FC 103PROTEOME WIDE OXIDATIVE STRESS PROFILING IN MESOTHELIAL CELLS INDUCED BY PERITONEAL DIALYSIS FLUID

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
Rebecca Herzog ◽  
Anja Wagner ◽  
Klaus Kratochwill
Keyword(s):  
Oxidative Stress ◽  
Superoxide Dismutase ◽  
Peritoneal Dialysis ◽  
Pathway Analysis ◽  
Cell Damage ◽  
Mesothelial Cells ◽  
Superoxide Dismutase Activity ◽  
H2o2 Treatment ◽  
Redox Proteomics ◽  
Intracellular Ros

Abstract Background and Aims Reactive oxygen species (ROS) in the peritoneal cavity may result both from CKD and the specific composition of peritoneal dialysis fluids (PDF). Elevated cellular oxidative stress is defined as a cellular oxidant/antioxidant imbalance which impairs not only peritoneal cell viability but also contributes to progression of local and systemic PD-related pathomechanisms. So far only single targets or mediators of oxidative stress were investigated in mesothelial cells exposed to PD fluids. Here, we aim to analyze the broad impact and also identify individual targets of ROS during PD. Using the developed technique the anti-oxidative effect of alanyl-glutamine (AlaGln) supplementation of PDF was characterized on the proteome level. Method To establish a redox-proteomics workflow for studying oxidative stress in peritoneal mesothelial cells we used a gold-standard model of redox-stress (100 µm hydrogen peroxide (H2O2)) and PD-fluid induced stress. Levels of oxidative stress were first evaluated by intracellular ROS levels and superoxide dismutase activity. Oxidative stress levels induced by PDF were titrated to comparable levels of H2O2 treatment to be able to characterize redox modifications and the effect of addition of 8 mM AlaGln. To detect alterations of the redox proteome we adapted and refined an approach combining redox-sensitive isobaric mass tags and high-performance liquid chromatography coupled to mass spectrometry (LC/MS). We used a sequential combination of direct and indirect labeling of redox-sensitive cysteine residues. Results Exposure to PDF increased intracellular ROS production and accumulation as well as cell damage assessed by LDH-release compared to control cells. Cells exposed to AlaGln supplemented PDF showed less cell damage compared to PDF alone. Addition of AlaGln not only reduced the overall redox status (intracellular ROS and superoxide dismutase activity) but also led to different proteins being affected by redox modifications. The carefully optimized highly sensitive LC/MS-based redox proteomics workflow allowed identification of 5537 proteins of which 2614 contained a labeled cysteine. H2O2 treatment resulted in a shift of median oxidation from 11% under control conditions to 36%. While PDF alone increased the oxidation level to 31%, AlaGln supplemented PDF only led to 15% oxidation. Pathway analysis of proteins that changed their oxidation level >50% following the treatment were subjected to molecular pathway analysis revealing distinct differences. PDF exposure leads to regulation of general cell processes like regulation of glucokinase, RNA-binding and SUMOylation, addition of AlaGln regulated more specific signaling pathways for example fibrosis related pathways like TGF-ß and SMAD signaling. Conclusion Redox proteomics of peritoneal cells could represent a novel tool for the identification of mediators of uraemia and PD-induced pathomechanisms, and also to evaluate anti-oxidant pharmacological interventions to improve PD outcomes.

scholarly journals P1143REDOX PROFILING IN MESOTHELIAL CELLS IN EXPERIMENTAL PERITONEAL DIALYSIS

2020 ◽  
Vol 35 (Supplement_3) ◽  
Author(s):  
Rebecca Herzog ◽  
Anja Wagner ◽  
Klaus Kratochwill
Keyword(s):  
Oxidative Stress ◽  
Superoxide Dismutase ◽  
Fluorescent Dyes ◽  
Mesothelial Cells ◽  
Superoxide Dismutase Activity ◽  
Cysteine Oxidation ◽  
Redox Stress ◽  
Redox Proteomics ◽  
Intracellular Ros ◽  
The Impact

Abstract Background and Aims PD-fluids lead to generation of reactive oxygen species (ROS) in the peritoneal cavity. The caused oxidative stress, defined as a cellular oxidant-antioxidant imbalance impairs not only peritoneal cell viability but also contributes to progression of local and systemic PD-related pathomechanisms. We aim to analyze the impact and specific targets of ROS during PD and the anti-oxidative mechanism of supplementation of PD-fluid with alanyl-glutamine (AlaGln) on a global proteome-wide level. Method To establish a redox-proteomics workflow for studying oxidative stress in peritoneal mesothelial cells we used a gold-standard model of redox-stress (H2O2) and PD-fluid induced stress. Levels of oxidative stress were first validated by increased intracellular ROS and superoxide dismutase activity with PD-fluid and H2O2 treatment and a reduction of these parameters by the addition of AlaGln. To detect alterations of the redox proteome, cysteine residues were either directly or indirectly labeled with fluorescent dyes (redox-2D-DiGE) or isobaric tags (iodo-TMT). Results: The gel-based approach allowed global visualization of the reduced and oxidized cysteines and revealed redox profiles of 540 protein spots. Compared to control, we found an increase in oxidized and decrease in reduced cysteines in all PD treatments. The development of a highly sensitive LC/MS-based redox proteomics workflow allowed identification of ∼950 proteins affected by redox-stress in mesothelial cells and confirmed the quantitative levels seen on cysteine oxidation. The addition of AlaGln reduced the overall redox status (intracellular ROS and superoxide dismutase activity) but further showed different proteins to be affected by redox modifications. Conclusion: Redox proteomics of peritoneal cells could represent a novel approach for the identification of mediators of PD-induced pathomechanisms, but also to evaluate effects of novel anti-oxidant therapeutical or pharmacological interventions.

Diazoxide Modulates Cardiac Hypertrophy by Targeting H2O2 Generation and Mitochondrial Superoxide Dismutase Activity

2020 ◽  
Vol 13 (1) ◽  
pp. 76-83
Author(s):  
Aline Maria Brito Lucas ◽  
Joana Varlla de Lacerda Alexandre ◽  
Maria Thalyne Silva Araújo ◽  
Cicera Edna Barbosa David ◽  
Yuana Ivia Ponte Viana ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Superoxide Dismutase ◽  
Cardiac Hypertrophy ◽  
Superoxide Dismutase Activity ◽  
Heart Weight ◽  
Pharmacological Intervention ◽  
Wall Thickening ◽  
H2o2 Production ◽  
Mitochondrial Superoxide ◽  
Mitochondrial Superoxide Dismutase

Background: Cardiac hypertrophy involves marked wall thickening or chamber enlargement. If sustained, this condition will lead to dysfunctional mitochondria and oxidative stress. Mitochondria have ATP-sensitive K+ channels (mitoKATP) in the inner membrane that modulate the redox status of the cell. Objective: We investigated the in vivo effects of mitoKATP opening on oxidative stress in isoproterenol- induced cardiac hypertrophy. Methods: Cardiac hypertrophy was induced in Swiss mice treated intraperitoneally with isoproterenol (ISO - 30 mg/kg/day) for 8 days. From day 4, diazoxide (DZX - 5 mg/kg/day) was used in order to open mitoKATP (a clinically relevant therapy scheme) and 5-hydroxydecanoate (5HD - 5 mg/kg/day) or glibenclamide (GLI - 3 mg/kg/day) were used as mitoKATP blockers. Results: Isoproterenol-treated mice had elevated heart weight/tibia length ratios (HW/TL). Additionally, hypertrophic hearts had elevated levels of carbonylated proteins and Thiobarbituric Acid Reactive Substances (TBARS), markers of protein and lipid oxidation. In contrast, mitoKATP opening with DZX avoided ISO effects on gross hypertrophic markers (HW/TL), carbonylated proteins and TBARS, in a manner reversed by 5HD and GLI. Moreover, DZX improved mitochondrial superoxide dismutase activity. This effect was also blocked by 5HD and GLI. Additionally, ex vivo treatment of isoproterenol- induced hypertrophic cardiac tissue with DZX decreased H2O2 production in a manner sensitive to 5HD, indicating that this drug also acutely avoids oxidative stress. Conclusion: Our results suggest that diazoxide blocks oxidative stress and reverses cardiac hypertrophy. This pharmacological intervention could be a potential therapeutic strategy to prevent oxidative stress associated with cardiac hypertrophy.

Absence of Modulating Effects of Cytokines on Antioxidant Enzymes in Peritoneal Mesothelial Cells

1997 ◽  
Vol 17 (5) ◽  
pp. 455-466 ◽  
Author(s):  
Jinn-Yang Chen ◽  
An-Hang Yang ◽  
Yao-Ping Lin ◽  
Jen-Kou Lin ◽  
Wu-Chang Yang ◽  
...  
Keyword(s):  
Superoxide Dismutase ◽  
Antioxidant Enzymes ◽  
Glutathione Peroxidase ◽  
Human Liver ◽  
Mesothelial Cells ◽  
Superoxide Dismutase Activity ◽  
Antioxidant Enzyme Activities ◽  
Peritoneal Mesothelial Cells ◽  
Liver And Kidney ◽  
Human Peritoneal Mesothelial Cells

Objective To investigate the modulation of superoxide dismutase, glutathione peroxidase, and catalase by cytokines and endotoxin in human peritoneal mesothelial cells. Design Cultured human peritoneal mesothelial cells were treated with various concentrations of interleu kin-1 α, tumor necrosis factor-α(TNFα), interleukin-6, interleukin-8, transforming growth factor-β (TGFβ), and lipopolysaccharide. Cell morphology was observed and the activities of superoxide dismutase, catalase, and glutathione peroxidase were assayed. The antioxidant enzyme activities of human peritoneal mesothelial cells were also compared with those of human liver and kidney tissues. Results Interleukin-1α, TNFα, TGFβ, and lipopolysaccharide caused dose-dependent cytotoxicities in mesothelial cells. The activities of these three antioxidant enzymes did not change after treatment with cytokines and endotoxin. The total superoxide dismutase activity of confluent human peritoneal mesothelial cells was found to be greater than that of human liver and kidney tissues and was composed mostly of manganese superoxide dismutase activity. Furthermore, glutathione peroxidase and catalase activities of human peritoneal mesothelial cells were lower than those of human liver and kidney tissues. Conclusion In human peritoneal mesothelial cells, lack of induction of antioxidant enzymes by inflammatory cytokines, as well as high superoxide dismutase activity accompanied by insufficient glutathione peroxidase and catalase activities may both contribute to the susceptibility of these cells to oxidative damage. Therefore, appropriate management to decrease oxidative injury to the peritoneum should be taken into consideration when treating long-term continuous ambulatory peritoneal dialysis patients.

scholarly journals MiR-122 Participates in Oxidative Stress and Apoptosis in STZ-Induced Pancreatic β Cells by Regulating PI3K/AKT Signaling Pathway

2021 ◽  
Vol 2021 ◽  
pp. 1-11
Author(s):  
Jing Wang ◽  
Zhichun Dong ◽  
Liyin Lou ◽  
Lijuan Yang ◽  
Jingying Qiu
Keyword(s):  
Oxidative Stress ◽  
Insulin Secretion ◽  
Caspase 3 ◽  
Cell Damage ◽  
Caspase 9 ◽  
Intracellular Ros ◽  
Reverse Transcription Quantitative Pcr ◽  
Apoptosis Detection ◽  
Dichlorofluorescein Diacetate ◽  
And Oxidative Stress

At present, there are few reports concerning the relationship between miR-122 and diabetes. In addition, the effect of miR-122 on streptozotocin- (STZ-) induced oxidative damage in INS-1 cells remains unclear. The present study aimed to investigate the role and modulatory mechanisms involving miR-122 in diabetes. STZ was used to induce INS-1 cell damage. Reverse transcription-quantitative PCR was used to investigate the expression of miR-122. A TUNEL cell apoptosis detection kit was used to detect apoptosis. Intracellular ROS levels were determined using dichlorofluorescein-diacetate. The activities of insulin secretion, superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-px) were measured using ELISA kits. Western blotting was used to measure the expression levels of Bax, Bcl-2, PI3K, p-PI3K, caspase-3 and caspase-9, cleaved-caspase-3 and cleaved-caspase-9, AKT, and p-AKT. Then, LY294002 (LY, PI3K inhibitor) was used to treat INS-1 cells, and oxidative stress and apoptosis were measured. The results showed that STZ-induced inhibitory effects on insulin secretion were mitigated by miR-122 inhibitor, and the activities of SOD, CAT, and GSH-px were also increased. Furthermore, miR-122 inhibitor inhibited apoptosis and oxidative stress in STZ-induced INS-1 cells. Finally, the addition of LY increased insulin levels; reduced the activities of SOD, CAT, and GSH-px; and promoted apoptosis in STZ-induced INS-1 cells. In conclusion, interference with miR-122 can inhibit oxidative stress and apoptosis in STZ-induced INS-1 cells, involving a mechanism of action related to the PI3K/AKT pathway.

Biocompatibility of Peritoneal Dialysis Fluids: Long-term Exposure of Nonuremic Rats

2004 ◽  
Vol 24 (1) ◽  
pp. 37-47 ◽  
Author(s):  
Barbara Musi ◽  
Magnus Braide ◽  
Ola Carlsson ◽  
Anders Wieslander ◽  
Ann Albrektsson ◽  
...  
Keyword(s):  
Peritoneal Dialysis ◽  
Growth Factor ◽  
Solute Transport ◽  
Transforming Growth Factor ◽  
Cell Damage ◽  
Mesothelial Cell ◽  
Mesothelial Cells ◽  
Vascular Density ◽  
Tissue Thickness

Objectives Long-term peritoneal dialysis (PD) leads to structural and functional changes in the peritoneum. The aim of the present study was to investigate the long-term effects of PD fluid components, glucose and glucose degradation products (GDP), and lactate-buffered solution on morphology and transport characteristics in a nonuremic rat model. Methods Rats were subjected to two daily intraperitoneal injections (20 mL/day) during 12 weeks of one of the following: commercial PD fluid (Gambrosol, 4%; Gambro AB, Lund, Sweden), commercial PD fluid with low GDP levels (Gambrosol trio, 4%; Gambro AB), sterile-filtered PD fluid (4%) without GDP, or a glucose-free lactate-buffered PD fluid. Punctured and untreated controls were used. Following exposure, the rats underwent a single 4-hour PD dwell (30 mL, 4% glucose) to determine peritoneal function. Additionally, submesothelial tissue thickness, percentage of high mesothelial cells (perpendicular diameter > 2 μm), vascular density, vascular endothelial growth factor (VEGF), and transforming growth factor (TGF) β1 mRNA expression were determined. Submesothelial collagen concentration was estimated by van Gieson staining. Results Submesothelial tissue thickness and vascular density, mediated by VEGF and TGFβ production, in the diaphragmatic peritoneum increased significantly in rats exposed to any PD fluid. Gambrosol induced a marked increased fibrosis of the hepatic peritoneum. A significant increase in high mesothelial cells was observed in the Gambrosol group only. Net ultrafiltration was reduced in the Gambrosol and in the glucose-free groups compared to untreated controls. Small solute transport was unchanged, but all groups exposed to fluids showed significantly increased lymph flow. Conclusions Our results show that long-term exposure to different components of PD fluids leads to mesothelial cell damage, submesothelial fibrosis, and neoangiogenesis. Mesothelial cell damage could be connected to the presence of GDP; the other changes were similar for all fluids. Peritoneal transport characteristics did not change in any consistent way and the neoangiogenesis observed was not paralleled by increased solute transport.

Activity of erythrocyte antioxidant enzymes in healthy women depends on age, BMI, physical activity and diet

2020 ◽  
Author(s):  
Elżbieta Cecerska-Heryć ◽  
Klaudia Krauze ◽  
Angelika Szczęśniak ◽  
Aleksandra Goryniak Mikołajczyk ◽  
Natalia Serwin ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Physical Activity ◽  
Hydrogen Peroxide ◽  
Superoxide Dismutase ◽  
Antioxidant Enzymes ◽  
Catalase Activity ◽  
Smoking Status ◽  
Superoxide Dismutase Activity ◽  
Antioxidant Systems ◽  
Healthy Women

Abstract Introduction Antioxidant enzymes protect the human body against the harmful effects of oxidative stress. The activity of antioxidant enzymes changes with age, and depends on dietary nutrients such as fats and vitamins, which can have a significant impact on minimizing or exacerbating oxidative stress. Aim Examine the effect of age, BMI, diet, physical activity and smoking status on the activity of erythrocyte antioxidant enzymes catalase, glutathione reductase, glutathione peroxidase glutathione S-transferase, superoxide dismutase and glutathione concentrations in healthy women. Material and methods This study included 98 healthy women aged between 20 and 65 years. All women underwent anthropometric tests: body weight, height, hip and waist circumference. Antioxidant activity in erythrocytes was measured by spectrophotometric methods. Results Catalase activity increased significantly with age (p<0.001), while superoxide dismutase activities and glutathione decreased with age (p =0.008, p =0.023, respectively). Women with a lower BMI (emaciation) had higher superoxide dismutase activity than those in the first degree of obesity (p = 0.009 Conclusions 1. Increased catalase activity with age may be a sign of a large amount of hydrogen peroxide, resulting from poorly functioning antioxidant systems in older age. 2. Decreased superoxide dismutase activity with age may indicate inactivation of this enzyme by excessive hydrogen peroxide, as well as glycation of superoxide dismutase molecules or reactions with lipid peroxidation products, the intensity of which increases with age. 3. The negative correlation between superoxide dismutase activity and BMI index indicates reduced enzymatic activity in obese subjects, despite increased ROS production by adipose tissue.

scholarly journals Biochemical and Cellular Biomarkers in Brown Trout (Salmo trutta f. fario) in Response to the Antidepressants Citalopram and Venlafaxine

2020 ◽  
Author(s):  
Michael Ziegler ◽  
Helene Eckstein ◽  
Shannon Ottmann ◽  
Lukas Reinelt ◽  
Sabine Stepinski ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Superoxide Dismutase ◽  
Reuptake Inhibitor ◽  
Brown Trout ◽  
Salmo Trutta ◽  
Life Stage ◽  
Stress Protein ◽  
Superoxide Dismutase Activity ◽  
Control Fish ◽  
Histopathological Alterations

Abstract Background: During the last decades, a worldwide increase in the number of cases of depression accompanied by rising prescription rates of antidepressants was recorded. In Germany, the two most prescribed antidepressants are the selective serotonin reuptake inhibitor (SSRI) citalopram and the serotonin and noradrenalin reuptake inhibitor (SNRI) venlafaxine, taking about 30 % of the market share. Both antidepressants are found frequently in surface waters and have the potential of adversely affecting aquatic organisms. Most studies dealing with antidepressants address apical endpoints and behaviour, however, only few studies investigate biochemical biomarkers and histopathological alterations. We conducted citalopram and venlafaxine exposure experiments with brown trout eggs in the eyed ova stage for five months, as well as with juvenile brown trout for four weeks. Exposure concentrations ranged from environmentally relevant 1 µg/L up to 1 mg/L. In this study, we investigated the effects of the antidepressants on b-esterase activity (neurotoxicity), stress protein level (proteotoxicity) and superoxide dismutase activity (oxidative stress). Additionally, we assessed the health status of the liver by means of histopathological analyses.Results: We were able to show that both antidepressants did neither induce proteotoxic nor neurotoxic effects in brown trout. But for the antidepressant venlafaxine, the biochemical biomarker for oxidative stress (superoxide dismutase activity) was significantly increased in larvae exposed to at least 10 µg/L venlafaxine at 7°C. With regard to liver histopathology, fish exposed to higher citalopram concentrations were in a worse health condition than control fish irrespective of their life stage. Also the energy storage of fish exposed to 1 mg/L citalopram was reduced. Conclusion: Thus, we here report citalopram-dependent histopathological alterations in brown trout liver, and the induction of oxidative stress by venlafaxine.

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