p < 0.05), and they were lower in LPS+AG vs LPS (p < 0.05). The Cbfa1 in the groups LPS and LPS+BGF also resulted in a higher value compared to CTL (p < 0.05), and in LPS+AG it was lower compared to LPS (p < 0.05). NO was higher in LPS and LPS+BGF compared to CTL group (p < 0.05) and lower in LPS + AG compared to LPS group (p < 0.05). Cellular viability showed no statistical difference among groups. CONCLUSION: This study showed that increased NO/iNOS expression causes an increase in cbfa1 expression in VSMC.

P2X7 receptor and klotho expressions in diabetic nephropathy progression.

Diabetes mellitus is characterized by increased levels of reactive oxygen species (ROS), leading to high levels of adenosine triphosphate (ATP) and the activation of purinergic receptors (P2X7), which results in cell death. Klotho was recently described as a modulator of oxidative stress and as having anti-apoptotic properties, among others. However, the roles of P2X7 and klotho in the progression of diabetic nephropathy are still unclear. In this context, the aim of the present study was to characterize P2X7 and klotho in several stages of diabetes in rats. Diabetes was induced in Wistar rats by streptozotocin, while the control group rats received the drug vehicle. From the 1st to 8th weeks after the diabetes induction, the animals were placed in metabolic cages on the 1st day of each week for 24 h to analyze metabolic parameters and for the urine collection. Then, blood samples and the kidneys were collected for biochemical analysis, including Western blotting and qPCR for P2X7 and klotho. Diabetic rats presented a progressive loss of renal function, with reduced nitric oxide and increased lipid peroxidation. The P2X7 and klotho expressions were similar up to the 4th week; then, P2X7 expression increased in diabetes mellitus (DM), but klotho expression presented an opposite behavior, until the 8th week. Our data show an inverse correlation between P2X7 and klotho expressions through the development of DM, which suggests that the management of these molecules could be useful for controlling the progression of this disease and diabetic nephropathy.

Short-term l-arginine supplementation attenuates elevation of interleukin 6 level after resistance exercise in overweight men.

BACKGROUND AND AIM: l-Arginine (l-arg) supplementation and resistance exercise can induce changes in inflammatory and anti-inflammatory cytokines; however, it has not been investigated in obese hypertensive men. This study examines the effects of short-term l-arg supplementation and acute resistance exercise (AREX) on cytokine levels in obese hypertensive men. METHODS: Eight obese hypertensive men aged 46 ± 6 yrs. with an average body weight of 92.56 ± 9.9 kg and a BMI of 31.68 ± 2.18 kg/m2 participated in a randomized, double-blinded, crossover study. The patients were distributed into exercise groups based on the type of supplementation (6 g/day of placebo or l-arg for 7 days). Supplementation periods were separated by a seven-day washout period. The AREX regimen consisted of eight exercises with an exercise intensity of 60% of 1 repetition maximum. The interleukins IL-1ra, IL-6, and IL-10 and the IL-6/IL10 ratio were determined at rest, immediately after exercise and 1 h after exercise sessions. RESULTS: IL-1ra levels exhibited a significant difference both immediately and 1 h after exercise when the l-arg and placebo groups were compared (P < 0.05). IL-6 levels increased significantly after exercise in the placebo group compared with the l-arg group (P < 0.05). The placebo group showed a decrease in the IL-10 levels 1 h after exercise compared with resting levels (P < 0.05). The IL-6/IL-10 ratio showed a statistically significant increase in the placebo group after exercise compared to the l-arg group (P < 0.05). CONCLUSIONS: LARG supplementation attenuates the cytokine increase after AREX, in particular peak IL-6 levels decrease and exercise induced decreases in IL-10 levels are attenuated.

Exercise capacity in polycystic kidney disease.

BACKGROUND: Reports about exercise performance in autosomal dominant polycystic kidney disease (ADPKD) are scarce. We aimed to evaluate exercise capacity and levels of nitric oxide and asymmetric dimethylarginine (ADMA) in normotensive patients with ADPKD. STUDY DESIGN: Prospective controlled cohort study. SETTING & PARTICIPANTS: 26 patients with ADPKD and 30 non-ADPKD control participants (estimated glomerular filtration rate>60 mL/min/1.73 m2, aged 19-39 years, and blood pressure [BP]<140/85 mmHg). We excluded smokers, obese people, and individuals with associated diseases. PREDICTOR: ADPKD versus control. OUTCOMES: Exercise capacity and nitric oxide and ADMA levels in response to exercise. MEASUREMENTS: Cardiopulmonary exercise testing and serum and urinary nitric oxide, plasma ADMA, and BP levels before and after exercise. RESULTS: Mean basal systolic and diastolic BP, estimated glomerular filtration rate, and age did not differ between the ADPKD and control groups (116±12 vs. 110±11 mmHg, 76±11 vs 71±9 mmHg, 113±17 vs. 112±9.6 mL/min/1.73 m2, and 30±8 vs. 28.9±7.3 years, respectively). Peak oxygen uptake and anaerobic threshold were significantly lower in the ADPKD group than in controls (22.2±3.3 vs. 31±4.8 mL/kg/min [P<0.001] and 743.6±221 vs. 957.4±301 L/min [P=0.01], respectively). Postexercise serum and urinary nitric oxide levels in patients with ADPKD were not significantly different from baseline (45±5.1 vs. 48.3±4.6 µmol/L and 34.7±6.5 vs. 39.8±6.8 µmol/mg of creatinine, respectively), contrasting with increased postexercise values in controls (63.1±1.9 vs. 53.9±3.1 µmol/L [P=0.01] and 61.4±10.6 vs. 38.7±5.6 µmol/mg of creatinine [P=0.01], respectively). Similarly, whereas postexercise ADMA level did not change in the ADPKD group compared to those at rest (0.47±0.04 vs. 0.45±0.02 µmol/L [P=0.6]), it decreased in controls (0.39±0.02 vs. 0.47±0.02 µmol/L [P=0.006]), as expected. A negative correlation between nitric oxide and ADMA levels after exercise was found in only the control group (r = -0.60; P<0.01). LIMITATIONS: Absence of measurements of flow-mediated dilatation and oxidative status. CONCLUSIONS: We found lower aerobic capacity in young normotensive patients with ADPKD with preserved kidney function and inadequate responses of nitric oxide and ADMA levels to acute exercise, suggesting the presence of early endothelial dysfunction in this disease.

Nitric oxide levels in cochlear fluids of guinea pig

PURPOSE: To describe a technique to obtain guinea pigs cochlear fluids and measure nitric oxide (NO) concentration. METHODS: Six guinea pigs were used and sacrificed. The cochlear fluids collected for measurement of NO, performed by chemiluminescence (NOA 280). RESULTS: Through the chemiluminescence was possible to analyze the concentration of NO in cochlear fluids obtained. Average levels of nitric oxide from guinea pigs was 12.55 µM. CONCLUSION: It is possible to obtain nitric oxide cochlear fluids, with this technique and nitric oxide concentration measure by chemiluminescence, a quantitative and more precise method.

Sintomas depressivos em pacientes com síndrome coronariana aguda / Depressive symptoms in patients with acute coronary syndrome

Objective: To trace an epidemiological profile, to verify presence of depressive symptoms in patients with previous diagnosis of acute coronary syndrome and to identify factors that contribute to maintenance of depressive symptoms in the sample. Methods: A cross-section study carried out at the Cardiology Outpatients Clinics of Universidade Federal de São Paulo. An instrument prepared by the authors was used, which was based on similar studies with patient identification data, questions related to the psychological follow-up, relationship with family members and friends, in addition to use of the Beck Depression Inventory. Results: A total of 200 patients were interviewed, 127 (63.5%) were male. The mean age was 60.19 years with a standard deviation of 9.38, minimum age of 36 years and maximum of 81 years; 164 (82%) denied any follow-up with a psychologist or psychiatrist in the phase after acute coronary syndrome diagnosis and treatment. In the utilization of Beck Depression Inventory, 67 (33.5%) presented scores between 0 and 4, indicating mild depressive symptoms; 72 (36%) had scores between 5 and 9, indicating mild to moderate depressive symptoms, and 61 (30.5%) presented scores greater than 9, which point out moderate to severe depressive symptoms. Conclusion: The evaluation and multiprofessional follow-up can help patients cope with the illness in addition to providing greater compliance to drug therapy and beginning changes in life habits.

Objetivo: Traçar o perfil epidemiológico da amostra, verificar a presença de sintomas de depressão em pacientes com diagnóstico prévio de síndrome coronariana aguda e identificar os fatores contribuintes para a manutenção dos sintomas de depressão naamostra estudada. Métodos: Estudo transversal realizado no Ambulatório de Cardiologia da Universidade Federal de São Paulo. Foi aplicado um instrumento elaborado pelos autores, baseado em estudos semelhantes, com dados de identificação dos pacientes, questões relacionadas ao acompanhamento psicológico, relacionamento com familiares e amigos, além da aplicação do Inventário de Depressão de Beck. Resultados: A amostra constou de 200 pacientes, sendo 127 (63,5%) do sexo masculino. A média de idade foi de 60,19 anos com um desvio padrão de 9,38, com idade mínima de 36 e máxima de 81 anos; 164 (82%) negaram ter feito qualquer acompanhamento com psicólogo ou psiquiatra na fase após o diagnóstico e tratamento da síndrome coronariana aguda. Na aplicação do Inventário de Depressão de Beck, 67 (33,5%) apresentaram escores entre 0 e 4, indicando sintomas leves de depressão; 72 (36%) apresentaram escores entre 5 e 9, indicando sintomas leves a moderados; e 61 (30,5%) apresentaram escores maiores que 9, indicando sintomas moderados a graves. Conclusão: Avaliação e acompanhamento multiprofissional podem ajudar o paciente no enfrentamento da doença além de proporcionar maior adesão à terapia farmacológica e início das mudanças de hábitos de vida.

Relationship between fluid-attenuated inversion-recovery (FLAIR) signal intensity and inflammatory mediator's levels in the hippocampus of patients with temporal lobe epilepsy and mesial temporal sclerosis.

We investigated a relationship between the FLAIR signal found in mesial temporal sclerosis (MTS) and inflammation. Twenty nine patients were selected through clinical and MRI analysis and submitted to cortico-amygdalo-hippocampectomy to seizure control. Glutamate, TNFα, IL1, nitric oxide (NO) levels and immunostaining against IL1ß and CD45 was performed. Control tissues (n=10) were obtained after autopsy of patients without neurological disorders. The glutamate was decreased in the temporal lobe epilepsy (TLE) -MTS group (p<0.001), suggesting increased release of this neurotransmitter. The IL1ß and TNFα were increased in the hippocampus (p<0.05) demonstrating an active inflammatory process. A positive linear correlation between FLAIR signal and NO and IL1ß levels and a negative linear correlation between FLAIR signal and glutamate concentration was found. Lymphocytes infiltrates were present in hippocampi of TLE patients. These data showed an association between hippocampal signal alteration and increased inflammatory markers in TLE-MTS.

Relationship between fluid-attenuated inversion-recovery (FLAIR) signal intensity and inflammatory mediator's levels in the hippocampus of patients with temporal lobe epilepsy and mesial temporal sclerosis / Correlação da intensidade do sinal em FLAIR e os níveis de mediadores inflamatórios no hipocampo de pacientes com epilepsia do lobo temporal e esclerose mesial temporal

We investigated a relationship between the FLAIR signal found in mesial temporal sclerosis (MTS) and inflammation. Twenty nine patients were selected through clinical and MRI analysis and submitted to cortico-amygdalo-hippocampectomy to seizure control. Glutamate, TNFα, IL1, nitric oxide (NO) levels and immunostaining against IL1β and CD45 was performed. Control tissues (n=10) were obtained after autopsy of patients without neurological disorders. The glutamate was decreased in the temporal lobe epilepsy (TLE) -MTS group (p<0.001), suggesting increased release of this neurotransmitter. The IL1β and TNFα were increased in the hippocampus (p<0.05) demonstrating an active inflammatory process. A positive linear correlation between FLAIR signal and NO and IL1β levels and a negative linear correlation between FLAIR signal and glutamate concentration was found. Lymphocytes infiltrates were present in hippocampi of TLE patients. These data showed an association between hippocampal signal alteration and increased inflammatory markers in TLE-MTS.

Este estudo foi delineado para investigar a presença de relação entre a intensidade de sinal em FLAIR e níveis de citocinas, óxido nítrico (NO) e glutamato no hipocampo de pacientes com epilepsia do lobo temporal refratária, associada com esclerose mesial (TLE-MTS). Vinte e nove pacientes foram selecionados através de análise clínica e de ressonância magnética (RM) que foram submetidos a cortico-amigdalo-hipocampectomia para o controle das crises. Os níveis de glutamato foram avaliados por HPLC, as citocinas TNFα e IL1β por ELISA e os níveis de NO via NO system. Avaliamos também por imuno-histoquímica a expressão de IL1β e CD45 em tecidos controles e com esclerose. Tecido controle foi obtido após autópsia de indivíduos mortos sem disfunções inflamatórias e neurológicas (n=10). A concentração de glutamato se mostrou reduzida no tecido TLE-MTS (p<0,001) sugerindo aumento na liberação desse neurotransmissor. TNFα e IL1β também apresentaram níveis elevados no hipocampo dos pacientes (p<0,05), demonstrando um processo inflamatório crônico. Houve uma correlação linear positiva entre a intensidade do sinal em FLAIR e os níveis de NO e IL1β. Em contraste, uma correlação linear negativa foi encontrada entre a intensidade do sinal em FLAIR e níveis de glutamato no hipocampo com esclerose. Infiltrado linfocitário hipocampal também foi visualizado pela imuno-marcação com CD45 em pacientes com TLE-MTS. Esses dados mostraram uma associação entre alteração de sinal na RM e marcadores inflamatórios em pacientes com TLE-MTS.

Suplementação proteica não aumenta a concentração plasmática de óxido nítrico em homens saudáveis / Protein supplementation does not increase nitric oxide plasmatic concentration in healthy men

Suplementos nutricionais, supostamente, capazes de potencializar a produção endógena de óxido nítrico (NO) têm experimentado crescente popularidade entre os indivíduos fisicamente ativos. Diante da carência de informações sobre o assunto, o objetivo do presente estudo foi avaliar o efeito de um suplemento comercial à base de proteínas e aminoácidos sobre a produção endógena de NO. MÉTODOS: A amostra foi constituída de 12 homens sedentários, mas sem fatores de risco para doenças cardiovasculares. O protocolo de suplementação foi conduzido conforme o arranjo experimental duplo-cego cruzado. Os participantes receberam, aleatoriamente, placebo (PLA) ou suplemento proteico (SP), em dois momentos diferentes, separados por uma semana. Com o intuito de determinar a concentração plasmática de NO, amostras de sangue foram coletadas antes (24h e imediatamente antes) e depois (30 e 60 minutos) do consumo da substância PLA ou do SP. RESULTADOS: Não foi observada alteração na concentração plasmática de NO após a ingestão do SP em comparação com o PLA (pós- suplementação 30min - PLA: 19,3 ± 4,7µmol.L- 1 vs. SP: 18,9 ± 4,4µmol.L-1 e pós-suplementação 60min - PLA: 21,3 ± 6,5µmol.L-1 vs. SP: 20,3 ± 4,9µmol.L-1). Também não foi verificada alteração da pressão arterial. CONCLUSÃO: O suplemento nutricional à base de proteínas e aminoácidos, testado no presente estudo, não potencializou a produção endógena de NO.

Nutritional supplements, theoretically able to increase endogenous nitric oxide (NO) production have experienced great popularity among physically active individuals. AIM: scientific evidence available regarding this issue is scarce. Therefore, the purpose of this study was to evaluate the effect of a dietary supplement commercialized as a nitric oxide booster. MATERIALS AND METHODS: twelve sedentary men with no risk factors for cardiovascular diseases were supplemented with placebo or protein in two different occasions. The present study was conducted in a cross double-blind design. In order to assess plasmatic NO concentration, blood samples were obtained before (24hs and immediately before) and after (30 and 60 minutes) consumption of placebo (PLA) or protein supplement (SP). RESULTS: there was no difference in plasmatic nitric oxide concentration between both trails (Post-supplementation 30 min - PLA: 19.3±4.7 µmol.L-1 vs. SP: 18.9±4.4 µmol.L-1 and Post-supplementation 60 min - PLA: 21.3±6.5 µmol.L-1 vs. SP: 20.3±4.9 µmol.L-1). In addition, no difference was detected for arterial blood pressure. CONCLUSION: the dietary supplement analyzed in the present study failed to increase nitric oxide endogenous production.

Cyclosporine A and NAC on the inducible nitric oxide synthase expression and nitric oxide synthesis in rat renal artery cultured cells.

BACKGROUND: The immunosuppressor cyclosporine A (CsA) presents the nephrotoxicity as its major side effect that is mostly attributed to a renal vasoconstriction. This may be due to an excessive generation of vasoconstrictors like reactive oxygen species (ROS), or due to a reduction of vasodilators such as the nitric oxide, which in turn, can be caused by increased amounts of ROS. We evaluated the effect of CsA and the antioxidant N-acetylcysteine (NAC) on inducible nitric oxide synthase (iNOS) mRNA expression and nitric oxide synthesis, in rat renal artery vascular smooth muscle cells (rVSMCs) primary culture. METHODS: In cells treated during 72 hours with CsA (10 microg/mL), its vehicle (control) (10 microL/mL), Escherichia coli lipopolysaccharide (LPS) (100 microg/mL), CsA + LPS, NAC (6.13 mmol/L), or CsA + NAC, we determined the nitric oxide synthesis (Griess and chemiluminescence methods), iNOS expression [reverse transcription-polymerase chain reaction (RT-PCR)] and cell viability (acridine orange method). RESULTS: In rVSMCs, LPS increased nitric oxide and iNOS expression; CsA decreased basal and LPS-induced nitric oxide and iNOS expression; NAC increased nitric oxide and blunted the nitric oxide reduction caused by CsA, with no effect on iNOS. CsA reduced cell viability. CONCLUSION: In this study, CsA reduced nitric oxide synthesis in rVSMCs, both through iNOS down-regulation and reduction of cell viability, which could be responsible for the vasoconstrictive effect of the CsA. In the effect of CsA on nitric oxide, probably a role is also played by free radical production, as this effect was blunted by NAC.