Red Blood Cell Precursors Research Articles

Seeing EPO

Modeling and experiments help explain responsiveness of red blood cell precursors to very large changes in a proliferative signal.

α-Hemoglobin Stabilizing Protein (AHSP), a Kinetic Scheme of the Action of a Human Mutant, AHSPV56G

A kinetic analysis has been made of the interaction of alpha-Hb chains with a mutant alpha-hemoglobin stabilizing protein, AHSP(V56G), which is the first case of an AHSP mutation associated with clinical symptoms of mild thalassemia syndrome. The chaperone AHSP is thought to protect nascent alpha chains until final binding to the partner beta-Hb. Rather than protecting alpha chains, the mutant chaperone is partially unfolded but recovers its secondary structure via interaction with alpha-Hb. For both AHSP(WT) and AHSP(V56G), the binding to alpha-Hb is quite rapid relative to the alpha-beta reaction, as expected because the chaperone binding must be quite competitive to complete the alpha chain folding process before alpha-Hb binds irreversibly to beta-Hb. The main kinetic difference is a dissociation rate of AHSP(V56G).alpha-Hb some four times faster relative to AHSP.alpha-Hb. Considering a role of protein folding, the AHSP(V56G) apparently does not bind long enough (0.5 s versus 2 s for the WT) to complete the structural modifications. The overall replacement reaction (AHSP.alpha-Hb + beta-Hb --> AHSP + alphabeta) can be quite long, especially if there is an excess of AHSP relative to beta-Hb monomers.

Expression of microRNA-451 in normal and thalassemic erythropoiesis

MicroRNAs (miRNAs) are negative regulators of gene expression that play an important role in hematopoiesis. Thalassemia, a defective globin synthesis leading to precipitate of excess unbound globins in red blood cell precursors, results in defective erythroid precursors and ineffective erythropoiesis. Expression pattern of miR-451, an erythroid-specific miRNA, was analyzed during differentiation of erythroid progenitors derived from normal and thalassemic peripheral blood CD34-positive cells, after 14 days of culture. A biphasic expression with transient up-regulation of miRNA-451 on day 3 of cultures was observed during thalassemic erythroid differentiation. In contrast, the expression pattern of the miR-451 in erythroid cells obtained from the other extravascular hemolytic anemia, i.e., hereditary spherocytosis patients showed no transient up-regulation of miR-451 on day 3 of cultures. Our results suggest that early erythroid progenitors in beta-thalassemia have a dysregulated miRNA-451 expression program, and analysis of microRNA is a relevant approach to determine abnormalities of erythropoiesis.

Anti-glycophorin single-chain Fv fusion to low-affinity mutant erythropoietin improves red blood cell-lineage specificity

The presence of erythropoietin (Epo) receptors on cells besides red blood cell precursors, such as cancer cells or megakaryocyte precursors, can lead to side effects during Epo therapy including enhanced tumor growth and platelet production. It would be ideal if the action of Epo could be limited to erythroid precursors. To address this issue, we constructed single-chain variable fragment (scFv)-Epo fusion proteins that used the anti-glycophorin 10F7 scFv amino-terminal to Epo analogues that would have minimal activity alone. We introduced the Epo mutations N147A, R150A and R150E, which progressively weakened receptor affinity in the context of Epo alone, as defined by cell proliferation assays using TF-1 or UT-7 cells. Fusion of these mutant proteins to the 10F7 scFv significantly rescued the activity of the mutant proteins, but had a relatively small effect on wild-type Epo. For example, fusion to the 10F7 scFv enhanced the activity of Epo(R150A) by 10- to 27-fold, while a corresponding fusion to wild-type Epo enhanced its activity only up to 2.7-fold. When glycophorin was blocked by antibody competition or reduced by siRNA-mediated inhibition of expression, the activity of 10F7 scFv-Epo(R150A) was correspondingly reduced, while such inhibition had essentially no effect on the activity of 10F7 scFv-Epo(wild-type). In addition, potent stimulation of Epo receptors by 10F7 scFv-Epo(R150A) was observed in long-term proliferation and viability assays. Taken together, these results indicate that a combination of targeting and affinity modulation can be used to engineer forms of Epo with enhanced cell-type specificity.

Érythropoïétine et radiothérapie

Erythropoietin (EPO) is a glycoprotein hormone. This hormone is a growth factor for red blood cells precursors in the bone marrow. The decrease of oxygen partial pressure, a reduced number of erythrocytes caused by bleeding or excessive destruction, or increased tissues oxygen requirements lead to increased secretion of EPO. Its action takes place on bone marrow erythroblastic cells through specific receptors. EPO stimulates the proliferation of red cell precursors stem cells in the bone marrow, thus increasing their production in one to two weeks. The effectiveness of EPO at increasing haemoglobin and improving patients’ quality of life has been demonstrated by several studies. However, its use in radiotherapy remains controversial. While tumour hypoxia caused by anaemia is a factor of radio resistance and thus a source of local failure, tumour expression of EPO receptors presents a significant risk for tumour progression and neo-angiogenesis, which would be increased during the administration of EPO. The purpose of this article is to answer the question: is there a place for EPO in combination with radiotherapy in the management of cancer?

Neuroprotective role of erythropoietin by antiapoptosis in the retina

Erythropoietin (EPO) stimulates red blood cell production, in part by inhibiting apoptosis of the red blood cell precursors. The erythropoietic effects of EPO are circadian stage dependent. Retinal injury due to light occurs through oxidative mechanisms and is manifest by retinal and retinal pigment epithelium (RPE) cells apoptosis. The visual cycle might be circadian coordinated as a means of effectively protecting the retina from the detrimental effects of light-induced, oxygen-dependent, free radical-mediated damage, especially at the times of day when light is more intense. We show that the retinal expression of EPO and its receptor (EPOR), as well as subsequent Janus kinase 2 (Jak2) phosphorylations, are each tightly linked to a specific time after oxidative stress and in anticipation of daily light onset. This is consistent with physiological protection against daily light-induced, oxidatively mediated retinal apoptosis. In vitro, we verify that EPO protects RPE cells from light, hyperoxia, and hydrogen peroxide-induced retinal cell apoptosis, and that these stimuli increase EPO and EPOR expression in cultured RPE cells. Together, these data support the premise that EPO and its EPOR interactions represent an important retinal shield from physiologic and pathologic light-induced oxidative injury.

Current Understanding of the Red Blood Cell Diseases

The human red blood cell is produced continuously from hematopoietic stem cells in the bone marrow. These hematopoietic stem cells can differentiate into red blood cell precursors, which continuously produce hemoglobin until it accounts for roughly 90% of the dry cell weight. The red blood cell precursors then undergo enucleation, a process in which the nucleus of the precursor cell is squeezed out to generate a mature red blood cell. 1 The mature red blood cell takes the rather unique shape of a biconcave disk, which is maintained by structure proteins in the cell membrane. The life span of a mature erythroblast is roughly 120 days in humans. A variety of red blood cell diseases are known, such as anemia, sickle cell diseases, malaria, hemolysis and polycythemias. Diseases involving red blood cells are generally due to three factors: genetic deficiency, poor nutrition or parasitic infection. In the past years, progress in genetic and molecular research tools has allowed for a deeper understanding of red blood cell diseases. In this paper, we’ll provide a review of advances in understanding and treatment of the most common red blood cell diseases: namely Malaria, Sickle Cell Disease, and Iron Deficiency Anemia.

Treatment of anemia by recombinant human erythropoietin in cancer patients undergoing radiotherapy

Anemia is a deficiency in red blood cells or in the hemoglobin (Hb) levels that leads to a decrease in the transport capacity of oxygen in the blood, which can reduce tolerance in radiotherapy (RT) and chemotherapy. The relationship between anemia and hypoxia, however, is complex and influenced by multiple variables. Although the blood Hb values that might develop hypoxia in tumors were not described clearly, optimal oxygen pressure was accepted in patients with an Hb value of 12–14 g/dL. Erythropoietin is a glycoprotein, which acts via EPOR to stimulate the growth, to prevent apoptosis, and to induce differentiation of red blood cell precursors. RhuEPO-α and -β are classically administered subcutaneously three times per week at doses ranging from 150 to 300 IU/kg. Darbepoetin-α has been shown to exhibit a longer elimination half-life, thus allowing a once-weekly administration at the dose of 2.25 µg/kg. Side-effects related to rhuEPO include hypertension and thromboembolic events. RhuEPO can be used effectively in the treatment of anemia in patients with solid tumor being treated by RT or chemoradiotherapy. Furthermore, the use of rhuEPO has been demonstrated to have a sustained beneficial impact on quality of life in cancer patients. However, the role of combination of rhuEPO with external RT still remains inconclusive and several clinical trials have been pointed increased mortality in patients treated with rhuEPO. In this paper, the probable radiobiological effects of anemia in patients treated with RT, the beneficial and adverse effects of rhuEPO, and related studies are reviewed. Future directions for the use of rhuEPO are proposed.

ATP8A1 activity and phosphatidylserine transbilayer movement

The asymmetric distribution of the amino-containing phospholipids, phosphatidyl-serine (PS) and phosphatidyl-ethanolamine (PE), across the two leaflets of red blood cell (RBC) membrane is essential to the function and survival of the cell. PS and PE are sequestered in the inner leaflet by an ATP-dependent transport activity of a membrane protein known as the RBC flippase that specifically moves amino-phospholipids from the outer to the inner leaflet. The enucleated RBC lacks the means to replace damaged enzymes and inactivation of the flippase can lead to the unwarranted exposure of PS on the cell surface. Loss in the ability to maintain phospholipid asymmetry is exacerbated in RBC disorders and PS-exposing RBCs present in the circulation play a significant role in the pathology of hemoglobinopathies. We identified the Atp8a1 protein, a member of the family of the P(4)-type ATPases, as a RBC flippase candidate. Atp8a1 is expressed in RBC precursors and is present in the membrane of mature red cells. The flippase activity of the protein was established in purified secretory vesicles of Saccharomyces cerevisiae. ATPase activity was stimulated by PS and PE. In addition, Atp8a1 can move PS molecules across the leaflets of the vesicle membrane in presence of ATP.

Erythropoietin and Iron

SUMMARY The US blood supply is the safest it has ever been, due to evolution of a combination of donor education, donor screening, and new laboratory assays. Currently, the most significant risk of mortality from blood transfusion is administrative error resulting in an ABO mismatch between blood unit and transfusion recipient, with hemolysis (one in 60,000) and death (one in 600,000). Knowledge gained regarding the relationship between erythropoietin, iron, and erythropoiesis suggests that under conditions of maximal stimulation of red blood cell precursors, iron restricted erythropoiesis occurs. The emergence of safer iron preparations makes the administration of intravenous iron a possible solution to the problem of iron restricted erythropoiesis. The value of this approach has been best demonstrated in patients with chronic kidney disease undergoing dialysis. Clinical trials are needed to establish the value of parenteral iron supplementation in other clinical settings employing erythropoietin therapy.

Obsessive compulsive disorder comorbidity in DBA

Diamond-Blackfan Anemia (DBA) is a congenital erythroid aplasia characterized as a normochromic macrocytic anemia with a selective deficiency in red blood cell precursors in otherwise normocelullar bone marrow. DBA is known to be associated with mental retardation and learning disabilities. Although comorbidities with other psychiatric conditions have not been reported in the existing literature, we report in this paper a case of a DBA patient with previously undiagnosed comorbidity of obsessive compulsive disorder (OCD), successfully treated with sertaline 200 mg/day and valproic acid 600 mg/day. This case of comorbid presentation has clinical, therapeutic and pathophysiological implications. Given the difficulty of distinguishing among mental retardation, learning disabilities and OCD and the importance of precocious diagnosis in treating OCD especially since there are treatment methods interfering with anemia symptoms, physicians should adapt an adequate screening tool treating a child with DBA and comorbid mental disorder.

High-performance liquid chromatography separation of aminopterin-polyglutamates within red blood cells of children treated for acute lymphoblastic leukemia

Aminopterin (AMT), like the related compound methotrexate (MTX), is a drug with anticancer and antiinflammatory efficacy that works by interfering with synthetic reactions dependent on the vitamin folic acid. Red blood cell (RBC) precursors will accumulate antifolates like AMT and MTX through the same mechanism by which they take up folate. Intracellular folate and antifolates are then metabolized to polyglutamates that remain within the mature RBCs. RBC MTX has been correlated with toxicity and/or treatment efficacy among patients with acute lymphoblastic leukemia (ALL) or rheumatoid arthritis. Because AMT may offer clinically relevant advantages over MTX, we are testing whether it can be administered safely in multiagent therapy to children with ALL. Total RBC AMT was measured to monitor compliance with this oral, outpatient regimen, and to estimate AMT exposure to the bone marrow. Here we describe methods for quantifying each AMT-polyglutamate species within the RBCs of patients. The assay was linear over a concentration range of 62.5-500 nmol/L. Recovery of individual AMT-polyglutamates ranged from 85% to 92%, and the intraday coefficients of variation were 1.3% to 3.6%. Long-chain AMT-polyglutamates (triglutamate and tetraglutamate forms) accounted for over 40% of intracellular AMT within the RBCs of patients. Patients with long-chain AMT polyglutamate concentrations above the median tended to have lower mean neutrophil counts during weekly AMT therapy, which suggests that RBC AMT polyglutamate accumulation may correlate with hematologic toxicity. As AMT continues to be tested in clinical trials, the methods described here will be useful to define relationships between clinical response to AMT and RBC accumulation of AMT-polyglutamates.

Transient Erythroblastopenia of Childhood in Siblings: Case Report and Review of the Literature

Transient erythroblastopenia of childhood is characterized by anemia due to decreased production of red blood cell precursors. It is almost always self-resolving and requires clinical intervention only in severe cases. This article describes 2 cases in half-siblings diagnosed approximately 10 years apart. A review of the literature identifies 11 other sibling pairs. Our case suggests an autosomal dominant pattern of inheritance. To date, the gene involved in the development of transient erythroblastopenia of childhood has not been identified.

Diseased red blood cells topple iron balance

In the inherited anemia β-thalassemia, diseased red blood cell precursors release a blood-borne signal that promotes excessive intestinal iron absorption, predisposing affected individuals to multiorgan damage (pages 1096–1101).

Production of erythropoietic cells in vitro for continuous culture of Plasmodium vivax

Plasmodium vivax cannot be maintained in a continuous culture. To overcome this major obstacle to P. vivax research, we have developed an in vitro method to produce susceptible red blood cell (RBC) precursors from freshly isolated human cord hematopoietic stem cells (HSCs), which were activated with erythropoietin to differentiate into erythroid cells. Differentiation and maturation of erythroid cells were monitored using cell surface markers (CD71, CD36, GPA and Fy6). Duffy + reticulocytes appeared after 10 days of erythroid cell culture and exponentially increased to high numbers on days 14–16. Beginning on day 10 these erythroid cells, referred to as growing RBCs (gRBCs), were co-cultured with P. vivax-infected blood directly isolated from patients. Parasite-infected gRBCs were detected by Giemsa staining and a P. vivax-specific immunofluorescence assay in 11 out of 14 P. vivax isolates. These P. vivax cultures were continuously maintained for more than 2 weeks by supplying fresh gRBCs; one was maintained for 85 days before discontinuing the culture. Our results demonstrate that gRBCs derived in vitro from HSCs can provide susceptible Duffy + reticulocytes for continuous culture of P. vivax. Of particular interest, we discovered that parasites were able to invade nucleated erythroid cells or erythroblasts that are normally in the bone marrow. The possibility that P. vivax causes erythroblast destruction and hence inflammation in the bone marrow needs to be addressed.

Ribosomal protein S17 gene (RPS17) is mutated in Diamond-Blackfan anemia

Diamond-Blackfan anemia (DBA) is a congenital erythroid aplasia characterized as a normochromic macrocytic anemia with a selective deficiency in red blood cell precursors in otherwise normocellular bone marrow. In 40% of DBA patients, various physical anomalies are also present. Currently two genes are associated with the DBA phenotype--the ribosomal protein (RP) S19 mutated in 25% of DBA patients and RPS24 mutated in approximately 1.4% of DBA patients. Here we report the identification of a mutation in yet another ribosomal protein, RPS17. The mutation affects the translation initiation start codon, changing T to G (c.2T>G), thus eliminating the natural start of RPS17 protein biosynthesis. RNA analysis revealed that the mutated allele was expressed, and the next downstream start codon located at position +158 should give rise to a short peptide of only four amino acids (Met-Ser-Arg-Ile). The mutation arose de novo, since all healthy family members carry the wild-type alleles. The identification of a mutation in the third RP of the small ribosomal subunit in DBA patients further supports the theory that impaired translation may be the main cause of DBA pathogenesis.

What's Good for the Marrow Is Bad for the Heart: Improving Anemia in a Cardiac Iron Overload Model of Hbbth1/th1 Mice.

β-thalassemia is a β-globin gene mutation that leads to ineffective erythropoiesis, increased apoptosis, shortened red blood cell (RBC) survival, and anemia. The severity of anemia is variable, ranging from β-thalassemia major patients requiring transfusion therapy to β-thalassemia intermedia patients who are transfusion independent but absorb more iron from the gastrointestinal tract. Both types develop iron overload, with cardiac complications accounting for the majority of deaths. We hypothesized that cardiac iron overload in β-thalassemia intermedia occurs as a consequence of increased iron demand in the hematopoietic organs which, when not met, unexpectedly leads to relative iron deficiency and increased iron absorption. Iron is not present in the hearts of Hbbth1/th1 mice at baseline. However, abnormal echocardiogram results were observed in our mice relative to wild type, with lower ejection fractions (P=0.004, n=6) and a proportionally larger left ventricular mass (P=0.005, n=6) at baseline. We also developed a mouse model of cardiac iron overload in Hbbth1/th1 mice using iron dextran injections. After iron dextran, we demonstrated a significant decrease in left ventricular mass (P=0.008, n=4) compared to baseline and in a pilot experiment, observed a 150-fold increase in non-heme iron in heart as measured by spectrophotometry,. Furthermore, 130 mg of iron dextran injected over four weeks increases the reticulocyte count (P=0.03, n=5) as well as the hematocrit (P=0.01, n=5). Transferrin saturation with this method of iron loading increased from 35% to 100% (P=6x10−9 relative to baseline, n=6). Extramedullary hematopoiesis in the liver increased proportionally to the amount of iron administered and could be visualized near iron-laden Kupffer cells. Due to the increased iron demand in β-thalassemia and improved reticulocyte count and hemoglobin with the administration of iron, we postulated that Hbbth1/th1 mice are relatively iron deficient and set out to analyze the physiological mechanism of improved hematopoiesis after iron. With ter-119 and CD-71 to identify RBC precursors, and Annexin-V to identify apoptotic cells, we used flow cytometry to show that iron dextran administration decreases the degree of apoptosis in RBC precursors (from 21% to 8.5%) in Hbbth1/th1 mice to levels measured in wildtype mice at baseline. Preliminary results on RBC lifespan show that there is a shortened RBC survival in Hbbth1/th1 mice, with 50% of Hbbth1/th1 RBCs destroyed at 10.1 days relative to 21.8 days in C57 controls (P<0.0001, n=5). This analysis confirms the utility of this method for measuring the effects of iron on RBC survival in Hbbth1/th1 mice. In conclusion, the administration of iron dextran leads to clear improvement in erythropoiesis in Hbbth1/th1 mice and is in part due to increased extramedullary hematopoiesis, decreased apoptosis of RBC precursors, and possibly improved RBC survival. The loss of left ventricular mass after iron dextran administration implies myocyte death and supports our use of Hbbth1/th1 mice as a model of cardiac iron overload in β-thalassemia intermedia.

Impaired binding of AHSP to α chain variants: Hb Groene Hart illustrates a mechanism leading to unstable hemoglobins with α thalassemic like syndrome

Alpha hemoglobin stabilizing protein (AHSP) is a small protein of 102 residues induced by GATA-1, Oct-1- and EKLF. It is synthesized at a high level in the red blood cell precursors and acts as a chaperone protecting the alpha hemoglobin (α-Hb) chains against precipitation. AHSP and α-Hb form a heterodimer complex. In the absence of AHSP, α-Hb oxidizes and precipitates within the erythrocyte precursors of the bone marrow leading to apoptosis and defective erythropoiesis. In vitro the binding of AHSP to ferrous α-Hb accelerates oxidation of the heme iron in α-Hb, but the complex is more resistant to protein unfolding. AHSP could act as a modulating factor in beta-thalassemia. Recent studies showed more severe thalassemic syndromes in patients with decreased levels of AHSP and in one patient who carried a structurally abnormal AHSP. Some α-Hb variants with structural abnormality located in the contact area between α-Hb and AHSP exhibit an instability and a thalassemic like syndrome. We suggest that this could result from a disturbed interaction between α-Hb variants and AHSP. To study this interaction, we constructed the pGEX-α-AHSP vector that co-expressed human α-Hb and AHSP. Using this approach, we investigated the α42 (C7), α104 (G11) and α119 (H2) sites, where variants with some thalassemic features have been described. Results obtained with recombinant Groene Hart α-Hb and Diamant α-Hb, in which proline 119 is replaced by a serine and a leucine, respectively, showed clearly an impaired interaction with AHSP. In contrast, the α mutants at the sites 42 and 104 exhibit a normal interaction with AHSP. The CO rebinding kinetics of the AHSP/α-Hb 42mutant complexes were similar to those previously obtained with the AHSP/α-Hb WT complex, which shows a modified rate that is intermediate to the classical Hb allosteric states.

Parvovirus Infection, Malaria, and Anemia in the Tropics—a New Hidden Enemy?

Anemia takes an exacting toll on the lives of individuals, particularly children living in tropical areas where malaria is endemic. In such areas, there is general agreement that the major causes of anemia are malaria, iron deficiency, hookworm infection, and the hemoglobinopathies, especially sickle-cell disease. In general, anemia is caused by factors that decrease hemoglobin production and those that lead to increased blood loss. In this respect, iron deficiency represents a major cause of decreased hemoglobin production, whereas worm infection (via fecal blood loss), hemoglobinopathies, and malaria (mainly via hemolysis) relate to blood loss. Parvovirus B19 (B19) infection in humans is unique in that it can specifically and abruptly lead to a transitory cessation of erythropoiesis [1]. Parvovirus has specificity for red blood cell precursors and can totally suppress erythropoiesis for ~37 days. The bone marrow of patients with B19 infection reveals an absence of ma-

Anemia in Chronic Heart Failure

Anemia occurs commonly in patients with chronic heart failure (CHF) and has been proposed as a novel therapeutic target in this patient population. The present review will summarize the current knowledge of the prevalence, causative factors, and pathophysiological correlates of anemia in CHF. Potential risks and benefits of therapy with erythropoietic agents for the treatment of anemia in CHF will also be discussed. Prevalence of Anemia in CHF Estimates of the prevalence of anemia in patients with CHF and low ejection fraction range widely from 4% to 61% (median 18%).1–15 Variability in estimated prevalence is partly attributable to use of inconsistent definitions of anemia in individual reports. The World Health Organization definition of anemia (hemoglobin concentration 13.0 g/dL in men and 12.0 g/dL in women) takes into account known gender differences in distribution of hemoglobin values, 16 whereas the National Kidney Foundation defines anemia as hemoglobin 12 g/dL in men and postmenopausal women. 17 These standard definitions of anemia are not based on wellestablished physiological or population norms. Published reports in CHF populations have used these and other study-specific definitions of anemia (including other arbitrary or statistically defined hemoglobin and hematocrit categories and administrative diagnostic codes from hospital records). Despite these inconsistencies in the definition of anemia cases, most studies indicate that the prevalence of anemia is increased in CHF populations with comorbid kidney disease, advanced age, and more severe symptoms (range, 30% to 61%) when compared with less symptomatic ambulatory populations (range, 4% to 23%). In patients with CHF and preserved ejection fraction, the few published reports indicate that anemia is also highly prevalent in this group.18–20 Underlying Cause of Anemia in CHF Anemia occurs when there is a deficiency in new erythrocyte production relative to the rate of removal of aged erythrocytes. Erythropoietin, a 30.4-kDa glycoprotein growth factor produced primarily by kidney, is the key component of the homeostatic system for regulation of red blood cell mass and tissue oxygen delivery.21–24 Erythropoietin prevents the programmed cell death of erythrocyte progenitor cells and thereby stimulates their proliferation, maturation, and terminal differentiation. 23 Any abnormality that reduces renal secretion of or bone marrow response to erythropoietin may result in anemia. Iron deficiency is present in 30% of anemic patients with CHF, so the majority of observed anemia is normocytic, often classified as anemia of chronic disease. Clinical characteristics commonly associated with increased risk of anemia in CHF populations are listed in the Table. Although risk factors for anemia identified in cross-sectional studies do not provide evidence of a causal link, these observations suggest that several distinct mechanisms may commonly contribute to anemia in patients with CHF. Several of the most important potential causal pathways will be discussed briefly below and are summarized in Figure 1. Chronic kidney disease is a common comorbidity in patients with CHF and is a strong independent predictor of increased risk of anemia in several studies. In chronic kidney disease populations without heart failure, moderate to severe kidney disease (defined as glomerular filtration rate [GFR] 60 mL/min) is associated with diminished erythropoietin production and a progressive decrease in hemoglobin values that is linearly related to reduction in GFR.25 The estimated prevalence of at least moderate chronic kidney disease (defined as GFR 60 mL/min) in CHF populations is 20% to 40%. 4,9,26,27 Anemia is frequently associated with decreased body mass index in published reports, a finding that suggests that patients with cachexia are at greater risk for anemia. Serum levels of proinflammatory cytokines are increased in cachectic patients with CHF and may contribute to development of anemia by several mechanisms. Proinflammatory cytokines including tumor necrosis factor- (TNF-), interleukin-1, and interleukin-6 have been shown to disrupt multiple aspects of erythropoiesis, including reduction of renal erythropoietin secretion, suppression of erythropoietin activity in red blood cell precursors in the bone marrow level, and reduction of bioavailability of iron stores for hemoglobin synthesis.28–31 Proinflammatory cytokines also increase levels of the liverderived peptide hormone, hepcidin.32,33 Hepcidin interacts with ferroportin and other iron transport proteins in the enterocyte to inhibit gut iron absorption and thereby reduces iron bioavailability for hemoglobin synthesis. 32,33 In a mouse