Lia S. Nakao

Increased plasma and endothelial cell expression of chemokines and adhesion molecules in chronic kidney disease.

Chemokines and adhesion molecules are involved in early events of atherogenesis. In the present study, we investigated the effects of the uremic milieu on the expression of monocyte chemoattractant protein-1 (MCP-1), interleukin-8 (IL-8), soluble vascular adhesion molecule-1 (sVCAM-1) and soluble intercellular adhesion molecule-1 (sICAM-1) and their relationship to cardiovascular status. Plasma samples were obtained from patients in different stages of chronic kidney disease (CKD). Cardiovascular status was evaluated by intima-media thickness and endothelial dysfunction by flow mediation dilatation and proteinuria. In vitro studies were performed using human umbilical endothelial cells exposed to uremic plasma or plasma from healthy subjects. MCP-1, IL-8, sVCAM-1 and sICAM-1 levels in plasma and in supernatant were analyzed by enzyme-linked immunosorbent assay. The population consisted of 73 (mean age 57 years; 48% males) CKD patients with glomerular filtration rate (GFR) of 37 ± 2 ml/min. MCP-1 and sVCAM-1 plasma levels were negatively correlated with GFR (ρ = –0.40, p < 0.0005 and ρ = –0.42, p < 0.0005, respectively). Fibrinogen was positively correlated with MCP-1, sICAM-1 and sVCAM-1 (ρ = 0.33, p < 0.005, ρ = 0.32, p < 0.05 and ρ = 0.25, p < 0.05, respectively) and ultra-high-sensitivity C-reactive protein was positively correlated with sICAM-1 (ρ = 0.25, p < 0.0005). Plasma IL-8 had a significant positive correlation with proteinuria (ρ = 0.31, p < 0.01). There was a time- and CKD-stage-dependent MCP-1, IL-8 and sVCAM-1 endothelial expression (p < 0.05). In summary, plasma levels of markers of endothelial cell activation (MCP-1 and sVCAM-1) are increased in more advanced CKD. Exposure of endothelial cells to uremic plasma results in a time- and CKD-stage-dependent increased expression of MCP-1, IL-8 and sVCAM-1, suggesting a link between vascular activation, systemic inflammation and uremic toxicity. Future studies are necessary to investigate whether these biomarkers add predictive value in comparison to the previously described ones. Also, endothelial response to uremic toxicity should be viewed as a potential target for intervention in order to reduce morbidity and mortality in CKD-related cardiovascular disease.

Toxic effects of DDT and methyl mercury on the hepatocytes from Hoplias malabaricus

Here, we examined the impact of dichlorodiphenyltrichloroethane (DDT) and monomethyl mercury (MeHg) on the redox milieu and survival of hepatocytes from Hoplias malabaricus (traíra). After isolation and attachment of cells, we established one control and four treatments: DDT (50nM of DDT), MeHg I (0.25microM of MeHg), MeHg II (2.5microM of MeHg) and DDT * MeHg I (combination of 50nM of DDT and 0.25microM of MeHg). After four days the exposed hepatocytes presented significantly increased damage in lipids (all treatments), proteins (DDT * MeHg I and MeHg II) and reduced cell viability (all treatments). Also the antioxidant enzymes catalase, glucose-6-phosphate dehydrogenase (G6PDH), glutathione reductase and superoxide dismutase were affected. The current data showed that despite of some protective responses, the increased disturbs on membrane lipids and proteins, increased hydrogen peroxide levels, and decreased glutathione concentration and cell viability strongly indicate oxidative stress as the reason of hepatotoxicity due to DDT and MeHg exposure. In addition, DDT and MeHg together had greater effect than alone when G6PDH and glutathione-S-transferase activities and lipids damage were considered. These findings are indicative of hepatotoxicity occurring at realistic concentrations of DDT and MeHg found in Amazonian fish tissues.

Association between Biomarkers of Carbonyl Stress with Increased Systemic Inflammatory Response in Different Stages of Chronic Kidney Disease and after Renal Transplantation

Background: Chronic kidney disease (CKD) is characterized by progressive kidney dysfunction accompanied by accumulation of uremic toxins and a potential disequilibrium between the redox status and the generation of prooxidants, resulting in oxidative stress and chronic inflammation which is associated with complications (particularly cardiovascular disease) in this population. We aimed to analyze the concentration of total plasma thiols (indicator of antioxidant capacity) and the protein carbonyl content (a marker of carbonyl stress) in relation to kidney function and inflammation in a group of patients with CKD. Patients and Methods: A group of 68 patients with CKD (stages 2–5; mean age 57 ± 12 years, 46% male, 34% diabetics) and another group of 21 patients who underwent living donor kidney transplantation (mean age 36 ± 17 years, 50% male, 10% diabetics, and 9 ± 2 months after renal transplantation) were included in the study. Total plasma thiol and protein carbonyl levels were determined by the DTNB and DNPH methods, respectively, and were adjusted to the plasma albumin concentrations. Plasma levels of fibrinogen and C-reactive protein (CRP) were measured by routine methods and used as markers of inflammation. Results: Mean glomerular filtration rate (GFR) was 48 ml/min, and there was a positive correlation between GFR and thiol (r = 0.25, p < 0.05) and a negative correlation between GFR and carbonyl (r = –0.26, p < 0.05), fibrinogen (r = –0.45, p < 0.0001) and CRP (r = –0.14, p = ns). Carbonyl strongly correlated with CRP (0.49, p < 0.0001) and fibrinogen (0.30, p < 0.01). There was a significant reduction in plasma carbonyl after renal transplantation (1.4 ± 0.4 nmol/mg albumin), compared with the levels before the procedure (2.0 ± 1.4 nmol/mg albumin, p < 0.05), which parallels an improvement in thiol levels (15 ± 4 vs. 21 ± 5 nmol/mg albumin, p < 0.001). In addition, there was a significant correlation between CRP and carbonyl after the transplantation (r = 0.65; p < 0.005). Conclusion: We observed that patients with CKD present an altered redox status and increased signs of carbonyl stress and inflammatory activity as kidney function deteriorates, which was partially but significantly improved after renal transplantation. These findings indicate the importance of renal function in the complications of CKD related to oxidative stress and inflammation.

Plasma Cysteine/Cystine Reduction Potential Correlates with Plasma Creatinine Levels in Chronic Kidney Disease

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.

Unravelling the cardiovascular effects induced by α-terpineol: a role for the nitric oxide-cGMP pathway.

1. α‐Terpineol is a monoterpene found in the essential oils of several aromatic plant species. In the present study, we investigated the mechanisms underlying the cardiovascular changes induced by α‐terpineol in rats.

Tissue distribution of quiescin Q6/sulfhydryl oxidase (QSOX) in developing mouse

Quiescin Q6/sulfhydryl oxidases (QSOX) are revisited thiol oxidases considered to be involved in the oxidative protein folding, cell cycle control and extracellular matrix remodeling. They contain thioredoxin domains and introduce disulfide bonds into proteins and peptides, with the concomitant hydrogen peroxide formation, likely altering the redox environment. Since it is known that several developmental processes are regulated by the redox state, here we assessed if QSOX could have a role during mouse fetal development. For this purpose, an anti-recombinant mouse QSOX antibody was produced and characterized. In E13.5, E16.5 fetal tissues, QSOX immunostaining was confined to mesoderm- and ectoderm-derived tissues, while in P1 neonatal tissues it was slightly extended to some endoderm-derived tissues. QSOX expression, particularly by epithelial tissues, seemed to be developmentally-regulated, increasing with tissue maturation. QSOX was observed in loose connective tissues in all stages analyzed, intra and possibly extracellularly, in agreement with its putative role in oxidative folding and extracellular matrix remodeling. In conclusion, QSOX is expressed in several tissues during mouse development, but preferentially in those derived from mesoderm and ectoderm, suggesting it could be of relevance during developmental processes.

Expression of cardiac function genes in adult stem cells is increased by treatment with nitric oxide agents.

Mesenchymal stem cells (MSCs) have received special attention for cardiomyoplasty because several studies have shown that they differentiate into cardiomyocytes both in vitro and in vivo. Nitric oxide (NO) is a free radical signaling molecule that regulates several differentiation processes including cardiomyogenesis. Here, we report an investigation of the effects of two NO agents (SNAP and DEA/NO), able to activate both cGMP-dependent and -independent pathways, on the cardiomyogenic potential of bone marrow-derived mesenchymal stem cells (BM-MSCs) and adipose tissue-derived stem cells (ADSCs). The cells were isolated, cultured and treated with NO agents. Cardiac- and muscle-specific gene expression was analyzed by indirect immunofluorescence, flow cytometry, RT-PCR and real-time PCR. We found that untreated (control) ADSCs and BM-MSCs expressed some muscle markers and NO-derived intermediates induce an increased expression of some cardiac function genes in BM-MSCs and ADSCs. Moreover, NO agents considerably increased the pro-angiogenic potential mostly of BM-MSCs as determined by VEGF mRNA levels.

Ação da uleína sobre a produção de óxido nítrico em células RAEC e B16F10

The influence of the rich alkaloidal fraction and of the major substance in this fraction, uleine, isolated from the barks of Himatanthus lancifolius (Muell. Arg.) Woodson, Apocynaceae, popularly known as agoniada, on the nitric oxide production in RAEC and B16F10 cells and its correlation with the antioxidant activity, were investigated. For the antioxidant activity the methods of formation of a phosphomolybdenum complex and the reduction of the free radical DPPH were used. The results demonstrated an antioxidant activity of 59.3 ± 0.8% for the alkaloidal fraction, while for uleine the effect was of 0.5 ± 0.1% in the reduction of the phosphomolibdenium method. In the assay of DPPH, the alkaloidal fraction presented IC50 = 196.3 ± 8.9 µg/mL and for uleine, 6475.0 ± 25.0 µg/mL. Uleine also stimulated a maximum nitric oxide production in the concentrations of 0.1 µg/mL (20.9 ± 1.4 µM) and 1 µg/mL (41.1 ± 0.2 µM) using RAEC and B16F10 cells, respectively, demonstrating that the effect of uleine in the cells occurs by promoting the nitric oxide production pathway, but not through a free radical scavenger effect.

The 2-nitrate-1,3-dibuthoxypropan, a new nitric oxide donor, induces vasorelaxation in mesenteric arteries of the rat ☆

The reduced availability of nitric oxide (NO) is associated with cardiovascular diseases. Therefore, NO donors such as organic nitrates are useful for the treatment of these disorders. The 2-nitrate-1,3-dibuthoxypropan (NDBP) is an organic nitrate synthesized from glycerin, which the pharmacological effects have not been investigated. In this study we evaluated the vasorelaxant effect induced by NDBP in superior mesenteric artery from rats. In phenylephrine pre-contracted artery rings, NDBP (10(-8)-10(-4)M) elicited concentration-dependent and endothelium-independent relaxation, which were attenuated by hydroxocobalamin-HDX (30 μM), a NO extracellular scavenger, and 1-H-[1,2,4] oxadiazolo [4,3-a] quinoxalin-1-one-ODQ (10 μM), an inhibitor of soluble guanylyl cyclase (sGC). In addition, the NDBP-induced relaxation was reduced by non-selective K(+) channels blocker KCl (20 mM) or selective K(+) channels blockers such as tetraethylammonium-TEA (B(KCa), 1 mM), charybdotoxin-ChTX (B(KCa), 100 nM), glibenclamide (K(ATP), 1μM) and 4-aminopyridine-4-AP (K(V), 1mM). In preparations with ODQ (10 μM) plus TEA (1 mM), the response was virtually abolished. In rat smooth muscle cells culture, NDBP (10(-6)-10(-4)M) caused concentration-dependent increases in NO levels. These findings suggest that NDBP causes vasorelaxation through NO generation and activation of the sCG/cGMP/PKG pathway.

Transplantation of SNAP-treated adipose tissue-derived stem cells improves cardiac function and induces neovascularization after myocardium infarct in rats

Stem cell therapy has been considered a promise for damaged myocardial tissue. We have previously shown that S-nitroso-N-acetyl-D,L-penicillamine (SNAP) increases the expression of several muscular markers and VEGF in mesenchymal stem cells, indicating that transplantation of SNAP-treated cells could provide better functional outcomes. Here, we transplanted SNAP-treated adipose tissue-derived stem cells (ADSCs) in rat infarcted myocardium. After 30days, we observed a significant improvement of the ejection fraction in rats that received SNAP-treated ADSCs, compared with those that received untreated cells (p=0.008). Immunohistochemical reactions showed an increased expression of troponin T-C and von Willebrand factor, and organized vascular units in the infarcted area of tissue transplanted with treated ADSCs. SNAP exposure induced intracellular S-nitrosation, a decreased GSH/GSSG ratio, but did not increase cGMP levels. Collectively, these results indicate that SNAP alters the redox environment of ADSCs, possibly associated with a pre-differentiation state, which may improve cardiac function after transplantation.