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- Effect of hemodialysis procedure in prohepcidin serum levels in regular hemodialysis patientsPublication . Costa, Elísio; Rocha, S.; Rocha-Pereira, P.; Reis, F.; Castro, E.; Teixeira, F.; Miranda, V.; Faria, M. do Sameiro; Loureiro, A.; Quintanilha, A.; Belo, L.; Santos-Silva, A.
- Linkage of cytosolic peroxiredoxin 2 to erythrocyte membrane imposed by hydrogen peroxide-induced oxidative stressPublication . Rocha, Susana; Costa, Elísio; Coimbra, Susana; Nascimento, Henrique; Catarino, Cristina; Rocha-Pereira, Petronila; Quintanilha, Alexandre; Belo, Luís; Santos-Silva, AliceHuman erythrocyte peroxiredoxin 2 (Prx2) is a typical 2-cys cytosolic peroxiredoxin with thiol-dependent hydrogen peroxide scavenger activity. In a previous work, we reported Prx2 erythrocyte membrane linkage in some Hereditary Spherocytosis patients and that it seemed to be related to oxidative stress. The aim of the present work was to determine if Prx2 linkage to erythrocyte membrane could be induced by oxidative stress mediated by H2O2 and to further understand how and why this process occurs. We performed in vitro assays in which catalase or both Hb autoxidation and catalase were inhibited, under H2O2-induced oxidative stress conditions. Erythrocyte membrane linked Prx2 was detected by immunoblotting and quantified by densitometry. As oxidative stress markers, we determined membrane bound hemoglobin and lipid peroxidation, and we found that their values increased with H2O2 concentration. Prx2 linkage to the membrane also rose with increasing H2O2 concentration, and was only observed when the oxidized form of the enzyme was present in the cytosol. Oxidized Hb and Prx2 membrane linkages appear to be independent processes, although, both result from oxidative stress and may be useful as oxidative stress and/or erythrocyte damage/senescence markers.
- Recombinant human erythropoietin therapy has beneffical cardio-renal effects on moderate stages of chronic renal failure in the ratPublication . Garrido, P.; Reis, F.; Costa, Elísio; Parada, B.; Piloto, N.; Sereno, J.; Teixeira, A.; Pinto, R.; Figueiredo, A.; Alves, R.; Rocha-Pereira, P.; Belo, L.; Santos-Silva, A.; Teixeira, F.This study aimed to assess the cardio-renal effects of rhEPO therapy on an animal model of moderate chronic renal failure (CRF). Four groups (n =7) of male rat were evaluated during a 12-week follow up period: control; rhEPO: 50 IU/Kg/wk; CRF: two-stage 3/, nephrectomy; CRF+ rhEPO (start after the 3'd wk of surgery). Renal function, haematology and serum inflammation and redox status were assessed. rhEPO treatment was able to partially attenuate renal function markers, totally correct anaemia, also showing a proliferative and antioxidant action, due to increased serum TGF-13I and decreased 3-NT. In conclusion, rhEPO therapy might be recommended in moderate CRF stages in order to efficiently correct not only the underlying anaemia but also the deleterious cardio-renal effects, due to a proliferative and antioxidant renoprotective action.
- Resistance to recombinant human erythropoietin therapy in haemodialysis patientsPublication . Costa, Elísio; Lima, Margarida; Rocha, Susana; Pereira, Petronila Rocha; Reis, Flávio; Castro, Elisabeth; Teixeira, Frederico; Miranda, Vasco; Faria, Maria do Sarmento; Loureiro, Alfredo; Quintanilha, Alexandre; Belo, Luís; Santos-Silva, AliceTo better clarify the mechanism of resistance to recombinan thuman erythropoietin (rhEPO) therapy in haemodialysis patients, we studied systemic changes associated with resistance to rhEPO therapy in haemodialysis patients under rhEPO therapies, with particular interest on inflammation, leukocyte activation, iron status, oxidative stress and erythrocyte damage. We studied 63 chronic kidney disease (CKD) patients under haemodialysis and rhEPO therapies (32 responders and 31 non-responders to rhEPO therapy) and 26 healthy volunteers. In 20 of the CKD patients (10 responders and 10 non-responders to rhEPO therapy), blood samples were also collected immediately after dialysis to study the effect of the haemodialysis procedure. When compared to controls, haemodialysis patients presented lymphopenia, which results, at least in part, from a decrease in total circulating CD3+ T-lymphocytes and affect both the CD4+ and the CD8+ T-cell subsets. These lymphocytes presented markers of enhanced continuous activation state and enhanced ability to produce Th1 related cytokines. Furthermore, haemodialysis patients presented raised markers of an inflammatory process, and of an enhanced neutrophil activation, as showed by the high serum levels of elastase. Concerning to iron status, patients showed increased ferritin and prohepcidin serum levels, and a decrease in transferrin. Furthermore, some changes were observed in erythrocyte membrane protein composition and in band 3 profile, being the decrease in spectrin the most significant change. Higher plasma levels of total antioxidant status (TAS), lipidic peroxidation (TBA) and TBA/TAS ratio were also found. When comparing the two groups of patients, we found that non-responders presented a significant decrease in total lymphocyte and CD4+ T-cell counts, a more accentuated inflammatory process and indicators of enhanced neutrophil activation. No significant differences were found in serum iron status markers between the two groups of patients, except for the soluble transferrin receptor, which was higher among nonresponders. Prohepcidin serum levels were significantly lower in non-responders, but were higher than those in the control group. An accentuated decrease in erythrocyte membrane spectrin, alterations in band 3 profile [decrease in band 3 proteolytic fragments (Pfrag) and in Pfrag/band 3 monomer ratio], and a trend to higher values of membrane bound haemoglobin were also found in non-responders patients. In conclusion, although the etiology of resistance to rhEPO therapy is still unknown, our work confirms that inflammationseems to have an important role in its pathophysiology. We also showed that resistance to rhEPO therapy is associated with “functional” iron deficiency, lymphopenia and CD4+ lymphopenia, higher elastase plasma levels, increased interleukin-7 serum levels, and alterations in erythrocyte membrane protein structure and in band 3 profile. Further studies are needed to understand the rise in inflammation with the associated need in higher doses of rhEPO and the reduced iron availability.
- Hemorheological and cardiovascular effects of erythropoietin in a rat model of sports dopingPublication . Piloto, N.; Teixeira, HM; Garrido, P.; Teixeira-Lemos, E.; Teixeira, M.; Parada, B.; Sereno, J.; Pinto, R .; Alves, R.; Santos, P.; Romão, AM; Nunes, S.; Neto, P.; Carvalho, L.; Couceiro, P.; Xavier, F.; Rocha-Pereira, P.; Costa, Elísio; Belo, L.; Santos-Silva, A.; Teixeira, F.; Reis, F.Recombinant human erythropoietin (rhEPO) has been therapeutically used for correction of anaemia. However, due to the increase in circulating red blood cells (RBCs) it promotes, thus increasing oxygen delivery to muscles and improving performance in sport, it has been also illegally used as sports doping. Besides the well known increase of hematocrit and blood viscosity; which might cause serious complications for the athletes, other disturbances could occur, whose mechanisms remain to be fully elucidated. This study aimed to evaluate the hemorheological and cardiovascular effects of administration of rhEPO to rats under chronic aerobic exercise. A ten week-protocol was performed in four male Wistar rat groups: control — sedentary; rhEPO — 50 IV/kg, 3 times/wk; exercised (EX) — swimming for 1 hr, 3 times/ wk; EX+rhEPO. rhEPO in trained rats promoted erythrocyte count increase, hypertension, heart hypertro-phy, sympathetic and serotonergic overactivation, as well as a trend to increased oxidative stress. In conclusion, rhEPO doping in rats under chronic exercise promotes not only the expected increased hematocrit, but also other serious deleterious cardiovascular and thromboembolic modifications, including live risk, which might be known and assumed by all sports authorities, including athletes and their physicians.
- Band 3 profile as a marker of erythrocyte changes in chronic kidney disease patientsPublication . Costa, Elísio; Rocha, Susana; Rocha-Pereira, Petronila; Castro, Elisabeth; Miranda, Vasco; Faria, Maria do Sameiro; Loureio, Alfredo; Quintanilha, Alexandre; Belo, Luís; Santos-Silva, AliceOur aim was to study changes in red blood cell (RBC) membrane band 3 profile, as a cumulative marker of RBC changes, in chronic kidney disease (CKD) patients under haemodialysis and recombinant human erythropoietin (rhEPO) therapy and its linkage with resistance to this therapy. We studied 63 CKD patients, 32 responders and 31 non-responders to rhEPO therapy, and 26 healthy individuals. We evaluated the band 3 profile [% of band 3 monomer, high molecular weight aggregates (HMWAg), and proteolytic fragments (Pfrag)], membrane-bound haemoglobin (MBH), haematological data, total serum bilirubin, glutathione peroxidase (GPx) and superoxide dismutase activities, total antioxidant status (TAS) and plasma lipid peroxidation (TBA). Compared to controls, band 3 profile presented by CKD patients showed statistically significant lower HMWAg and Pfrag values and a significant higher value in band 3 monomer. GPx, TBA and TAS activities, and TBA/TAS ratio were also significantly higher in CKD patients. Comparing responders to non-responders CKD patients, significantly lower value in Pfrag and a trend for a higher value in MBH were found in non-responders. Our data suggest that CKD patients present younger RBC population, which could be related to the rhEPO therapy. The adverse plasma environment associated to CKD patients under hemodialysis imposes changes in band 3 profile, particularly in non-responders, suggesting that resistance to rhEPO therapy in CKD patients seems to be associated to an increase in RBC damage.
- Recombinant human erythropoietin treatment protects the cardio-renal axis in a model of moderate chronic renal failurePublication . Teixeira, Ana Margarida; Garrido, Patrícia; Santos, Paulo; Alves, Rui; Parada, Belmiro; Costa, Elísio; Almeida, Anabela; Teixeira-Lemos, Edite; Sereno, José; Pinto, Rui; Belo, Luís; Santos-Silva, Alice; Teixeira, Frederico; Reis, FlávioChronic kidney disease (CKD) patients develop anemia because of the low kidney erythropoietin (EPO) production, thus promoting cardiovascular complications. The degree of renal insufficiency might determine the moment to start recombinant human erythropoietin (rhEPO) therapy, but the molecular basis for these options deserves better elucidation. This study aimed to clarify the cardio-renal effects of earlier rhEPO ther- 20 apy in rats with moderate chronic renal failure (CRF). Four groups of rats were evaluated for 15 weeks (control; rhEPO - 50 Ill/kg/week; CRF - 3/4 nephrectomy; CRF + rhEPO) to assess renal and hematology data, EPO levels, blood pressure, heart rate, peripheral catecholamines contents, serum-transforming growth factor-31 (TGF-f11), kidney gene expression of EPO, Caspase 9 (Casp9), and vascular endothelial growth factor (Vegf). This model of moderate CRF showed moderate and corrected anemia, hypertension, tachycardia, sympa- 25 thetic overactivity, and increased serum TGF-31 content. The remnant kidney showed a proliferative profile, with hypertrophy, downregulated gene expression of EPO, and upregulated gene expression of Vegf and Casp9. rhEPO treatment promoted erythrocytosis and prevented tachycardia and catecholamines increment, with a rise of serum TGF-$1. Furthermore, the decreased kidney gene expression of EPO and the overexpression of Casp9 were prevented, demonstrating a renoprotective action on the remnant kidney. In conclusion, 30 rhEPO therapy promotes a protective effect on the cardio-renal axis, which might be mainly attributed to its pro-proliferative and anti-apoptotic properties. These findings might recommend its use in earlier stages of CRF, acting as an erythropoiesis stimulating agent, to efficiently correct not only the anemia, one of the major complications in these patients, but also the succeeding adverse cardio-renal effects.
- DMT1 (NRAMP2/DCT1) genetic variability and resistance to recombinant human erythropoietin therapy in chronic kidney disease patients under haemodialysisPublication . Costa, Elísio; Rocha, Susana; Rocha-Pereira, Petronila; Reis, Flávio; Castro, Elisabeth; Teixeira, Frederico; Miranda, Vasco; Faria, Maria do Sameiro; Loureio, Alfredo; Quintanilha, Alexandre; Belo, Luís; Santos-Silva, Alice
- Plant aqueous extracts: antioxidant capacity via haemolysis and bacteriophage P22 protectionPublication . Gião, Maria S.; Leitão, Isabel; Pereira, Ana; Borges, André B.; Guedes, Catarina J.; Fernandes, João C.; Belo, Luís; Santos-Silva, Alice; Hogg, Tim A.; Pintado, Manuela E.; Malcata, F. XavierThe bacteriophage P22/Salmonella Typhimurium system, as well as human erythrocytes have been used to assay for protection, against forced oxidation caused by hydrogen peroxide, brought about by several aqueous extracts of selected adventitious plants grown in Portugal. This study proved, for the first time, that the aforementioned bacteriophage-based system is a suitable method to assess the antioxidant activity of plant extracts; among the 12 plants tested, raspberry (Rubus idaeus), sage (Salvia sp.), savory (Satureja montana) and yarrow (Achillea millefolium) were found to effectively protect against oxidative damage caused by H2O2. Haemolysis was inhibited via pre-treatment with every plant extract tested, except heath at 0.1% (w/v). The two analytical methods produced different results – and for some plants, there was a dependence (either direct or inverse) of the quantitative protection effect on extract concentration, whereas for others no significant dependence was found at all. Savory yielded the most promising results, using either method. Therefore, the P22/Salmonella system can be used as a suitable in vivo assay, and human erythrocytes as a suitable in vitro assay to confirm (or not) the antioxidant capacity of plant extracts in biological matrices.
- Cardiac antiapoptotic and proproliferative effect of recombinant human erythropoietin in a moderate stage of chronic renal failure in the ratPublication . Teixeira, M.; Rodrigues-Santos, P.; Garrido, P.; Costa, E.; Parada, B.; Sereno, J.; Alves, R.; Belo, L.; Teixeira, F.; Santos-Silva, A.; Reis, F.Objective: Recombinant human erythropoietin (rhEPO) therapy under circumstances of moderate chronic renal failure (CRF), with yet lower kidney and heart lesion, may have a protective cardiac effect beyond the correction of anemia, whose mechanism deserves better elucidation, namely by clarifying the impact on gene expression profile of markers of apoptosis, inflammation, proliferation, angiogenesis, and lesion/stress in the heart. Materials and Methods: Four groups of rats were studied over a period of 15 weeks (n=7 each): control-without surgery and without drug treatment; rhEPO-treated with 50 IU/kg/week of rhEPO-beta; CRF-submitted to partial nephrectomy (3/4); CRF + rhEPO-CRF with rhEPO treatment after the 3rd week of surgery. The heart was collected in order to evaluate the gene expression, by real-time qPCR, of markers of apoptotic machinery, inflammation/immunology, proliferation/ angiogenesis, and lesion/stress. Results: The main findings obtained were (a) CRF rats have demonstrated overexpression of EPO-R in the heart without changes on EPO expression, together with overexpression of Bax/Bcl2 ratio, PCNA, and IL-2; (b) rhEPO therapy on the heart of the rats with CRF induced by partial 3/4 nephrectomy promoted nonhematopoietic protection, demonstrated by the apoptosis prevention, viewed by the Bax/Bcl2 balance, by the promotion of proliferation, due to PCNA increment, and by the immunomodulatory action, expressed by a trend to prevent the IL-2 increment. Conclusion: In this model of moderate CRF, rhEPO treatment showed important cardiac nonhematopoietic effects, expressed mainly by the antiapoptotic and the proproliferative action, suggesting that early rhEPO therapy in moderate stages of CRF might have further therapeutic benefits.
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