RESUMO
Quiescin/sulfhydryl oxidase (QSOX1) is a secreted flavoprotein that modulates cellular proliferation, migration and adhesion, roles attributed to its ability to organize the extracellular matrix. We previously showed that exogenously added QSOX1b induces smooth muscle cells migration in a process that depends on its enzymatic activity and that is mediated by hydrogen peroxide derived from Nox1, a catalytic subunit of NAD(P)H oxidases. Here, we report that exogenous QSOX1b also stimulates the migration of L929 fibroblasts and that this effect is regulated by its endocytosis. The use of endocytosis inhibitors and caveolin 1-knockdown demonstrated that this endocytic pathway is caveola-mediated. QSOX1b colocalized with Nox1 in intracellular vesicles, as detected by confocal fluorescence, suggesting that extracellular QSOX1b is endocytosed with the transmembrane Nox1. These results reveal that endosomal QSOX1b is a novel intracellular redox regulator of cell migration.
Assuntos
Cavéolas , NADPH Oxidases , Fibroblastos , Endocitose , Proliferação de CélulasRESUMO
BACKGROUND: Patients with chronic kidney disease (CKD) have reduced expression of erythroid nuclear factor-related factor 2 (NRF2) and increased nuclear factor κB (NF-κB). "Food as medicine" has been proposed as an adjuvant therapeutic alternative in modulating these factors. No studies have investigated the effects of sulforaphane (SFN) in cruciferous vegetables on the expression of these genes in patients with CKD. OBJECTIVE: The study aimed to evaluate the effects of SFN on the expression of NRF2 and NF-κB in patients on hemodialysis (HD). DESIGN AND METHODS: A randomized, double-blind, crossover study was performed on 30 patients on regular HD. Fourteen patients were randomly allocated to the intervention group (1 sachet/day of 2.5 g containing 1% SFN extract with 0.5% myrosinase) and 16 patients to the placebo group (1 sachet/day of 2.5 g containing corn starch colored with chlorophyll) for 2 months. After a washout period of 2 months, the groups were switched. NRF2 and NF-κB mRNA expression was evaluated by real-time quantitative polymerase chain reaction, and tumor necrosis factor alpha and interleukin-6 levels were quantified by enzyme-linked immunosorbent assay. Malondialdehyde was evaluated as a marker of lipid peroxidation. RESULTS: Twenty-five patients (17 women, 55 [interquartile range = 19] years and 55 [interquartile range = 74] months on HD) completed the study. There was no significant difference concerning the expression of mRNA NRF2 (P = .915) and mRNA NF-κB (P = .806) after supplementation with SFN. There was no difference in pro-inflammatory and oxidative stress biomarkers. CONCLUSION: 150 µmol of SFN for 2 months had no antioxidant and anti-inflammatory effect in patients with CKD undergoing HD.
Assuntos
Isotiocianatos , NF-kappa B , Insuficiência Renal Crônica , Sulfóxidos , Humanos , Feminino , NF-kappa B/genética , NF-kappa B/metabolismo , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Estudos Cross-Over , Estresse Oxidativo , Diálise Renal/efeitos adversos , Insuficiência Renal Crônica/tratamento farmacológico , Insuficiência Renal Crônica/etiologia , RNA Mensageiro/metabolismo , RNA Mensageiro/farmacologia , Suplementos NutricionaisRESUMO
A Disintegrin And Metalloprotease 23 (ADAM23) is a member of the ADAMs family of transmembrane proteins, mostly expressed in nervous system, and involved in traffic and stabilization of Kv1-potassium channels, synaptic transmission, neurite outgrowth, neuronal morphology and cell adhesion. Also, ADAM23 has been linked to human pathological conditions, such as epilepsy, cancer metastasis and cardiomyopathy. ADAM23 functionality depends on the molecule presence at the cell surface and along the secretory pathway, as expected for a cell surface receptor. Because endocytosis is an important functional regulatory mechanism of plasma membrane receptors and no information is available about the traffic or turnover of non-catalytic ADAMs, we investigated ADAM23 internalization, recycling and half-life properties. Here, we show that ADAM23 undergoes constitutive internalization from the plasma membrane, a process that depends on lipid raft integrity, and is redistributed to intracellular vesicles, especially early and recycling endosomes. Furthermore, we observed that ADAM23 is recycled from intracellular compartments back to the plasma membrane and thus has longer half-life and higher cell surface stability compared with other ADAMs. Our findings suggest that regulation of ADAM23 endocytosis/stability could be exploited therapeutically in diseases in which ADAM23 is directly involved, such as epilepsy, cancer progression and cardiac hypertrophy.
Assuntos
Proteínas ADAM/metabolismo , Endocitose , Membrana Celular/metabolismo , Células Cultivadas , Endossomos/metabolismo , Meia-Vida , Humanos , Microdomínios da Membrana/metabolismoRESUMO
Oxidative stress (OS) is essential in uremia-associated comorbidities, including renal anemia. Complications experienced by hemodialysis (HD) patients, such as hypoxemia and uremic toxins accumulation, induce OS and premature death of red blood cells (RBC). We aimed to characterize reactive oxygen species (ROS) production and antioxidant pathways in HD-RBC and RBC from healthy controls (CON-RBC) and evaluate the role of uremia and hypoxia in these pathways. ROS production, xanthine oxidase (XO) and superoxide dismutase (SOD) activities, glutathione (GSH), and heme oxygenase-1 (HO-1) levels were measured using flow cytometry or spectrophotometry in CON-RBC and HD-RBC (pre- and post-HD), at baseline and after 24 h incubation with uremic serum (S-HD) and/or under hypoxic conditions (5% O2 ). Ketoprofen was used to inhibit RBC uremic toxins uptake. HD-RBC showed higher ROS levels and lower XO activity than CON-RBC, particularly post-HD. GSH levels were lower, while SOD activity and HO-1 levels of HD-RBC were higher than control. Hypoxia per se triggered ROS production in CON-RBC and HD-RBC. S-HD, on top of hypoxia, increased ROS levels. Inhibition of uremic toxins uptake attenuated ROS of CON and HD-RBC under hypoxia and uremia. CON-RBC in uremia and hypoxia showed lower GSH levels than cells in normoxia and non-uremic conditions. Redox mechanisms of HD-RBC are altered and prone to oxidation. Uremic toxins and hypoxia play a role in unbalancing these systems. Hypoxia and uremia participate in the pathogenesis of OS in HD-RBC and might induce RBC death and thus compound anemia.
Assuntos
Anemia , Uremia , Humanos , Eritrócitos/metabolismo , Uremia/metabolismo , Diálise Renal , Estresse Oxidativo , Glutationa/metabolismo , Hipóxia/metabolismo , Anemia/metabolismoRESUMO
OBJECTIVES: Uremic toxins such as indoxyl sulfate (IS), p-cresyl sulfate (pCS), and indole-3-acetic acid (IAA) produced by the gut microbiota are recognized as risk factors for many comorbidities, including cardiovascular diseases. Patients with chronic kidney disease (CKD) have an accumulation of these toxins, and nutritional strategies have been proposed to mitigate gut dysbiosis and, consequently, reduce these toxins. This study aimed to evaluate the effects of resveratrol supplementation on the plasma levels of IS, pCS, and IAA in nondialyzed patients with CKD. METHODS: In this placebo-controlled crossover study, twenty nondialyzed patients were randomly divided into two groups: they received either one capsule/day containing 500 mg of trans-resveratrol (63 ± 7.5 years, glomerular filtration rate [GFR]: 34 ± 14 mL/min, body mass index: 26.8 ± 5.6 kg/m2) or a placebo containing 500 mg wheat flour (62 ± 8.4 years, GFR: 34 ± 13 mL/min, body mass index: 28.6 ± 4.4 kg/m2) during 4 weeks. After 8 weeks of washout (no supplementation), another 4 weeks of supplementation with crossover was initiated. IS, IAA, and pCS plasma levels were quantified by the reverse phase high-efficiency liquid chromatography method with fluorescent detection. The mRNA expression of nuclear factor erythroid 2-related factor 2 and nuclear factor kappa B in peripheral blood mononuclear cells was evaluated by polymerase chain reaction. C-reactive protein plasma levels were also evaluated. RESULTS: As expected, the uremic toxin levels were negatively correlated with the GFR, but no effect of trans-resveratrol supplementation was found on levels of IS, IAA, and pCS. There was a positive correlation between IS and nuclear factor erythroid 2-related factor 2 (r = 0.24, P = .03) and also between IS and C-reactive protein (r = 0.21, P = .05). CONCLUSION: Supplementation with trans-resveratrol did not reduce the plasma levels of IS, pCS, and IAA in nondialyzed patients with CKD. The interactions among uremic toxins and anti-inflammatory and proinflammatory pathways deserve more studies.
Assuntos
Microbioma Gastrointestinal , Insuficiência Renal Crônica , Humanos , Resveratrol , Toxinas Urêmicas , Proteína C-Reativa , Leucócitos Mononucleares/metabolismo , Estudos Cross-Over , Farinha , Triticum , IndicãRESUMO
BACKGROUND/AIMS: Chronic kidney disease is frequently accompanied by anemia, hypoxemia, and hypoxia. It has become clear that the impaired erythropoietin production and altered iron homeostasis are not the sole causes of renal anemia. Eryptosis is a process of red blood cells (RBC) death, like apoptosis of nucleated cells, characterized by Ca2+ influx and phosphatidylserine (PS) exposure to the outer RBC membrane leaflet. Eryptosis can be induced by uremic toxins and occurs before senescence, thus shortening RBC lifespan and aggravating renal anemia. We aimed to assess eryptosis and intracellular oxygen levels of RBC from hemodialysis patients (HD-RBC) and their response to hypoxia, uremia, and uremic toxins uptake inhibition. METHODS: Using flow cytometry, RBC from healthy individuals (CON-RBC) and HD-RBC were subjected to PS (Annexin-V), intracellular Ca2+ (Fluo-3/AM) and intracellular oxygen (Hypoxia Green) measurements, at baseline and after incubation with uremic serum and/or hypoxia (5% O2), with or without ketoprofen. Baseline levels of uremic toxins were quantified in serum and cytosol by high performance liquid chromatography. RESULTS: Here, we show that HD-RBC have less intracellular oxygen and that it is further decreased post-HD. Also, incubation in 5% O2 and uremia triggered eryptosis in vitro by exposing PS. Hypoxia itself increased the PS exposure in HD-RBC and CON-RBC, and the addition of uremic serum aggravated it. Furthermore, inhibition of the organic anion transporter 2 with ketoprofen reverted eryptosis and restored the levels of intracellular oxygen. Cytosolic levels of the uremic toxins pCS and IAA were decreased after dialysis. CONCLUSION: These findings suggest the participation of uremic toxins and hypoxia in the process of eryptosis and intracellular oxygenation.
Assuntos
Eriptose , Eritrócitos/metabolismo , Oxigênio/sangue , Insuficiência Renal Crônica/sangue , Uremia/sangue , Adolescente , Adulto , Idoso , Anexina A5/sangue , Cálcio/sangue , Hipóxia Celular , Eritrócitos/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Insuficiência Renal Crônica/patologia , Uremia/patologiaRESUMO
Chronic kidney disease (CKD) is associated with high mortality rates, mainly due to cardiovascular diseases (CVD). Uremia has been considered a relevant risk factor for CVD in CKD patients, since uremic toxins (UTs) promote systemic and vascular inflammation, oxidative stress and senescence. Here, we demonstrate that uremic toxins indoxyl sulfate (IxS), p-cresyl sulfate (pCS) and indole acetic acid (IAA) are incorporated by human endothelial cells and inhibit the autophagic flux, demonstrated by cellular p62 accumulation. Moreover, isolated and mixed UTs impair the lysosomal stage of autophagy, as determined by cell imaging of the mRFP-GFP-LC3 protein. Endothelial cells exposed to UTs display accumulation of carbonylated proteins and increased sensitivity to hydrogen peroxide. Rapamycin, an autophagy activator which induces both autophagosome formation and clearance, prevented these effects. Collectively, our findings demonstrate that accumulation of oxidized proteins and enhanced cell sensitivity to hydrogen peroxide are consequences of impaired autophagic flux. These data provide evidence that UTs-induced impaired autophagy may be a novel contributor to endothelial dysfunction.
Assuntos
Cresóis/farmacologia , Peróxido de Hidrogênio/farmacologia , Indicã/farmacologia , Ácidos Indolacéticos/farmacologia , Proteínas de Ligação a RNA/metabolismo , Ésteres do Ácido Sulfúrico/farmacologia , Toxinas Biológicas/farmacologia , Animais , Apoptose/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Humanos , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Camundongos , Células NIH 3T3 , Estresse Oxidativo/efeitos dos fármacosRESUMO
Apoptosis-inducing factor (AIF) is a flavoprotein and essential partner of the CHCHD4 redox protein during the mitochondrial intermembrane space import machinery. Mammalian AIF has three cysteine residues, which have received little attention. Previous reports have evidenced a redox interaction between AIF and thioredoxin 1 (Trx1), particularly after oxidant conditions. Therefore, we asked whether the cysteine residues of the human AIF could be oxidized. Our data showed that endogenous AIF could be oxidized to disulfide-linked conjugates (DLC). Overexpressed WT AIF in HEK293T cells, as well as recombinant WT AIF, formed DLC. Expression of C256S, C317S or C441S AIF mutants severely inhibited DLC formation in cells exposed to oxidants. In vitro, DLC formation was completely precluded with C256S and C441S AIF mutants and partially inhibited with the C317S mutant. DLC was shown to enhance cellular susceptibility to apoptosis induced by staurosporine, likely by preventing AIF to maintain mitochondrial oxidative phosphorylation. Cells with decreased expression of Trx1 produced more AIF DLC than those with normal Trx1 levels, and in vitro, Trx1 was able to decrease the amount of AIF DLC. Finally, confocal analysis, as well as immunoblotting of mitochondrial fraction, indicated that a fraction of Trx1 is present in mitochondria. Overall, these data provide evidence that all three cysteine residues of AIF can be oxidized to DLC, which can be disrupted by mitochondrial Trx1.
Assuntos
Fator de Indução de Apoptose , Apoptose , Dissulfetos , Substituição de Aminoácidos , Fator de Indução de Apoptose/química , Fator de Indução de Apoptose/genética , Fator de Indução de Apoptose/metabolismo , Cisteína/química , Cisteína/genética , Cisteína/metabolismo , Dissulfetos/química , Dissulfetos/metabolismo , Células HEK293 , Células HeLa , Humanos , Mutação de Sentido Incorreto , Oxirredução , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Estaurosporina/farmacologiaRESUMO
Quiescent and contractile VSMC can switch to proliferative and migratory phenotype in response to growth factors and cytokines, an effect underscored by Nox family NADPH oxidases, particularly Nox1. We previously showed that quiescin/sulfhydryl oxidase 1 (QSOX1) has a role in neointima formation in balloon-injured rat carotid. Here, we investigated the intracellular redox mechanisms underlying these effects in primary VSMC. Our results show that exogenous incubation with wild type QSOX1b (wt QSOX), or with secreted QSOX1, but not with the inactive C452S QSOX 1b (C452S QSOX) or secreted inactive C455S QSOX1, induces VSMC migration and chemotaxis. PEG-catalase (PEG-CAT) prevented, while PEG-superoxide dismutase (PEG-SOD) increased migration induced by wt QSOX. Moreover, wt QSOX-induced migration was abrogated in NOX1-null VSMC. In contrast, both wt QSOX and C452S QSOX, and both secreted QSOX1 and C455S QSOX1, induce cell proliferation. Such effect was unaltered by PEG-CAT, while being inhibited by PEG-SOD. However, QSOX1-induced proliferation was not significantly affected in NOX1-null VSMC, compared with WT VSMC. These results indicate that hydrogen peroxide and superoxide mediate, respectively, migration and proliferation. However, Nox1 was required only for QSOX1-induced migration. In parallel, QSOX1-induced proliferation was independent of its redox activity, although mediated by intracellular superoxide.
Assuntos
Movimento Celular , Músculo Liso Vascular/citologia , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/metabolismo , Animais , Proliferação de Células , Células HEK293 , Humanos , Peróxido de Hidrogênio/metabolismo , Espaço Intracelular/metabolismo , Camundongos , NADPH Oxidase 1/metabolismo , Oxirredução/efeitos dos fármacos , Superóxidos/metabolismoRESUMO
OBJECTIVE: The objective of the study was to evaluate the effects of probiotic supplementation on the gut microbiota profile and inflammatory markers in chronic kidney disease patients undergoing maintenance hemodialysis (HD). DESIGN AND METHODS: This was a randomized, double-blind, placebo-controlled study. Forty-six HD patients were assigned to receive 1 of 2 treatments: probiotic (n = 23; Streptococcus thermophilus, Lactobacillus acidophilus e Bifidobacterialongum, 90 billion colony-forming units per day) or placebo (n = 23) daily for 3 months. Blood and feces were collected at baseline and after intervention. The inflammatory markers (C-reactive protein and interleukin-6) were analyzed by immunoenzymatic assay (enzyme-linked immunosorbent assay). Uremic toxins plasma levels (indoxyl sulfate, p-cresyl sulfate, and indole-3-acetic acid) were obtained by Reversed-Phase High-Performance Liquid Chromatography. Routine laboratory parameters were measured by standard techniques. Fecal pH was measured by the colorimetric method, and the gut microbiota profile was assessed by Denaturing Gradient Gel Electrophoresis analysis. RESULTS: Sixteen patients remained in the probiotic group (11 men, 53.6 ± 11.0 year old, 25.3 ± 4.6 kg/m2) and 17 in the placebo group (10 men, 50.3 ± 8.5 year old, 25.2 ± 5.7 kg/m2). After probiotic supplementation there was a significant increase in serum urea (from 149.6 ± 34.2 mg/dL to 172.6 ± 45.0 mg/dL, P = .02), potassium (from 4.4 ± 0.4 mmol/L to 4.8 ± 0.4 mmol/L, P = .02), and indoxyl sulfate (from 31.2 ± 15.9 to 36.5 ± 15.0 mg/dL, P = .02). The fecal pH was reduced from 7.2 ± 0.8 to 6.5 ± 0.5 (P = .01). These parameters did not change significantly in placebo group. Changes in the percentage delta (Δ) between groups were exhibited with no statistical differences observed. The inflammatory markers and gut profile were not altered by supplementation. CONCLUSIONS: Aprobiotic supplementation failed to reduce uremic toxins and inflammatory markers. Therefore, probiotic therapy should be chosen with caution in HD patients. Further studies addressing probiotic therapy in chronic kidney disease patients are needed.
Assuntos
Probióticos/administração & dosagem , Insuficiência Renal Crônica/terapia , Adulto , Bifidobacterium , Biomarcadores/sangue , Glicemia/metabolismo , Índice de Massa Corporal , Proteína C-Reativa/metabolismo , Cresóis/sangue , Método Duplo-Cego , Fezes/química , Fezes/microbiologia , Feminino , Microbioma Gastrointestinal , Humanos , Concentração de Íons de Hidrogênio , Indicã/sangue , Ácidos Indolacéticos/sangue , Interleucina-6/sangue , Lactobacillus acidophilus , Masculino , Pessoa de Meia-Idade , Avaliação Nutricional , Insuficiência Renal Crônica/microbiologia , Streptococcus thermophilus , Ésteres do Ácido Sulfúrico/sangue , Circunferência da CinturaRESUMO
OBJECTIVES: To evaluate the effects of low-protein diet (LPD) on uremic toxins and the gut microbiota profile in nondialysis chronic kidney disease (CKD) patients. DESIGN AND METHODS: Longitudinal study with 30 nondialysis CKD patients (stage 3-4) undergoing LPD for 6 months. Adherence to the diet was evaluated based on the calculation of protein equivalent of nitrogen appearance from the 24-hour urine analysis. Good adherence to LPD was considered when protein intake was from 90% to 110% of the prescribed amount (0.6 g/kg/day). Food intake was analyzed by the 24-hour recall method. The anthropometric, biochemical and lipid profile parameters were measured according to standard methods. Uremic toxin serum levels (indoxyl sulfate, p-cresyl sulfate, indole-3-acetic acid) were obtained by reversed-phase high-performance liquid chromatography (RP-HPLC). Fecal samples were collected to evaluate the gut microbiota profile through polymerase chain reaction and denaturing gradient gel electrophoresis. Statistical analysis was performed by the SPSS 23.0 program software. RESULTS: Patients who adhered to the diet (n = 14) (0.7 ± 0.2 g/kg/day) presented an improvement in renal function (nonsignificant) and reduction in total and low-density lipoprotein cholesterol (183.9 ± 48.5-155.7 ± 37.2 mg/dL, P = .01; 99.4 ± 41.3-76.4 ± 33.2 mg/dL, P = .01, respectively). After 6 months of nutricional intervention, p-cresyl sulfate serum levels were reduced significantly in patients who adhered to the LPD (19.3 [9.6-24.7] to 15.5 [9.8-24.1] mg/L, P = .03), and in contrast, the levels were increased in patients who did not adhere (13.9 [8.0-24.8] to 24.3 [8.1-39.2] mg/L, P = .004). In addition, using the denaturing gradient gel electrophoresis technique, it was observed change in the intestinal microbiota profile after LPD intervention in both groups, and the number of bands was positively associated with protein intake (r = 0.44, P = .04). CONCLUSION: LPD seems be a good strategy to reduce the uremic toxins production by the gut microbiota in nondialysis CKD patients.
Assuntos
Cresóis/sangue , Dieta com Restrição de Proteínas , Microbioma Gastrointestinal/fisiologia , Indicã/sangue , Ácidos Indolacéticos/sangue , Insuficiência Renal Crônica/dietoterapia , Ésteres do Ácido Sulfúrico/sangue , Adulto , Idoso , Fezes/microbiologia , Feminino , Humanos , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Cooperação do Paciente , Insuficiência Renal Crônica/sangueRESUMO
Thioredoxin (Trx)-fold proteins are protagonists of numerous cellular pathways that are subject to thiol-based redox control. The best characterized regulator of thiols in proteins is Trx1 itself, which together with thioredoxin reductase 1 (TR1) and peroxiredoxins (Prxs) comprises a key redox regulatory system in mammalian cells. However, there are numerous other Trx-like proteins, whose functions and redox interactors are unknown. It is also unclear if the principles of Trx1-based redox control apply to these proteins. Here, we employed a proteomic strategy to four Trx-like proteins containing CXXC motifs, namely Trx1, Rdx12, Trx-like protein 1 (Txnl1) and nucleoredoxin 1 (Nrx1), whose cellular targets were trapped in vivo using mutant Trx-like proteins, under conditions of low endogenous expression of these proteins. Prxs were detected as key redox targets of Trx1, but this approach also supported the detection of TR1, which is the Trx1 reductant, as well as mitochondrial intermembrane proteins AIF and Mia40. In addition, glutathione peroxidase 4 was found to be a Rdx12 redox target. In contrast, no redox targets of Txnl1 and Nrx1 could be detected, suggesting that their CXXC motifs do not engage in mixed disulfides with cellular proteins. For some Trx-like proteins, the method allowed distinguishing redox and non-redox interactions. Parallel, comparative analyses of multiple thiol oxidoreductases revealed differences in the functions of their CXXC motifs, providing important insights into thiol-based redox control of cellular processes.
Assuntos
Proteoma/metabolismo , Proteômica/métodos , Tiorredoxinas/metabolismo , Fator de Indução de Apoptose/genética , Fator de Indução de Apoptose/metabolismo , Sítios de Ligação/genética , Western Blotting , Cromatografia Líquida , Células HEK293 , Células HeLa , Humanos , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Oxirredução , Oxirredutases/genética , Oxirredutases/metabolismo , Ligação Proteica , Proteoma/genética , Interferência de RNA , Proteínas Recombinantes/metabolismo , Espectrometria de Massas em Tandem , Tiorredoxinas/genéticaRESUMO
Quiescin sulfhydryl oxidase 1 (QSOX1) is a flavoenzyme largely present in the extracellular milieu whose physiological functions and substrates are not known. QSOX1 has been implicated in the regulation of tumor cell survival, proliferation and migration, in addition to extracellular matrix (ECM) remodeling. However, data regarding other pathophysiological conditions are still lacking. Arterial injury by balloon catheter is an established model of post-angioplasty restenosis. This technique induces neointima formation due to migration and proliferation of vascular smooth muscle cells (VSMC), followed by ECM synthesis and remodeling. Here, we show that QSOX1 knockdown inhibited VSMC migration and proliferation in vitro. In contrast, QSOX1 overexpression stimulated these processes. While migration could be induced by the incubation of cells with the active recombinant QSOX1, proliferation was induced by addition of the active and also of an inactive mutant QSOX1 protein. The proliferation induced by both recombinants was independent of intracellular hydrogen peroxide and dependent of the MEK/ERK pathway. To recapitulate in vivo VSMC pathophysiology, balloon-induced arterial injury was performed. The expression of QSOX1 in the neointimal layer of balloon-injured rat carotids was high and peaked at 14 days post-injury. In vivo QSOX1 knockdown led to a significant decrease in PCNA expression at day 14 post-injury and a decreased intima/media area ratio at day 21 post-injury, compared with scrambled siRNA transfection. In summary, our findings demonstrate that QSOX1 induces VSMC migration and proliferation in vitro and contributes to neointima thickening in balloon-injured rat carotids.
Assuntos
Movimento Celular , Proliferação de Células , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Neointima/metabolismo , Tiorredoxinas/metabolismo , Animais , Artérias Carótidas/patologia , Artérias Carótidas/cirurgia , Células Cultivadas , Peróxido de Hidrogênio/metabolismo , Sistema de Sinalização das MAP Quinases , Masculino , Camundongos , Músculo Liso Vascular/citologia , Músculo Liso Vascular/fisiologia , Miócitos de Músculo Liso/fisiologia , Ratos , Ratos Wistar , Tiorredoxinas/genéticaRESUMO
OBJECTIVE: Protein-bound uremic toxins from gut microbiota tend to accumulate in chronic kidney disease (CKD) patients and are poorly removed by current dialysis techniques. These toxins induce inflammation and are associated with cardiovascular disease (CVD). The aim of this study was to report the relationship between uremic toxins and inflammatory and cardiovascular markers in CKD patients. DESIGN: This was a cross sectional study. SUBJECTS: Twenty-one nondialysis patients were included (43% men, 63.0 ± 7.8 years, glomerular filtration rate: 34.4 ± 12.5 mL/min) as well as 29 hemodialysis (HD) patients [58% men, 52.7 ± 10.3 years, time on dialysis 54 (31-94.5 months)]. MAIN OUTCOME MEASURE: Total levels of uremic toxins (IS, p-CS, and IAA) were assessed by high-performance liquid chromatography with fluorescence detection. C-reactive protein, Interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), and calprotectin plasma levels were determined by immunometric assays. RESULTS: HD patients presented higher inflammatory markers and uremic toxins levels than nondialysis patients. IL-6 levels were positively correlated with IS (r = 0.49; P = .03), p-CS (r = 0.35; P = .04) and IAA (r = 0.36; P = .03). A positive correlation was also observed between MCP-1 levels with IS (r = 0.72; P = .001), p-CS (r = 0.48; P = .001) and IAA (r = 0.75; P = .0001). Linear regression showed that IS was an independent predictor for IL-6 and MCP-1 levels after adjustment. CONCLUSION: Plasma uremic toxins were associated with higher IL-6 and MCP-1 levels in CKD patients, potentially playing a role in the development of CVD.
Assuntos
Microbioma Gastrointestinal , Diálise Renal , Insuficiência Renal Crônica/microbiologia , Idoso , Estudos Transversais , Feminino , Humanos , Interleucina-6 , Masculino , Pessoa de Meia-IdadeRESUMO
Chronic kidney disease (CKD) progression and complications are associated with increased oxidative stress, as well as with Nrf2 inactivation. Lipoic acid (LA) has been considered an inducer of Nrf2 antioxidant response. We tested whether oral administration of LA provides beneficial effects in experimental CKD in rats. Wistar rats underwent 5/6 nephrectomy (CKD group) or sham laparotomy. Seven days later, CKD group was divided into three subgroups that received: (i) LA continuously in the drinking water (100 mg/kg/day), (ii) LA by gavage every other day (100 mg/kg), or (iii) no LA treatment. LA treatment lasted until day 60. Plasma urea and creatinine, 24 h-proteinuria, glomerulosclerosis, interstitial fibrosis/tubular atrophy, and Nrf2 activation were analyzed. All parameters measured were significantly altered in the untreated CKD group, compared with the sham group, as expected. Oral LA administration, either in the drinking water or by gavage, did not improve significantly any parameter, comparing the treated-groups with the untreated CKD group. These results indicate that oral LA administration for 53 days was ineffective to reactivate Nrf2 in the remnant kidney of uremic rats, likely preventing improvements in biochemical and histopathological markers of renal function.
Assuntos
Rim , Fator 2 Relacionado a NF-E2 , Insuficiência Renal Crônica , Ácido Tióctico , Animais , Masculino , Ratos , Modelos Animais de Doenças , Rim/efeitos dos fármacos , Rim/fisiologia , Nefrectomia/métodos , Ratos Wistar , Insuficiência Renal Crônica/tratamento farmacológico , Ácido Tióctico/farmacologia , Ácido Tióctico/uso terapêutico , Fator 2 Relacionado a NF-E2/fisiologiaRESUMO
Sulforaphane (SFN), found in cruciferous vegetables, is a known activator of NRF2 (master regulator of cellular antioxidant responses). Patients with chronic kidney disease (CKD) present an imbalance in the redox state, presenting reduced expression of NRF2 and increased expression of NF-κB. Therefore, this study aimed to evaluate the effects of SFN on the mRNA expression of NRF2, NF-κB and markers of oxidative stress in patients with CKD. Here, we observed a significant increase in the mRNA expression of NRF2 (p = 0.02) and NQO1 (p = 0.04) in the group that received 400 µg/day of SFN for 1 month. Furthermore, we observed an improvement in the levels of phosphate (p = 0.02), glucose (p = 0.05) and triglycerides (p = 0.02) also in this group. On the other hand, plasma levels of LDL-c (p = 0.04) and total cholesterol (p = 0.03) increased in the placebo group during the study period. In conclusion, 400 µg/day of SFN for one month improves the antioxidant system and serum glucose and phosphate levels in non-dialysis CKD patients.
Assuntos
Isotiocianatos , NAD(P)H Desidrogenase (Quinona) , Fator 2 Relacionado a NF-E2 , Estresse Oxidativo , RNA Mensageiro , Insuficiência Renal Crônica , Sulfóxidos , Humanos , Isotiocianatos/farmacologia , Isotiocianatos/uso terapêutico , Fator 2 Relacionado a NF-E2/metabolismo , Fator 2 Relacionado a NF-E2/genética , NAD(P)H Desidrogenase (Quinona)/genética , NAD(P)H Desidrogenase (Quinona)/metabolismo , Insuficiência Renal Crônica/metabolismo , Insuficiência Renal Crônica/genética , Insuficiência Renal Crônica/tratamento farmacológico , Insuficiência Renal Crônica/patologia , Masculino , Pessoa de Meia-Idade , Feminino , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Antioxidantes/metabolismo , Antioxidantes/farmacologia , Triglicerídeos/sangue , Triglicerídeos/metabolismo , Glicemia/metabolismo , Regulação para Cima/efeitos dos fármacos , Adulto , Idoso , NF-kappa B/metabolismo , NF-kappa B/genéticaRESUMO
BACKGROUND & AIMS: Turmeric (a source of curcumin) is an excellent food to modulate oxidative stress, inflammation, and gut dysbiosis in patients with chronic kidney disease (CKD). However, no studies report the benefits of curcumin in patients undergoing peritoneal dialysis (PD). This study aims to evaluate the effects of curcuminoid supplementation on oxidative stress, inflammatory markers, and uremic toxins originating from gut microbiota in patients with CKD undergoing PD. METHODS: This longitudinal, randomized, single-blind, placebo-controlled trial evaluated 48 patients who were randomized into two groups: Curcumin (three capsules of 500 mg of Curcuma longa extract, with 98.42 % total curcuminoids) or placebo (three capsules of 500 mg of starch) for twelve weeks. In the peripheral blood mononuclear cells (PBMCs), the transcriptional expression levels of Nrf2, HOX-1 and NF-κB were evaluated by quantitative real-time PCR. Oxidative stress was evaluated by malondialdehyde (MDA) and total Thiol (T-SH). TNF-α and IL-6 plasma levels were measured by ELISA. P-cresyl sulphate plasma level, a uremic toxin, was evaluated by high-performance liquid chromatography (HPLC) with fluorescent detection. RESULTS: Twenty-four patients finished the study: 10 in the curcumin group (57.5 ± 11.6 years) and 14 in the placebo group (56.5 ± 10.0 years). The plasma levels of MDA were reduced after 12 weeks in the curcumin group (p = 0.01), while the placebo group remained unchanged. However, regarding the difference between the groups at the endpoint, no change was observed in MDA. Still, there was a trend to reduce the p-CS plasma levels in the curcumin group compared to the placebo group (p = 0.07). Likewise, the concentrations of protein thiols, mRNA expression of Nrf2, HOX-1, NF-κB, and cytokines plasma levels did not show significant changes. CONCLUSION: Curcuminoid supplementation for twelve weeks attenuates lipid peroxidation and might reduce uremic toxin in patients with CKD undergoing PD. This study was registered on Clinicaltrials.gov as NCT04413266.
Assuntos
Curcumina , Diálise Peritoneal , Insuficiência Renal Crônica , Uremia , Humanos , Curcumina/farmacologia , Curcumina/uso terapêutico , NF-kappa B/metabolismo , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Leucócitos Mononucleares/metabolismo , Método Simples-Cego , Inflamação , Estresse Oxidativo , Insuficiência Renal Crônica/complicações , Insuficiência Renal Crônica/terapia , Diarileptanoides/farmacologia , Diarileptanoides/uso terapêutico , Suplementos Nutricionais , Uremia/tratamento farmacológicoRESUMO
For many years, nitric oxide (NO) has been studied as an important mediator in the control of vascular tone. Endothelial deficiencies that diminish NO production can result in the development of several future cardiovascular diseases, such as hypertension and arteriosclerosis. In this context, new drugs with potential ability to donate NO have been studied. In this study, 3 aromatic oximes [benzophenone oxime, 4-Cl-benzophenone oxime, and E-cinnamaldehyde oxime (E-CAOx)] induced vasorelaxation in endothelium-denuded and intact superior mesenteric rings precontracted with phenylephrine. E-CAOx demonstrated the most potent effect, and its mechanism of action was evaluated. Vascular reactivity experiments demonstrated that the effect of E-CAOx was reduced by the presence of 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, 1H[1,2,4,]oxadiazolo[4,3-a]quinoxalin-1-one, and (Rp)-8-(para-chlorophenylthio)guanosine-3',5'-cyclic monophosphorothioate, suggesting the participation of NO/sGC/PKG pathway. NO donation seems to be mediated through nicatinamide adenine dinucleotide phosphate-dependent reductases because 7-ethoxyresorufin decreased the effect of E-CAOx on vascular reactivity and reduced NO formation as detected by flow cytometry using the NO indicator diaminofluorescein 4,5-diacetate. Further downstream of NO donation, K+ subtype channels were also shown to be involved in the E-CAOx vasorelaxant effect. The present study showed that E-CAOx acts like an NO donor, activating NO/sGC/PKG pathway and thus K+ channels.
Assuntos
Acroleína/análogos & derivados , Artéria Mesentérica Superior/efeitos dos fármacos , Óxido Nítrico/fisiologia , Transdução de Sinais/efeitos dos fármacos , Acroleína/farmacologia , Animais , Cálcio/metabolismo , Relação Dose-Resposta a Droga , Endotélio Vascular/fisiologia , Inibidores Enzimáticos/farmacologia , Técnicas In Vitro , Contração Isométrica/efeitos dos fármacos , Luminescência , Masculino , Relaxamento Muscular/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico/metabolismo , Doadores de Óxido Nítrico , Óxido Nítrico Sintase Tipo III/antagonistas & inibidores , Oximas/farmacologia , Canais de Potássio/efeitos dos fármacos , Canais de Potássio/fisiologia , Ratos , Ratos WistarRESUMO
BACKGROUND/AIMS: Oxidative stress has been considered a nontraditional risk factor for cardiovascular disease in the chronic kidney disease (CKD) population, possibly triggered by uremic toxicity. METHODS: A chromatographic method with coulometric detection was adapted to directly and simultaneously determine cysteine (Cys) and cystine (Cyss) in plasma samples. Healthy subjects and CKD subjects in different stages were analyzed. The free Cys and free Cyss levels in their plasma were determined, and the reduction potential [Eh(Cyss/2Cys)] was calculated with the Nernst equation. RESULTS: Healthy plasma presented Eh(Cyss/2Cys) of -123 ± 7 mV. Plasma Eh(Cyss/2Cys) correlated significantly with creatinine levels (p < 0.0001, r = 0.62). CONCLUSION: Plasma Eh(Cyss/2Cys) correlated with increased levels of plasma creatinine, supporting the view that uremia triggers oxidative stress. In addition, it may be used as a quantitative oxidative stress biomarker in uremic conditions.
Assuntos
Creatinina/sangue , Cisteína/sangue , Cistina/sangue , Insuficiência Renal Crônica/sangue , Adolescente , Adulto , Idoso , Biomarcadores/sangue , Cromatografia Líquida de Alta Pressão , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estresse Oxidativo , Adulto JovemRESUMO
BACKGROUND: Anemia is one of the most frequent complications in patients with chronic kidney disease (CKD). Despite being multifactorial, the relative or absolute deficiency of erythropoietin production is the leading cause. Recent studies have shown that uremic toxins produced by the gut microbiota also may play a role in the genesis of anemia in these patients. OBJECTIVE: To evaluate the possible association between uremic toxins plasma levels and anemia in patients with CKD on hemodialysis (HD). METHODS: This cross-sectional study evaluated one hundred fifty-four patients (53.2% men, 51.2 ± 11.2 years, hemoglobin (Hb) levels of 11.2 ± 1.6 g/dL). Biochemical variables such as urea, creatinine, hemoglobin, hematocrit, were measured according to standard methods and uremic toxins such as indoxyl sulfate (IS), indole-3-acetic acid (IAA), p-cresyl sulfate (p-CS) plasma levels were measured by reverse-phase high-performance liquid chromatography (RP-HPLC). RESULTS: The levels of uremic toxins such as IS, IAA, p-CS were increased in all patients. However, no correlation was found between uremic toxins plasma levels and anemia parameters. Only patients with Hb < 11 g/dL presented a negative correlation between hematocrit and IAA plasma levels. CONCLUSION: There is no strong evidence that uremic toxins produced by the gut microbiota may be associated with anemia in patients with CKD on HD.