EPO (Erythropoietin)

Erythropoietin (EPO) introduction

Erythropoietin (abbreviation EPO) is endogenous glycoprotein cytokine, with protein chemical formula C₈₁₅H₁₃₁₇N₂₃₃O₂₄₁S₅ and protein avarage weight 18396.1 Da. Erythropoietin is secreted mainly by the kidney in response to cellular hypoxia; it stimulates red blood cell production (erythropoiesis) in the red bone marrow. 

Red blood cells, Erythropoiesis and red bone marrow

Erythropoiesis is the process which produces red blood cells (RBCs; erythrocytes), which is the development from erythropoietic stem cell to mature red blood cell. It is stimulated by decreased O2 in circulation, which is detected by the kidneys, which then secrete the hormone erythropoietin (EPO). Erythropoietin stimulates proliferation and differentiation of red cell precursors, which activates increased erythropoiesis in the hemopoietic tissues, ultimately producing red blood cells (erythrocytes). 

In humans, erythropoiesis usually occurs within the red bone marrow. Increased level of physical activity can cause an increase in erythropoiesis. However, in humans with certain diseases and in some animals, erythropoiesis also occurs outside the bone marrow, within the spleen or liver. This is termed extramedullary erythropoiesis. 

The bone marrow of essentially all the bones produces red blood cells until a person is around five years old. The tibia and femur cease to be important sites of hematopoiesis by about age 25; the vertebrae, sternum, pelvis and ribs, and cranial bones continue to produce red blood cells throughout life. Up to the age of 20 years RBCs are produced from red bone marrow of all the bones (long bones and all the flat bones). After the age of 20 years, RBCs are produced from membranous bones such as vertebrae, the sternum, ribs, scapulas, and the iliac bones. After 20 years of age, the shaft of the long bones becomes yellow bone marrow because of fat deposition and loses the erythropoietic function.

Erythropoietin (EPO) History

In 1905, Paul Carnot proposed the idea that a hormone regulates the production of red blood cells. After conducting experiments on rabbits subject to bloodletting, Carnot and his graduate student Clotilde-Camille Deflandre attributed an increase in red blood cells in rabbit subjects to a hemotropic factor called hemopoietin. Eva Bonsdorff and Eeva Jalavisto called the hemopoietic substance ‘erythropoietin’. K.R. Reissman and Allan J. Erslev demonstrated that a certain substance, circulated in the blood, is able to stimulate red blood cell production and increase hematocrit. This substance was purified and confirmed as erythropoietin (EPO). In 1977, Goldwasser and Kung purified EPO. Pure EPO allowed the amino acid sequence to be partially identified and the gene to be isolated. Synthetic EPO was first successfully used to correct anemia in 1987. In 1985, Lin et al isolated the human erythropoietin gene from a genomic phage library and used it to produce EPO. In 1989, the US Food and Drug Administration approved the hormone Epogen for use in certain anemias. Gregg L. Semenza and Peter J. Ratcliffe studied the EPO gene and its oxygen-dependent regulation. Along with William Kaelin Jr., they were awarded the 2019 Nobel Prize in Physiology or Medicine for their discovery of hypoxia-inducible factor (HIF), which regulates the EPO gene, as well as other genes, in response to hypoxia. Recombinant human erythropoietin (rhEPO) is arguably the most successful therapeutic application of recombinant DNA technology till date. 

EPO Functions

1) Red blood cell production

Erythropoietin is an essential hormone for red blood cell production. Without it, definitive erythropoiesis does not take place. Under hypoxic conditions, the kidney will produce and secrete erythropoietin to increase the production of red blood cells by targeting CFU-E, proerythroblast and basophilic erythroblast subsets in the differentiation. Erythropoietin has its primary effect on red blood cell progenitors and precursors (which are found in the bone marrow in humans) by promoting their survival through protecting these cells from apoptosis, or cell death.

Erythropoietin is the primary erythropoietic factor that cooperates with various other growth factors (e.g., IL-3, IL-6, glucocorticoids, and SCF) involved in the development of erythroid lineage from multipotent progenitors. The burst-forming unit-erythroid (BFU-E) cells start erythropoietin receptor expression and are sensitive to erythropoietin. Subsequent stage, the colony-forming unit-erythroid (CFU-E), expresses maximal erythropoietin receptor density and is completely dependent on erythropoietin for further differentiation. Precursors of red cells, the proerythroblasts and basophilic erythroblasts also express erythropoietin receptor and are therefore affected by it.

2) Other possbile Non-hematopoietic roles

Erythropoietin was reported to have a range of actions beyond stimulation of erythropoiesis including vasoconstriction-dependent hypertension, stimulating angiogenesis, and promoting cell survival via activation of EPO receptors resulting in anti-apoptotic effects on ischemic tissues. However this proposal is controversial with numerous studies showing no effect. It is also inconsistent with the low levels of EPO receptors on those cells. Clinical trials in humans with ischemic heart, neural and renal tissues have not demonstrated the same benefits seen in animals. In addition some research studies have shown its neuroprotective effect on diabetic neuropathy, however these data were not confirmed in clinical trials that have been conducted on the deep peroneal, superficial peroneal, tibial and sural nerves.

EPO Synthesis and regulation

Endogenous erythropoietin is produced by interstitial fibroblasts in the kidney in close association with the peritubular capillary and proximal convoluted tubule. It is also produced in perisinusoidal cells in the liver. Liver production predominates in the fetal and perinatal period; renal production predominates in adulthood. It is homologous with thrombopoietin. Low levels of EPO (around 10 mU/mL) are constantly secreted sufficient to compensate for normal red blood cell turnover. However, in hypoxic stress, EPO production may increase up to 1000-fold, reaching 10 000 mU/mL of blood.

In adults, EPO is synthesized mainly by interstitial cells in the peritubular capillary bed of the renal cortex, with additional amounts being produced in the liver, and the pericytes in the brain. Regulation is believed to rely on a feedback mechanism measuring blood oxygenation and iron availability. Constitutively synthesized transcription factors for EPO, known as hypoxia-inducible factors, are hydroxylated and proteosomally digested in the presence of oxygen and iron. During normoxia GATA2 inhibits the promoter region for EPO. GATA2 levels decrease during hypoxia and allow the promotion of EPO production.

Mechanism of action

Erythropoietin or exogenous epoetin alfa binds to the erythropoietin receptor (EPO-R) and activates intracellular signal transduction pathways: EPO binds to the erythropoietin receptor on the red cell progenitor surface and activates a JAK2 signalling cascade. This initiates the STAT5, PIK3 and Ras MAPK pathways. This results in differentiation, survival and proliferation of the erythroid cell. SOCS1, SOCS3 and CIS are also expressed which act as negative regulators of the cytokine signal. The affinity (Kd) of EPO for its receptor on human cells is ∼100 to 200 pM.

High level erythropoietin receptor expression is localized to erythroid progenitor cells. While there are reports that EPO receptors are found in a number of other tissues, such as heart, muscle, kidney and peripheral/central nervous tissue, those results are confounded by nonspecificity of reagents such as anti-EpoR antibodies. In controlled experiments, a functional EPO receptor is not detected in those tissues. In the bloodstream, red cells themselves do not express erythropoietin receptor, so cannot respond to EPO. However, indirect dependence of red cell longevity in the blood on plasma erythropoietin levels has been reported, a process termed neocytolysis. In addition, there is conclusive evidence that EPO receptor expression is upregulated in brain injury.

Recombinant human EPO and medical use

Exogenous erythropoietins – recombinant human erythropoietins (rhEPO) available for use as therapeutic agents, are produced by recombinant DNA technology in cell culture and are collectively called erythropoiesis-stimulating agents (ESA).

Available types of Erythropoiesis-stimulating agents:

• • Erythropoietin (EPO)
  • Epoetin alfa (Procrit, Epogen)
  • Epoetin beta (NeoRecormon)
  • Epoetin zeta (Silapo, Retacrit)
  • Darbepoetin alfa (Aranesp)
  • Methoxy polyethylene glycol-epoetin beta (Mircera)

Erythropoiesis-stimulating agents (ESAs) are used in the treatment of anemia in chronic kidney disease, chemotherapy induced anemia in patients with cancer, inflammatory bowel disease (Crohn’s disease and ulcerative colitis) and myelodysplasia from the treatment of cancer (chemotherapy and radiation). Anemia is a condition in which you lack enough healthy red blood cells to carry adequate oxygen to your body’s tissues.

Pharmacodynamics

EPO is highly glycosylated (40% of total molecular weight), with half-life in blood around 5 h. EPO’s half-life may vary between endogenous and various recombinant versions. Additional glycosylation or other alterations of EPO via recombinant technology have led to the increase of EPO’s stability in blood (thus requiring less frequent injections).

Erythropoietin and epoetin alfa are involved in the regulation of erythrocyte differentiation and the maintenance of a physiological level of circulating erythrocyte mass. Epoetin alfa serves to restore erythropoietin deficiency in pathological and other clinical conditions where normal production of erythropoietin is impaired or compromised. In anemic patients with chronic renal failure (CRF), administration with epoetin alfa stimulated erythropoiesis by increasing the reticulocyte count within 10 days, followed by increases in the red cell count, hemoglobin, and hematocrit, usually within 2 to 6 weeks. Depending on the dose administered, the rate of hemoglobin increase may vary. In patients receiving hemodialysis, a greater biologic response is not observed at doses exceeding 300 Units/kg 3 times weekly.

Risks of therapy with EPO and ESA

Possible risks of therapy with EPO include also death, myocardial infarction, stroke, venous thromboembolism, and tumor recurrence. Risk increases when EPO treatment raises hemoglobin levels over 11 g/dL to 12 g/dL: this is to be avoided.

Research and EPO

Chronic kidney disease (CKD): Patients with CKD on dialysis have subnormal endogenous EPO production. Studies have shown that rhEPO treatment corrects anemia and improves quality of life (QOL) in patients with CKD. It also optimizes the patient’s hemodynamic status and minimizes the risk of left ventricular hypertrophy, along with improvement in physical performance and cognitive function.

Heart failure: Recombinant EPO therapy has been found to be useful in patients with heart failure, especially with the cardio-renal anemia syndrome. Some recent studies show reduction in cardiac remodeling, Brain Natriuretic eptide levels, and hospitalization rate, resulting in improvement in left and right ventricular systolic function.

Stroke: There is a lot of interest in the role of EPO as a neuroprotective agent in ischemic stroke based on preclinical studies and one pilot study; however, a recent study failed to show any benefit and raised some doubts regarding the safety of EPO in such patients.

Acute kidney injury: The role of EPO in acute kidney injury (AKI) is undergoing active research and animal studies have revealed a physiological basis for the use of erythropoietin in AKI; however, a recent study failed to show any benefit.

With the discovery of EPO-R in non-erythroid tissue, pleiotropic effects of EPO were understood. Some areas of research with EPO as a novel therapeutic agent:

Spinal cord injury (SCI): Recently, research has focused on rhEPO and its effects on SCI treatment as well as the mechanisms such as anti-apoptotic, anti-inflammatory, and edema reduction, leading to neuronal and oligodendrocytes’ survival and restoration of vascular integrity.

EPO in depression: A current study is underway to evaluate the potential for EPO to alleviate depression and neurocognitive deficits in affective disorders among treatment-resistant cases.

EPO in diabetes: EPO has been found to affect all phases of wound healing and shows encouraging results for chronic wound healing in experimental animal and human studies, especially in the management of patients with chronic diabetic wounds.

EPO as an immunomodulating agent: A recent article shows that macrophages act as direct targets of EPO which enhances the pro-inflammatory activity and function of these cells.

Doping – rhEPO as a performance-enhancing drug

Because of its capacity to improve oxygenation, EPO has been abused by athletes participating in endurance sports. Administration of rhEPO increases the body’s maximum oxygen consumption capacity, thus increasing endurance and physical fitness.

As a performance-enhancing drug, EPO has been banned since the early 1990s, but a first test was not available until the 2000 Summer Olympics. EPO can often be detected in blood, due to slight differences from the endogenous protein; for example, in features of posttranslational modification. Before this test was available, some athletes were sanctioned after confessing to having used EPO, for example in the Festina affair, when a car with doping products for the Festina cycling team was found.

The first doping test in cycling was used in the 2001 La Flèche Wallonne. The first rider to test positive in that race was Bo Hamburger, although he was later acquitted because his B-sample was not conclusive. The U.S. Postal Service Pro Cycling Team, under the leadership of Lance Armstrong and Johan Bruyneel, ran a sophisticated doping program that lasted for many years during the late 1990s and early 2000s. Erythropoietin was a common substance used by the cyclists.

A 2007 study showed that EPO has a significant effect on exercise performance, but a 2017 study showed that the effects of EPO administered to amateur cyclists was not distinguishable from a placebo. In March 2019, American mixed martial artist and former UFC Bantamweight Champion T.J. Dillashaw tested positive for EPO in a drug test administered by USADA, and he subsequently was stripped of the UFC bantamweight title and suspended for 2 years.

The most important recombinant EPOs and analogues misused in sports are:

• • rhEPO (recombinant human erythropoietins)
  • Darbepoetin alpha
  • CERA (continuous erythropoietin receptor activator; CERAs have an extended half-life and a mechanism of action that promotes increased stimulation of erythropoietin receptors compared with other ESAs)

The detection of EPO abuse has been challenging for the following reasons:

• • Timing of sampling and availability of specialized dedicated laboratories with immense infrastructure requirements are the major limiting factors in detecting EPO misuse. The other factors playing a role in the detection are follows:
  • It is difficult to discriminate between the endogenous EPO and recombinant exogenous hormone.
  • EPO has a relatively short half-life in serum (the half-life of rhEPO-a is 8.5 ± 2.4 hours when administered IV and 19.4 ± 10.7 hours when administered SC).
  • EPO is undetectable in urine after 3–4 days of injection.
  • Screening in large numbers may be difficult as it requires highly trained technicians and standardization between laboratories.

EPO can often be detected in blood, due to slight differences from the endogenous protein; for example, in features of posttranslational modification.

Scientifically investigated possible benefits of EPO and ESA

• • EPO use in the treatment of anemia in chronic kidney disease, chemotherapy induced anemia in patients with cancer, inflammatory bowel disease (Crohn’s disease and ulcerative colitis), anemia in HIV-infected patients, hemolysis and resultant anemia during the treatment of hepatitis C with Ribavirin and Interferon, and myelodysplasia from the treatment of cancer (chemotherapy and radiation)
  • EPO can useful in patients with heart failure, especially with the cardio-renal anemia syndrome
  • EPO can play role as a neuroprotective agent in ischemic stroke
  • Effects on Spinal cord injury (SCI) treatment as well as the mechanisms such as anti-apoptotic, anti-inflammatory, and edema reduction, leading to neuronal and oligodendrocytes’ survival and restoration of vascular integrity
  • Potential to alleviate depression and neurocognitive deficits in affective disorders among treatment-resistant cases
  • EPO affects all phases of wound healing and shows encouraging results for chronic wound healing in experimental animal and human studies, especially in the management of patients with chronic diabetic wounds
  • EPO can effect as an immunomodulating agent – a recent article shows that macrophages act as direct targets of EPO which enhances the pro-inflammatory activity and function of these cells

EPO and ESA possible side-effects

• • Allergic and anaphylactic reactions
  • A meta-analysis involving nearly 10,000 cancer patients indicates that treatment with rhEPO increases the risk of thrombosis
  • Hypertension
  • Possibility of cancer progression, rhEPO can enhance tumor progression (in patients who have cancer, ESAs may cause the tumor to grow)
  • Pure red cell aplasia (mainly reported in patients with CKD): Autoantibodies in the serum can neutralize both rhEPO and endogenous EPO. This was mainly observed in CKD patients, especially after SC injection
  • Dizziness, nausea, fever
  • Pain at the site of the injection
  • ESAs increase the risk of venous thromboembolism (blood clots in the veins). A blood clot can break away from one location and travel to the lung (pulmonary embolism), where it can block circulation. Symptoms of blood clots include chest pain, shortness of breath, pain in the legs, and sudden numbness or weakness in the face, arm, or leg.
  • ESAs can cause hemoglobin to rise too high, which puts the patient at higher risk for heart attack, stroke, heart failure, and death.

Possible risks of therapy with EPO include death, myocardial infarction, stroke, venous thromboembolism, and tumor recurrence. Risk increases when EPO treatment raises hemoglobin levels over 11 g/dL to 12 g/dL: this is to be avoided.

EPO FAQ

Is erythropoietin (EPO) a hormone?

Erythropoietin (EPO) is a hormone produced by the kidney that promotes the formation of red blood cells by the bone marrow. The kidney cells that make erythropoietin are sensitive to low oxygen levels in the blood that travels through the kidney.

Is EPO dangerous?

It is well known that EPO, by thickening the blood, leads to an increased risk of several deadly diseases, such as heart disease, stroke, and cerebral or pulmonary embolism. The misuse of recombinant human EPO may also lead to autoimmune diseases with serious health consequences.

What is normal erythropoietin level?

The normal range for EPO levels can vary from 3.7 to 36 international units per liter (IU/L). Higher-than-normal levels may mean you have anemia. In severe cases of anemia, EPO levels in the blood may be a thousand times higher than normal. Unusually low levels may be because of polycythemia vera.

What are common side effects of EPO (erythropoietin)?

Common side effects may include increased blood pressure; joint pain, bone pain, muscle pain; itching or rash; fever, chills, cough; mouth pain, trouble swallowing; nausea, vomiting; headache, dizziness or trouble sleeping.

Does erythropoietin increase blood pressure?

Chronic administration of erythropoietin (EPO) is associated with an increase in arterial blood pressure in patients and animals with chronic renal failure (CRF). Several mechanisms have been considered in the pathogenesis of EPO-induced hypertension.

Is EPO safe to use?

While proper use of EPO has an enormous therapeutic benefit in the treatment of anaemia related to kidney disease, its misuse can lead to serious health risks for athletes who use this substance simply to gain a competitive edge.

What kind of drug is EPO?

Erythropoietin (EPO) is a hormone naturally produced by the kidneys. However, this hormone can be artificially produced to improve the performance of, for example, athletes or cyclists by injection.

Is erythropoietin a protein?

Chemically, erythropoietin is a protein with an attached sugar (a glycoprotein). It is one of a number of similar glycoproteins that serve as stimulants for the growth of specific types of blood cells in the bone marrow.

What is a normal red blood cell count range?

The normal RBC range for men is 4.7 to 6.1 million cells per microliter (mcL). The normal RBC range for women who aren’t pregnant is 4.2 to 5.4 million mcL. The normal RBC range for children is 4.0 to 5.5 million mcL.

How is erythropoietin produced?

Erythropoietin is produced by interstitial fibroblasts in the kidney in close association with the peritubular capillary and proximal convoluted tubule. It is also produced in perisinusoidal cells in the liver. Liver production predominates in the fetal and perinatal period; renal production predominates in adulthood.

What are the benefits of erythropoietin?

Erythropoietin stimulates the bone marrow to produce more red blood cells. The resulting rise in red cells increases the oxygen-carrying capacity of the blood. As the prime regulator of red cell production, erythropoietin’s major function is promote the development of red blood cells.

Does EPO show up in urine?

EPO, or erythropoietin, is a natural substance produced within the kidneys that stimulates the creation of new red blood cells. Blood-boosting drugs like EPO, if injected, are only detectable in the urine or blood for a short window of time.

How does EPO work?

Erythropoietin (EPO) is a peptide hormone that is produced naturally by the human body. EPO is released from the kidneys and acts on the bone marrow to stimulate red blood cell production. An increase in red blood cells improves the amount of oxygen that the blood can carry to the body’s muscles

What is EPO used to treat?

Erythropoietin can be used to correct anemia by stimulating red blood cell production in the bone marrow in these conditions. The medication is known as epoetin alfa (Epogen, Procrit) or as darbepoietin alfa (Arnesp).

Why is EPO banned in sport?

The drug erythropoietin, often called EPO, is banned from sports because it is believed to enhance an athlete’s performance and give people who use it an unfair advantage over unenhanced competitors.

Why is EPO dangerous?

It is well known that EPO, by thickening the blood, leads to an increased risk of several deadly diseases, such as heart disease, stroke, and cerebral or pulmonary embolism. The misuse of recombinant human EPO may also lead to autoimmune diseases with serious health consequences.

Can you live without red blood cells?

No, because red blood cells carry oxygen throughout your body. When you don’t have enough red blood cells, your organs don’t get enough oxygen and can’t work properly.

EPO and ESA must be used carefully

Various types of rhEPO are commercially available today with different dosage schedules and modes of delivery. Their efficacy in stimulating erythropoiesis is dose dependent and differs according to the patient’s disease and nutritional status. EPO must be used carefully according to guidelines as unsolicited use can result in serious adverse effects. The health care provider must keep an eye on the patient’s blood cell counts to make sure they do not put him or her at a higher risk. The dosing may change, depending on the patient’s needs.

Dosage and use examples of EPO and ESA

Clinical Applications of Recombinant Human Erythropoietin at anemia associated with Chronic kidney disease on dialysis: The approved dosage in anemia of chronic kidney disease (CKD) for adult patients is 50 to 100 Units/kg IV (intravenous ) or SC (subcutaneous) 3 times weekly. Weekly monitoring of hemoglobin is suggested upon initiation of therapy and then to maintain hemoglobin levels <12 g/dl and to avoid increases of hemoglobin >1 g/dl over a 2-week period.

Patients on Cancer Chemotherapy: Initiate Epogen in patients on cancer chemotherapy only if the hemoglobin is less than 10 g/dL, and if there is a minimum of two additional months of planned chemotherapy. Use the lowest dose of Epogen necessary to avoid RBC transfusions. Recommended starting dose in adults is 150 Units/kg subcutaneously 3 times per week until completion of a chemotherapy course or 40,000 Units subcutaneously weekly until completion of a chemotherapy course. Reduce dose by 25% if Hemoglobin increases greater than 1 g/dL in any 2-week period or Hemoglobin reaches a level needed to avoid RBC transfusion. Withhold dose if hemoglobin exceeds a level needed to avoid RBC transfusion. Reinitiate at a dose 25% below the previous dose when hemoglobin approaches a level where RBC transfusions may be required. After the initial 4 weeks of Epogen therapy, if hemoglobin increases by less than 1 g/dL and remains below 10 g/dL, increase dose to 300 Units/kg three times per week in adults or 60,000 Units weekly in adults. After 8 weeks of therapy, if there is no response as measured by hemoglobin levels or if RBC transfusions are still required, discontinue Epogen.

In Surgery Patients: The recommended Epogen regimens are 300 Units/kg per day subcutaneously for 15 days total: administered daily for 10 days before surgery, on the day of surgery, and for 4 days after surgery; or 600 Units/kg subcutaneously in 4 doses administered 21, 14, and 7 days before surgery and on the day of surgery. Deep venous thrombosis prophylaxis is recommended during Epogen therapy.