The hypoxia inducible factor/erythropoietin (EPO)/EPO receptor pathway is disturbed in a rat model of chronic kidney disease related anemia.

OBJECTIVES: Anemia is a known driver for hypoxia inducible factor (HIF) which leads to increased renal erythropoietin (EPO) synthesis. Bone marrow (BM) EPO receptor (EPOR) signals are transduced through a JAK2-STAT5 pathway. The origins of anemia of chronic kidney disease (CKD) are multifactorial, including impairment of both renal EPO synthesis as well as intestinal iron absorption. We investigated the HIF- EPO- EPOR axis in kidney, BM and proximal tibia in anemic juvenile CKD rats. METHODS: CKD was induced by 5/6 nephrectomy in young (20 days old) male Sprague-Dawley rats while C group was sham operated. Rats were sacrificed 4 weeks after CKD induction and 5 minutes after a single bolus of IV recombinant human EPO. An additional control anemic (C-A) group was daily bled for 7 days. RESULTS: Hemoglobin levels were similarly reduced in CKD and C-A (11.4 ± 0.3 and 10.8±0.2 Vs 13.5±0.3 g/dL in C, p<0.0001). Liver hepcidin mRNA was decreased in CA but increased in CKD. Serum iron was unchanged while transferrin levels were mildly decreased in CKD. Kidney HIF2α protein was elevated in C-A but unchanged in CKD. Kidney EPO protein and mRNA levels were unchanged between groups. However, BM EPO protein (which reflects circulating EPO) was increased in C-A but remained unchanged in CKD. BM and proximal tibia EPOR were unchanged in C-A but decreased in CKD. Proximal tibial phospho-STAT5 increased after the EPO bolus in C but not in CKD. CONCLUSIONS: Compared to blood loss, anemia in young CKD rats is associated with inappropriate responses in the HIF-EPO-EPO-R axis: kidney HIF2α and renal EPO are not increased, BM and bone EPOR levels, as well as bone pSTAT5 response to EPO are reduced. Thus, anemia of CKD may be treated with additional therapeutic avenues beyond iron and EPO supplementation.

Diagnosis and Management of Iron Deficiency in CKD: A Summary of the NICE Guideline Recommendations and Their Rationale.

The UK-based National Institute for Health and Care Excellence (NICE) has updated its guidance on iron deficiency and anemia management in chronic kidney disease. This report outlines the recommendations regarding iron deficiency and their rationale. Serum ferritin alone or transferrin saturation alone are no longer recommended as diagnostic tests to assess iron deficiency. Red blood cell markers (percentage hypochromic red blood cells, reticulocyte hemoglobin content, or reticulocyte hemoglobin equivalent) are better than ferritin level alone at predicting responsiveness to intravenous iron. When red blood cell markers are not available, a combination of transferrin saturation < 20% and ferritin level < 100ng/mL is an alternative. In comparisons of the cost-effectiveness of different iron status testing and treatment strategies, using percentage hypochromic red blood cells > 6% was the most cost-effective strategy for both hemodialysis and nonhemodialysis patients. A trial of oral iron replacement is recommended in people not receiving an erythropoiesis-stimulating agent (ESA) and not on hemodialysis therapy. For children receiving ESAs, but not treated by hemodialysis, oral iron should be considered. In adults and children receiving ESAs and/or on hemodialysis therapy, intravenous iron should be offered. When giving intravenous iron, high-dose low-frequency administration is recommended. For all children and for adults receiving in-center hemodialysis, low-dose high-frequency administration may be more appropriate.

Erythropoietin regulates POMC expression via STAT3 and potentiates leptin response.

The arcuate nucleus of the hypothalamus is essential for metabolic homeostasis and responds to leptin by producing several neuropeptides including proopiomelanocortin (POMC). We previously reported that high-dose erythropoietin (Epo) treatment in mice while increasing hematocrit reduced body weight, fat mass, and food intake and increased energy expenditure. Moreover, we showed that mice with Epo receptor (EpoR) restricted to erythroid cells (ΔEpoRE) became obese and exhibited decreased energy expenditure. Epo/EpoR signaling was found to promote hypothalamus POMC expression independently from leptin. Herein we used WT and ΔEpoRE mice and hypothalamus-derived neural culture system to study the signaling pathways activated by Epo in POMC neurons. We show that Epo stimulation activated STAT3 signaling and upregulated POMC expression in WT neural cultures. ΔEpoRE mice hypothalamus showed reduced POMC levels and lower STAT3 phosphorylation, with and without leptin treatment, compared to in vivo and ex vivo WT controls. Collectively, these data show that Epo regulates hypothalamus POMC expression via STAT3 activation, and provide a previously unrecognized link between Epo and leptin response.

Genistein attenuates hypoxic pulmonary hypertension via enhanced nitric oxide signaling and the erythropoietin system.

Upregulation of the erythropoietin (EPO)/EPO receptor (EPOR) system plays a protective role against chronic hypoxia-induced pulmonary hypertension (hypoxic PH) through enhancement of endothelial nitric oxide (NO)-mediated signaling. Genistein (Gen), a phytoestrogen, is considered to ameliorate NO-mediated signaling. We hypothesized that Gen attenuates and prevents hypoxic PH. In vivo, Sprague-Dawley rats raised in a hypobaric chamber were treated with Gen (60 mkg/kg) for 21 days. Pulmonary hemodynamics and vascular remodeling were ameliorated in Gen-treated hypoxic PH rats. Gen also restored cGMP levels and phosphorylated endothelial NO synthase (p-eNOS) at Ser(1177) and p-Akt at Ser(473) expression in the lungs. Additionally, Gen potentiated plasma EPO concentration and EPOR-positive endothelial cell counts. In experiments with hypoxic PH rats' isolated perfused lungs, Gen caused NO- and phosphatidylinositol 3-kinase (PI3K)/Akt-dependent vasodilation that reversed abnormal vasoconstriction. In vitro, a combination of EPO and Gen increased the p-eNOS and the EPOR expression in human umbilical vein endothelial cells under a hypoxic environment. Moreover, Gen potentiated the hypoxic increase in EPO production from human hepatoma cells. We conclude that Gen may be effective for the prevention of hypoxic PH through the improvement of PI3K/Akt-dependent, NO-mediated signaling in association with enhancement of the EPO/EPOR system.

The anemia of inflammation/malignancy: mechanisms and management.

Anemia is a common complication in patients with inflammatory diseases of many kinds, including cancer. The mechanisms that have captured the most attention include cytokine-mediated changes in both the production of and the response to erythropoietin (Epo), as well as important alterations in iron metabolism. The last is brought about by the relatively recently recognized peptide hormone, hepcidin. The availability of recombinant human Epo and its derivatives (known by class as Erythropoietic Stimulating Agents, ESAs) has dramatically changed anemia management in patients with cancer but, in the process, has raised as many issues as have been answered. This chapter reviews the mechanisms resulting in anemia in inflammation, including cancer, and focuses on the controversies around management with the ESAs and the adjuvant use of iron in anemia management.

Erythropoietin: new approaches to improved molecular designs and therapeutic alternatives.

Erythropoietin (Epo) is a glycoprotein hormone that is the prime regulator of erythropoiesis. Recombinant Epo is a highly effective pharmaceutical used to correct anemias associated with renal insufficiency, cancer and other diseases. Efforts to increase its efficacy in vivo by manipulating the protein's structure have met with some success, and novel Epo-like agents are in development. Additionally, efforts to create Epo mimetic agents are underway, as is the design of agents to increase endogenous production. Because Epo has tissue protective actions outside of erythropoiesis, other designs have focused on producing erythropoietically inactive molecules that still retain extra-hematopoietic activity. The demonstration that Epo can trigger signaling in some cancer cells with, potentially, adverse effects on patient health has raised warning signs in the medical community and has gained the attention of regulatory authorities.

Erythropoietin inhibits basal and stimulated corticotropin-releasing hormone release from the rat hypothalamus via a nontranscriptional mechanism.

Brain hypoxia-ischemia induces a local increase in the levels of erythropoietin (EPO) and vascular endothelial growth factor (VEGF); this condition is also associated with acute activation of the hypothalamo-pituitary-adrenal (HPA) axis, suggesting that increased levels of EPO and VEGF in the hypothalamus may play a role in the control of HPA function. Thus, in this study we used rat hypothalamic explants to investigate whether EPO and VEGF can directly modulate CRH release; the latter was assessed by RIA measurement of the peptide in the incubation medium and hypothalamic tissue. EPO and VEGF effects were studied in short-term (1-3 h) experiments under basal conditions or after stimulation with 56 mM KCl or 10 microM veratridine. We observed that EPO (1-10 nm) significantly reduced CRH release and, in parallel, increased intrahypothalamic CRH content. VEGF tended to reduce CRH release without reaching statistical significance. Moreover, EPO, but not VEGF, inhibited KCl- and veratridine-stimulated CRH release and counteracted the parallel decrease in intrahypothalamic CRH induced by the two secretagogues. EPO effects were not mediated by modification of CRH gene expression, either in the absence or the presence of KCl or veratridine; in this paradigm, KCl and veratridine per se did not modify CRH gene expression. Our findings suggest that EPO contributes to the regulation of the HPA axis activation; in pathological conditions such as brain ischemia, this growth factor may control the HPA axis function, preventing possible detrimental effects of HPA overactivation.

Insulin-like growth factor-I stimulates erythropoiesis when administered enterally.

BACKGROUND: Insulin-like growth factors I and II (IGF-I and IGF-II) are potent growth factors involved in development. IGF-I stimulates proliferation of erythropoietic progenitors and parenteral IGF-I administration stimulates in vivo erythropoiesis in animals. IGF-I and IGF-II are both present in mammalian milks and when milk-borne, are resistant to neonatal gastrointestinal degradation. Whether milk-borne IGF-I or IGF-II regulates neonatal erythropoiesis in not known. We hypothesized that physiological doses of enteral IGFs stimulate erythropoiesis in suckling rats. METHODS: Eight day-old Sprague Dawley rats were artificially fed for 4 days with rat milk substitute (RMS) or RMS supplemented with physiological levels of IGF-I or IGF-II. Rats fed IGF-I and IGF-II were compared to control RMS. Blood and marrow were collected; measures of red cell mass, measures of erythropoietic stimulus, and indices of iron status were measured. RESULTS: Rats fed IGF-I had higher hemoglobin (Hb) levels (100 +/- 10 g/l), compared to those fed RMS (94 +/- 9) or IGF-II (91 +/- 6), p < 0.001. After IGF-I supplementation, red blood cell counts (RBC) (p < 0.04) and hematocrits (p < 0.002) were also higher. Plasma erythropoietin (Epo) levels, reticulocytes, plasma iron and erythrocyte iron incorporation were similar. CONCLUSION: Intact enteral IGF-I reaches distal erythropoietic tissue resulting in greater red cell mass, but not by increasing plasma Epo levels or by altering cellular iron transport.

[Iron metabolism pre and post the erythropoietin era]. / Métabolisme du fer avant et après I'ere de l'erythropoïétine.

Uraemic patients are exposed either to iron deficiency due to impaired digestive n associated with various blood losses (particularly in dialysis patients) or iron verload related to blood transfusions in the pre-erythropoetin era or excessive intravenous iron supplementation. The central role of hepcidin in the regulation of oral iron absorption d its effects in uraemia have been recently evidenced. The increased haemoglobin synthesis induced by erythropoiesis stimulating agents (ESA) enhances iron requirements. In case of exhaustion of tissue reserves and/or insufficient exogenous supply, iron deficiency develops which is the major limiting factor for ESA efficacy. Careful biological follow-up is mandatory to detect early iron deficiency or overload, the latter being considered as possibly increasing the uraemic patients' susceptibility to bacterial or viral infections. Intravenous administration of Vitamin C, by enhancing the release of iron from the reticuloendothelial system towards transferrin increases the circulating iron available for erythropoiesis and contributes to the optimisation of ESA efficacy.

Fetal globin induction--can it cure beta thalassemia?

The beta thalassemias are one of a few medical conditions in which reactivation of a gene product that is expressed during fetal life can functionally replace a deficiency of essential proteins expressed at a later developmental stage. The fetal globin genes are present and normally integrated in hematopoietic stem cells, and at least one fetal gene appears accessible for reactivation, particularly in beta degrees thalassemia. However, rapid cellular apoptosis from alpha globin chain precipitation, and relatively low levels of endogenous erythropoietin (EPO) in some beta(+) thalassemia patients contribute to the anemia in beta thalassemia syndromes. In clinical trials, three classes of therapeutics have demonstrated proof-of-principle of this approach by raising total hemoglobin levels by 1-4 g/dL above baseline in thalassemia patients: EPO preparations, short chain fatty acid derivatives (SCFADs), and chemotherapeutic agents. Although thalassemic erythrocytes survive only for a few days, the magnitude of these responses is similar to those induced by rhu-EPO in anemic conditions of normal erythrocyte survival. New oral therapeutic candidates, which stimulate both fetal globin gene expression and erythropoiesis, and combinations of therapeutics with complementary molecular actions now make this gene-reactivation approach feasible to produce transfusion independence in many patients. Development of the candidate therapeutics is hindered largely by costs of drug development for an orphan patient population.

Haemoglobin during pregnancy: relationship to erythropoietin and haematinic status.

OBJECTIVE: To investigate the effect of haematinic status on erythropoietin throughout pregnancy in order to assess whether haematinic supplementation is appropriate for all pregnant women or should be reserved for individuals in whom deficiency has been identified. METHODS: A prospective, repeated-measures, longitudinal study of 263 women enrolled at an antenatal clinic in a district general hospital who received standard obstetric management including haematinic supplements. Haematological indices, iron status, folate and B12 status, and erythropoietin levels were measured by standard methods at scheduled intervals throughout pregnancy. The relationships between the measurements were analysed using statistical models which took into account the serial correlation between repeated measurements on the same individuals. RESULTS: Erythropoietin levels rose throughout pregnancy from a mean of 22.8 mU/mL at booking to 43.7 mU/mL at 38 wk. There was a significant relationship between erythropoietin levels and haemoglobin, ferritin, TIBC and folate. The relationship was strongest between erythropoietin and haemoglobin. Erythropoietin levels were influenced by parity but not maternal age. CONCLUSION: Haematinic levels, particularly iron are related to the rise in serum EPO which occurs during pregnancy. Therefore, haematinic deficiency in pregnancy will result in increased stimulation of erythroid progenitor cells by erythropoietin. While haematinic supplementation is not always prescribed throughout pregnancy, haematinic status must be assessed on an individual basis and supplementation provided as required.

Bloodless medicine and surgery in the OR and beyond.

Ideas about bloodless surgery are changing. It now is more than transfusion-free surgery for Jehovah's Witnesses. Technological advances and concerns about the safety and availability of blood have led to interest in bloodless surgery among non-Witnesses. Perioperative nurses need to be aware of bloodless surgery technology, which typically incorporates the use of recombinant human erythropoietin and special surgical techniques and equipment. They must have a proper attitude toward blood conservation and respect patient autonomy. Bloodless surgery care extends across specialties and inpatient areas. A knowledge of bloodless surgery will facilitate clear communication between staff members, patients, and patients' family members.

The role of iron in erythropoiesis in the absence and presence of erythropoietin therapy.

Preoperative autologous blood donation has served as a model for blood loss anaemia. Studies in these patients, along with clinical trials of i.v. iron and recombinant human erythropoietin (rHuEPO) therapy, have furthered our understanding of the relationship between erythropoietin, iron, and erythropoiesis. With supplemental oral iron, the endogenous erythropoietic response to routine autologous blood donation and to the anaemia of chronic illness has been shown to be modest, but predictable. In more aggressive donation and more severe anaemia, the endogenous erythropoietic response is more substantial, but still predictable. Studies in patients undergoing aggressive phlebotomy whilst receiving rHuEPO demonstrate a wide variation in response to rHuEPO dose. This variability is not related to age or gender and suggests factors such as iron-restricted erythropoiesis may be responsible. Supporting evidence arises from the superior erythropoietic response observed in patients with haemochromatosis. These patients maintain very high serum iron and transferrin saturation levels. In response to serial phlebotomy these patients can mount an endogenous erythropoietin response up to five-times greater than healthy individuals. When treated with rHuEPO, patients with haemochromatosis respond with much greater RBC expansion volumes than patients receiving rHuEPO and iron supplementation. Studies show no difference in the degree of endogenously stimulated erythropoiesis between patients with measurable iron stores and those without. However, when treated with rHuEPO, increased erythropoiesis has been observed in patients with measurable iron stores compared with those without. This suggests that, while oral iron supplementation may be sufficient to keep pace with endogenously stimulated erythropoiesis, it may not be adequate to prevent iron-restricted erythropoiesis during rHuEPO therapy. Some studies have suggested that i.v iron may prevent iron-restricted erythropoiesis during rHuEPO therapy although further research is needed. The availability of better tolerated i.v. iron preparations provides an ideal opportunity to study the value of iron therapy in patients with acute blood loss, particularly those undergoing rHuEPO therapy.

Anemia in the critically ill: the role of erythropoietin.

Anemia is a common clinical problem in critically ill patients and is associated with substantial red blood cell (RBC) transfusion requirements. However, RBC transfusion has significant risks, including adverse effects on the immune system. Although a low hemoglobin concentration may be tolerable, it may not be optimal for the critically ill patient. Thus, alternative therapies that can increase hemoglobin and avoid complications of RBC transfusion are desirable. Critically ill patients appear to have anemia identical to the anemia of chronic inflammatory disease with blunted erythropoietin production. Results of a recent randomized controlled trial in critically ill patients demonstrated that recombinant human erythropoietin (r-HuEPO, epoetin alfa) significantly reduced (by approximately 50%) the number of RBC units transfused (P <.002) and significantly increased hematocrit (P <.01) compared with placebo. There was no increase in mortality or adverse clinical events with therapy. Epoetin alfa may be an effective therapeutic approach to anemia in critically ill patients, decreasing the need for transfusion and achieving higher hemoglobin concentrations than generally attained with transfusion.

Erythropoietin in obstetrics.

Our objective was to discuss the role of erythropoietin in fetal erythropoiesis and to review its clinical uses in perinatal medicine. All relevant articles compiled through a MEDLINE search (years 1986-1997) were reviewed. Erythropoietin is essential for fetal erythropoiesis and is produced in response to hypoxia and anemia. Cord blood erythropoietin is purely fetal and reflects tissue oxygenation. It has been found to be increased in many complicated pregnancies with underlying fetal hypoxia. Erythropoietin could be used as a marker of fetal hypoxia because its concentration rises rapidly by increased production in response to hypoxia. Its measurement might enable more accurate timing of hypoxic injury. In addition, erythropoietin levels have been well correlated with perinatal brain damage and may facilitate treatment of high risk neonates. Erythropoietin has also been used successfully in anemia of prematurity, decreasing the transfusion requirement. However, studies are still needed to determine the optimal doses of erythropoietin and iron supplementations required for maximizing the red blood cell response. Erythropoietin has been examined as potential maternal therapy in various disorders during pregnancy. These include end-stage renal disease, severe antepartum iron deficiency anemia, and postpartum anemia. Erythropoietin has been found to be effective and well tolerated in these conditions. An additional promising use lies in the optimization of maternal red blood cell mass to allow autologous blood donation. This may be critical in cases where a large amount of bleeding might be anticipated, as with placenta previa. This would also minimize the donor transfusion-related hazards. Erythropoietin with its wide clinical applications could improve maternal and neonatal outcome.

The use of iron in patients on chronic dialysis: mistake and misconceptions.

Anemia is the most common hematologic abnormality in patients with chronic renal failure. The reasons for anemia in chronic renal failure are many and include erythropoietin and iron deficiencies, inflammation, infection, aluminum toxicity, and hyperparathyroidism. Iron deficiency alone affects more than 50% of patients on dialysis, and the estimated iron loss for these patients is 1.5 to 3 grams per year. The use of erythropoietin has also uncovered iron deficiency in a multitude of patients. Iron and erythropoietin supplementation has often restored normal or near-normal levels of hematocrit in these patients and has therefore improved some of the symptoms classically connected with chronic renal failure, such as fatigue, cold intolerance, and mental sluggishness, among others. Resistance to erythropoietin is frequently observed in the maintenance care for dialysis patients, and the most common reason is iron deficiency. It is important to understand the physiology of renal anemia, erythropoiesis and iron metabolism in order to avoid mistakes and misconceptions in the management of iron in chronic dialysis patients. In this article, we review several mistakes, misconceptions, practices, and guidelines in iron supplementation therapy. We also review the physiology of anemia in renal disease and the importance of erythropoietin and iron in causing anemia and discuss recent Dialysis Outcomes Quality Initiative (DOQI) guidelines on the topic.

Purification and molecular cloning of SH2- and SH3-containing inositol polyphosphate-5-phosphatase, which is involved in the signaling pathway of granulocyte-macrophage colony-stimulating factor, erythropoietin, and Bcr-Abl.

Grb2/Ash and Shc are the adapter proteins that link tyrosine-kinase receptors to Ras and make tyrosine-kinase functionally associated with receptors and Ras in fibroblasts and hematopoietic cells. Grb2/Ash and Shc have the SH3, SH2, or phosphotyrosine binding domains. These domains bind to proteins containing proline-rich regions or tyrosine-phosphorylated proteins and contribute to the association of Grb2/Ash and Shc with other signaling molecules. However, there could remain unidentified signaling molecules that physically and functionally interact with these adapter proteins and have biologically important roles in the signaling pathways. By using the GST fusion protein including the full length of Grb2/Ash, we have found that c-Cbl and an unidentified 135-kD protein (pp135) are associated with Grb2/Ash. We have also found that they become tyrosine-phosphorylated by treatment of a human leukemia cell line, UT-7, with granulocyte-macrophage colony-stimulating factor (GM-CSF). We have purified the pp135 by using GST-Grb2/Ash affinity column and have isolated the full-length complementary DNA (cDNA) encoding the pp135 using a cDNA probe, which was obtained by the degenerate polymerase chain reaction based on a peptide sequence of the purified pp135. The cloned cDNA has 3,958 nucleotides that contain a single long open reading frame of 3,567 nucleotides, encoding a 1,189 amino acid protein with a predicted molecular weight of approximately 133 kD. The deduced amino acid sequence reveals that pp135 is a protein that has one SH2, one SH3, and one proline-rich domain. The pp135, which contains two motifs conserved among the inositol polyphosphate-5-phosphatase proteins, was shown to have the inositol polyphosphate-5-phosphatase activity. The pp135 was revealed to associate constitutively with Grb2/Ash and inducibly with Shc using UT-7 cells stimulated with GM-CSF. In the cell lines derived from human chronic myelogenous leukemia, pp135 was constitutively tyrosine-phosphorylated and associated with Shc and Bcr-Abl. These facts suggest that pp135 is a signaling molecule that has a unique enzymatic activity and should play an important role in the signaling pathway triggered by GM-CSF and in the transformation of hematopoietic cells caused by Bcr-Abl.

Response of the erythron and erythropoietin to autologous blood donations in paediatric subjects. Is erythropoietin supplement necessary?

OBJECTIVES: This study was undertaken to evaluate the need for erythropoietin (Epo) therapy to augment autologous blood collection in adolescents undergoing spinal corrective surgery. METHODS: We measured serum Epo and parameters of iron metabolism in 35 adolescents undergoing autologous blood collection for orthopaedic surgery. Ages of subjects ranged from 11 to 16 years (mean 15.5 years) with a female predominance. Generally, 10% of intravascular blood volume was collected once a week up to a total of three collections. RESULTS: There was an average 2.5-fold rise in serum Epo over the period of blood collection. Epo increased immediately after blood collection. There was a 1.4-fold rise in reticulocyte count, consistent with the Epo response, and an average of 1.5 units of red blood cells (200 ml/unit) being produced over this period. Despite this there was an average fall of 2 g/dl (15%) in haemoglobin level. Serum ferritin and transferrin saturation also fell. CONCLUSIONS: Paediatric subjects are able to donate the required units of blood as they have a good Epo response to mild anaemia. The amount of blood donated did not exceed their total mobilisable iron and the iron supplement was adequate for red cell synthesis.