Search

The widespread use of aluminum (Al) provides easy exposure of humans to the metal and its accumulation remains a potential problem. In vivo and in vitro assays have associated Al overload with anemia. To better understand the mechanisms by which Al affects human erythrocytes, morphological and biochemical changes were analyzed after long-term treatment using an in vitro model. The appearance of erythrocytes with abnormal shapes suggested metal interaction with cell surface, supported by the fact that high amounts of Al attached to cell membrane. Long-term incubation of human erythrocytes with Al induced signs of premature erythrocyte death (eryptosis), such as phosphatidylserine externalization, increased intracellular calcium, and band 3 degradation. Signs of oxidative stress, such as significant increase in reactive oxygen species in parallel with decrease in the amount of reduced glutathione, were also observed. These oxidative effects were completely prevented by the antioxidant N-acetylcysteine. Interestingly, erythrocytes were also protected from the prooxidative action of Al by the presence of erythropoietin (EPO). In conclusion, results provide evidence that chronic Al exposure may lead to biochemical and morphological alterations similar to those shown in eryptosis induced by oxidant compounds in human erythrocytes. The antieryptotic effect of EPO may contribute to enhance the knowledge of its physiological role on erythroid cells. Irrespective of the antioxidant mechanism, this property of EPO, shown in this model of Al exposure, let us suggest potential benefits by EPO treatment of patients with anemia associated to altered redox environment., (Copyright © 2011 Wiley Periodicals, Inc.)

The in-vivo survival of 51Cr-labelled murine red blood cells (RBCs) loaded with recombinant human erythropoietin (rhEpo-RBCs) was slightly lower than that of normal RBCs. Intravenous administration to normal mice of the encapsulated rhEpo shows the pharmacokinetic bicompartmental profile typical of the free rhEpo. Distribution and elimination half-life values for the RBC-entrapped rhEpo were no longer than those for the free protein. The area under the curve value was significantly increased for rhEpo-RBCs. Hypertransfused polycythaemic mice were evaluated as an adequate animal model to study the in vivo biological activity of encapsulated rhEpo. rhEpo-RBCs stimulate the erythropoiesis of polycythaemic mice in a linear dose-radio-iron incorporation response relationship. These results suggest that rhEpo-RBCs may behave as an alternative to the administration of free rhEpo in the clinical field.

Recombinant human erythropoietin (rhuEPO) therapy has been reported to maintain corrected hematocrit values by increasing the length of red blood cell (RBC) survival. This article presents a controlled study that assessed the RBC survival before, during, and after termination of prolonged rhuEPO treatment of chronic hemodialysis patients. Two groups of 20 patients were studied. The hematocrit value of each patient was below 28 vol%. One group (Group A) was treated with rhuEPO for 1 yr and then treatment was stopped because of the unavailability of the drug. The second group (Group B) was treated for 2 yr. Epoetin beta was administered subcutaneously. The initial dose was 20 U per kg body weight three times weekly. Upon reaching the target hematocrit value of 30 to 35 vol%, the dose was individualized for each patient, to maintain target range. RBC survival was determined by the chromium-51 technique. In Group A, RBC survival was determined: (1) before, (2) at 12 months, and (3) 1 yr after cessation of rhuEPO treatment. In Group B, RBC survival was determined: (1) at 24 months of therapy, and (2) 1 yr after cessation of rhuEPO treatment. RBC survival increased significantly in both patient groups under rhuEPO treatment. After cessation of therapy, the RBC survival decreased to pretreatment values. During the correction period, reticulocyte counts increased significantly in both groups. Over the maintenance period, they slightly decreased, and after termination of rhuEPO therapy, they decreased to the pretreatment values. The results of this study could suggest the possibility that RBC survival was prolonged by the action of EPO on the erythroid progenitors, resulting in the production of RBC with improved viability.

beta thalassemia (beta thal) in DBA/2J mice is a consequence of the spontaneous and complete deletion of the beta major globin gene. Homozygous beta thal mice have clinical and biological features similar to those observed in human beta thal intermedia. Erythrocytes in human beta thal are characterized by a relative cell dehydration and reduced K+ content. The role of this erythrocyte dehydration in the reduced erythrocyte survival, which typifies the disease, has not previously been evaluated. We examined for 1 month the effects on the anemia and the erythrocyte characteristics of beta thal mice of daily treatment with either clotrimazole (CLT), an inhibitor of red blood cell (RBC) dehydration via the Gardos channel, or human recombinant erythropoietin (r-HuEPO), or hydroxyurea (HU). The use of either r-HuEPO or HU induced a significant increase in hemoglobin (Hb), hematocrit (Hct), erythrocyte K+ and a decrease in percent reticulocytes, suggesting improved erythrocyte survival. CLT alone decreased only mean corpuscular hemoglobin concentration (MCHC) and cell density and increased cell K+. Thus, though the Gardos channel plays a major role in cell dehydration of murine beta thal erythrocyte survival. Combination therapy with r-HuEPO plus HU produced no incremental benefit beyond those of single drug therapy. However, addition of CLT to r-HuEPO, to HU, or to combined r-HuEPO plus HU led to statistically significant increase in Hb, Hct, and erythrocyte K+ compared with any of the regimens without CLT. These results suggest that CLT not only inhibits erythrocyte dehydration, but also potentiates the erythropoietic and cellular survival responses to r-HuEPO and HU.

Murine erythroid progenitors infected with the anemia-inducing strain of Friend virus (FVA cells) undergo apoptosis when deprived of erythropoietin (EPO). When cultured with EPO, they survive and complete terminal differentiation. Although cell volume is decreased and nuclear chromatin is condensed during both apoptosis and terminal differentiation, morphologic and biochemical distinctions between these two processes were observed. In apoptosis, homogeneous nuclear condensation with nuclear envelope loss occurred in cells that had not reached the stage of hemoglobin synthesis. In terminal erythroid differentiation, nuclear condensation with heterochromatin, euchromatin, and nuclear envelope preservation occurred simultaneously with hemoglobin synthesis. Cells with apoptotic morphology appeared asynchronously in EPO-deprived cultures, indicating that only a portion of the cells were undergoing apoptosis at any given time. The percentages of apoptotic cells and cleaved DNA increased with time in EPO-deprived cultures. Inhibition of DNA cleavage was directly proportional to EPO concentration over a wide physiologic range, demonstrating a heterogeneity in susceptibility to apoptosis based on variability in the EPO sensitivity of individual cells. A subpopulation of FVA cells with increased EPO sensitivity (decreased EPO requirement) was isolated from EPO-deprived cultures. This increased EPO sensitivity did not result from differences in EPO receptor number, affinity, or structure, suggesting that the differences are in the signal transduction pathway. These results indicate that control of red blood cell production involves both prevention of apoptosis by EPO and heterogeneity in the EPO requirement of individual progenitor cells.

The oxidative injury to erythrocytes, red blood cell (RBC) rigidity and splenic hemolysis was assayed in 17 chronically hemodialyzed patients before and during recombinant erythropoietin (EPO) treatment. When a stable hematocrit between 30 and 35% had been established for at least 4 months, a statistically significant increase in RBC volume, hemoglobin concentration, hematocrit, reticulocyte count, and several RBC enzymes (2,3-diphosphoglycerate, glucose 6-phosphate dehydrogenase, pyruvate kinase, hexokinase) was noted. This indicated significant RBC rejuvenation under the influence of EPO. However, no significant improvement in the RBC oxidative sensitivity, RBC deformability, splenic RBC volume, slow mixing splenic RBC volume, and the intrasplenic RBC transit time could be disclosed. These data confirm the existence of an extra-erythrocytic factor in uremic plasma, which is partly responsible for a reduced RBC life span in hemodialysis patients despite EPO treatment.

It is well known that red blood cell (RBC) and reticulocyte counts increase when anemic hemodialysis patients receive repeated administrations of recombinant human erythropoietin(rh-EPO). However, the early response of reticulocyte has not been reported precisely. In this study, reticulocyte counts and its fractions were observed in 8 maintenance hemodialysis patients after a single intravenous administration of 60 u/Kg of rh-EPO. The peripheral blood cells taken from the patients were stained with auramine 0, which bound to RNA and caused fluorescence. R-1000 (Sysmex, Kobe, Japan) analyzed the reticulocytes and classified the stained cells into three groups by the intensity of fluorescence. The number of highly fluorescent reticulocyte, which was young and large, increased and reached its highest level on day 4 after a single administration of rh-EPO. The reticulocyte counts paralleled these changes, but RBC counts did not change after administrations of rh-EPO. These data suggest that the observation of reticulocytes and its fractions in hemodialysis patients after a single administration of rh-EPO could tell us whether reticulocytes are responsive to rh-EPO or not, and this could be called as EPO test.

Fifty-seven patients receiving chronic high-flux hemodialysis began receiving recombinant alpha-human erythropoietin (rHuEPO). The mean initial rHuEPO dose used in 54 evaluable patients was 9963 +/- 4364 U/week; the final dose was 8972 +/- 4058 U/week. Treatment over a mean period of 154 +/- 40 days (84 to 224 days) resulted in an average increase in hematocrit from 24.7% +/- 3.7% to 32.5% +/- 4.4%. We present a model for these data that describes changes in hematocrit during rHuEPO therapy and that allows simultaneous estimation of red blood cell lifespan and rHuEPO-induced increases in red blood cell production rate. Analysis of the hematocrit values of the patients with the model, by use of NONMEM, a computer program for analysis of population data, reveals a nonlinear dose-response relationship with large interindividual variability (coefficient of variation) of about 50%. The estimated mean red blood cell lifespan is 64 days, with interindividual variability of about 30% (coefficient of variation). The intraindividual random variability in hematocrit about its prediction is +/- 5% of the prediction. For clinical dose adjustment, we present a method that uses only simple calculations.

Iron deficiency is the main reason for insufficient response to rEPO therapy. Serum ferritin and transferrin saturation give valuable information on storage iron and iron transport. Iron demand for correction of anemia can easily be estimated after HCT (vol%) x average blood volume (dl) = mg iron. Inadequate iron supply of the bone marrow in the presence of sufficient storage iron in the RES develops frequently under rEPO, possibly explaining the improvement of bone marrow response to rEPO by concomitant intravenous iron supply. The reasons of functional iron deficiency are still speculative.

The clinical and ferrokinetic effects of escalating doses of subcutaneously administered recombinant human erythropoietin (rh-EPO) were studied in ten patients with myelodysplastic syndromes and severe transfusion-dependent anemia. Red blood cell transfusion requirements diminished in four patients, and one of the patients eventually became transfusion independent with an EPO-induced rise of Hb from 7.7 g/dl to 12.3 g/dl. Endogenous serum levels of EPO were significantly increased in all patients (100-5700 mU/ml), but three of four responders had a relatively low baseline level. The effective red cell iron turnover (RCIT) improved in two responding patients and even normalized in one patient. This increase in RCIT was accompanied with a decline in the ineffective red cell iron turnover (IIT). The other responding patients had a relatively preserved RCIT before EPO treatment. EPO therapy further increased the fraction of IIT in the latter patients. Red cell survival time did not increase during EPO therapy, even in the responding patients. One transient and one maintained increase in platelet count were observed. Disease progression with a sustained increase in blast cells in one patient and a transient elevation of blasts in another patient was seen. No other side effects of EPO therapy were observed. These results suggest that anemic MDS patients with low serum EPO levels and relatively spared effective erythropoiesis as measured by ferrokinetic studies may be the best candidates for treatment with recombinant human EPO.

rHuEPO was administered to eight patients with chronic renal failure (CRF) and uremia (U) for 1 year. Creatinine Clearance (GFR) averaged 14.3 (9-25) ml/min at an Hct of 28% (26-30). Baseline RBC survival by 51Cr T1/2 was highly correlated with GFR (r = 0.66), p less than 0.05 (2), average 21.6 days. Repeat 51Cr T1/2 at 3 months with GFR 10 ml/min at Hct 38% was prolonged by 7 days to 28.6 (p less than 0.005), and at 1 year remained increased to 28 days (p less than 0.001) with Hct 39%, despite further decreased GFR to less than 4 ml/min and need for dialysis. Reticulocytes varied from 1.6% to 7.4 (3-6 weeks) to 3.1 (3 months) and 1.5% (1 year). Bone marrow cellularity increased from 36% to 47% (3 months) and 44% (1 year). M:E ratio decreased from 3.9:1 to 1.7:1 (3 months) to 1.6:1 (1 year). Marrow iron decreased from 4.1/6 to 2.4/6 (3 months) to 1.8/6 (1 year). Doses of rHuEPO had to be reduced to avoid polycythemia. rHuEPO stimulates erythropoiesis in pts with progressive CRF and U for 1 year. The initial increase in hematocrit is due to the early peak of reticulocytes. At 3 months, rHuEPO maintains the increased hematocrit by three mechanisms: 1) increased reticulocytosis, 2) a trend to increased bone marrow erythroid cellularity, and 3) lengthened RBC survival. At 1 year of rHuEPO therapy, the trend to increased marrow cellularity persists, however, the maintenance of target hematocrit is via a lengthened RBC survival. Despite progression of CRF and U, rHuEPO produced RBCs with longer survival than expected. RBC membranes may have been stabilized by rHuEPO.

The mechanism by which erythropoietin controls mammalian erythrocyte production is unknown. Labeling experiments in vitro with [3H]thymidine demonstrated DNA cleavage in erythroid progenitor cells that was accompanied by DNA repair and synthesis. Erythropoietin reduced DNA cleavage by a factor of 2.6. In the absence of erythropoietin, erythroid progenitor cells accumulated DNA cleavage fragments characteristic of those found in programmed cell death (apoptosis) by 2 to 4 hours and began dying by 16 hours. In the presence of erythropoietin, the progenitor cells survived and differentiated into reticulocytes. Thus, apoptosis is a major component of normal erythropoiesis, and erythropoietin controls erythrocyte production by retarding DNA breakdown and preventing apoptosis in erythroid progenitor cells.

Renal anemia is caused in part by a reduced life span of red blood cells (RBCs) and by reduced erythropoietin biosynthesis in the damaged kidney. The RBC age can be determined by density gradient centrifugation and estimation of cell-age-dependent enzyme activities, as aspartate aminotransferase. The RBC age distribution influences the median density (D50) of RBCs and the blood rheology in coherence with the hematocrit. In our study, the median density was determined by Percoll density gradient centrifugation in 18 healthy subjects (D50 = 1.0674 +/- 0.0016 g/ml) and in 14 hemodialysis patients (D50 = 1.0674 +/- 0.0016 g/ml in the course of recombinant human erythropoietin (rhEPO) therapy. During the first 4 weeks of therapy, a strong rejuvenation of RBCs was observed whereby the D50 reached a minimum after 2 weeks (D50 = 1.0655 +/- 0.0022 g/ml; p less than 0.05 vs. value before therapy) and a steady state after 4 weeks (D50 = 1.0658 +/- 0.0013 g/ml; p less than 0.1 vs. value before therapy). In 5 of the patients with elevated plasma parathyroid hormone (i-PTH) concentrations greater than 10 pmol/l, a significantly (p less than 0.05) reduced amount of younger RBCs (D50 = 1.0675 +/- 0.0016 g/ml) was observed in the first 2 weeks of rhEPO therapy as compared to patients with i-PTH less than 10 pmol/l (D50 = 1.0677 +/- 0.0019 g/ml). Thus, erythropoiesis in the early phase of rhEPO therapy is strongly influenced by elevated plasma i-PTH concentrations. Therefore, a gradual increase in rhEPO doses is preferable before therapy at elevated doses with an uncontrolled increase in RBC amount.

In eleven patients with uraemia on intermittent haemodialysis treatment, recombinant human erythropoietin (rHuEpo) was used at a dosage schedule of 100 IU/kg bodyweight thrice weekly. Erythrokinetic studies (blood volume, RBC survival and iron kinetics) were performed in nine cases before and after 6 months of treatment. The remaining two patients had only RBC and plasma volume determinations before and after treatment. Although total blood volume remained unchanged, RBC volume was increased in all cases. Red cell loss was not modified, and quantitative improvement of RBC production was noted in all cases. No qualitative defect of erythroid maturation or release was observed in the treated patients. In conclusion, rHuEpo treatment improves the anaemia of haemodialysis patients by normalising circulating RBC volume only through an increase in red cell production.

Study Objective: To determine the efficacy and safety of recombinant human erythropoietin (r-HuEPO) in predialysis renal patients., Design: Randomized, double-blind, placebo-controlled trial for 8 weeks., Setting: Inpatient and outpatient facility in the Clinical Research Center of a university-based hospital., Patients: Fourteen adult subjects with renal insufficiency (mean serum creatinine, 473 mumol/L +/- 61 [6.2 +/- 0.8 mg/dL]) and anemia (mean hematocrit, 0.27 +/- 0.01)., Interventions: Recombinant human erythropoietin, 50, 100, or 150 IU/kg body weight or placebo given intravenously three times per week., Measurements and Main Results: Subjects who received active r-HuEPO showed a dose-dependent rise in hematocrit; mean hematocrit increased 41% from 0.27 +/- 0.01 to 0.38 +/- 0.01. At the same time, erythrocyte mass rose 43% from 13.7 +/- 0.6 mL/kg in the baseline state to 19.6 +/- 1.0 mL/kg after treatment. Maximal oxygen consumption during exercise increased 9% from 16.0 mL/min.kg +/- 1.8 to 17.5 mL/min.kg +/- 1.9., Conclusions: Recombinant human erythropoietin is effective and safe in ameliorating the anemia of pre-dialysis patients.

Six anemic hemodialysis patients dependent on regular blood transfusions and with massive iron overload were treated with recombinant human erythropoietin (r-huEPO). The effect on absolute reticulocyte count, hemoglobin and serum ferritin was studied during a twenty-week period. Red-cell volume and red-cell life span were measured with 51Cr-tagged erythrocytes at baseline and after twenty weeks of r-huEPO. Absolute reticulocyte counts and hemoglobin concentration rose markedly (from 55.6 +/- 31.2 to a maximum of 174.9 +/- 31.0 x 10(9)/l at 4 weeks and from 6.8 +/- 0.3 to a maximum of 11.2 +/- 1.3 g/dl at 12 weeks, respectively, p less than 0.001) without any further need for transfusions. Red-cell volumes increased concomitantly (from 58 +/- 4 to 81 +/- 11% of normal, p less than 0.005), in spite of a persistent shortening of red-cell life span (45 +/- 18 and 47 +/- 4 days before and after r-huEPO). Markedly elevated serum ferritin concentrations indicating iron overload decreased slowly from 3,550 +/- 1,615 to 2,721 +/- 1,506 micrograms/l (p less than 0.05). It is concluded that r-huEPO is very effective in treating the anemia of patients maintained on hemodialysis. The favorable effects on hemoglobin and red-cell volumes occur in spite of persistent hemolysis and lead to a slow reduction of iron overload.