Anemia of Chronic Diseases: Wider Diagnostics—Better Treatment?

1 Department of Pharmacology and Therapeutics, Faculty of Medicine, Collegium Medicum in Bydgoszcz, Nicolaus Copernicus University, M. Curie 9, 85-090 Bydgoszcz, Poland; lp.pw@4iksniciw (M.W.); moc.liamg@zrogezrgrenzcil (G.L.); moc.liamg@iksledac.lorak (K.C.); lp.pw@rtoipkedat (T.K.)

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Grzegorz Liczner

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Karol Cadelski

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Tadeusz Kołnierzak

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Magdalena Nowaczewska

2 Department of Otolaryngology, Head and Neck Surgery, and Laryngological Oncology, Collegium Medicum in Bydgoszcz, Nicolaus Copernicus University, M. Curie 9, 85-090 Bydgoszcz, Poland; lp.kmu.mc@akswezcawon.m

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Bartosz Malinowski

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Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

Abstract

Anemia of chronic diseases is a condition that accompanies a specific underlying disease, in which there is a decrease in hemoglobin, hematocrit and erythrocyte counts due to a complex process, usually initiated by cellular immunity mechanisms and pro-inflammatory cytokines and hepcidin. This is the second most common type of anemia after iron deficiency anemia in the world. Its severity generally correlates with the severity of the underlying disease. This disease most often coexists with chronic inflammation, autoimmune diseases, cancer, and kidney failure. Before starting treatment, one should undertake in-depth diagnostics, which includes not only assessment of complete blood count and biochemical parameters, but also severity of the underlying disease. The differential diagnosis of anemia of chronic diseases is primarily based on the exclusion of other types of anemia, in particular iron deficiency. The main features of anemia of chronic diseases include mild to moderate lowering of hemoglobin level, decreased percentage of reticulocyte count, low iron and transferrin concentration, but increased ferritin. Due to the increasingly better knowledge of the pathomechanism of chronic diseases and cancer biology, the diagnosis of this anemia is constantly expanding with new biochemical indicators. These include: the concentration of other hematopoietic factors (folic acid, vitamin B₁₂), hepcidin, creatinine and erythropoietin. The basic form of treatment of anemia of chronic diseases remains supplementation with iron, folic acid and vitamin B₁₂ as well as a diet rich in the above-mentioned hematopoietic factors. The route of administration (oral, intramuscular or intravenous) requires careful consideration of the benefits and possible side effects, and assessment of the patient’s clinical status. New methods of treating both the underlying disease and anemia are raising hopes. The novel methods are associated not only with supplementing deficiencies, but also with the administration of drugs molecularly targeted to specific proteins or receptors involved in the development of anemia of chronic diseases.

Keywords: iron homeostasis, anemia, iron supplementation, oxidative stress, nutrition, hematological parameters, biochemical parameters, erythropoiesis

1. Introduction

Erythropoiesis is a multi-stage process of multiplication and erythrocyte differentiation from hematopoietic stem cells, which normally takes place in the bone marrow of flat bones and the epiphyses of human long bones. A unique feature of stem cells is their ability to self-renewal and differentiation. From the hematopoietic stem cell, a myelopoietic stem cell is formed, which subsequently undergoes a transformation to the erythropoietic progenitor cell. It matures through successive divisions and becomes a precursor cell, demonstrating at this stage some characteristics of the final cell. Further maturation occurs through changing the nature of the cell nucleus from basophilic to acidophilic, up to its loss in order to minimize metabolism and inhibit the possibility of division. Mature, enucleated erythrocytes are released into the blood through the selective bone marrow barrier, formed by endothelial cells of the marrow vessels. Under pathological conditions, erythropoiesis can occur in the liver and spleen. Consequently, immature forms of erythrocytes appear in the peripheral blood, including reticulocytes and erythroblasts containing the cell nucleus. Erythropoiesis is subject to both local and systemic regulation. Although erythrocyte maturation is tightly programmed in the genome of hematopoietic stem cells, there are a number of factors that modify the process. These include adhesion molecules, cytokines, ligands and receptors binding them, tyrosine kinases activating transcription factors in the cell nucleus. Adhesive molecules are responsible for the adhesion of blood cells to the medium, while hematopoietic cytokines determine their survival and multiplication. Normal cells require constant cytokine stimulation, since the lack of such signal causes direction of the cell to the apoptosis pathway. Proper cytokine supply is the basic mechanism that regulates cell homeostasis and ensures stability in the structure and number of specific blood cells at a given site. The factor that regulates erythropoiesis at the systemic level is glycoprotein peptide hormone secreted by the liver (20%) and, to a greater extent, by type I peritubular cells of the interstitial tissue of the kidney cortex (80%), called erythropoietin [1].

It stimulates various stages of erythropoiesis due to binding to transmembrane EPO-R receptors, present mainly on precursor cells of the erythropoietic lineal, i.e., proerythroblasts. After ligand attachment, it creates a homodimer receptor and then activates tyrosine kinases JAK (Janus-activated kinase) and other transcription factors. It is noted that the amount of erythropoietin receptors is inversely proportional to the degree of erythrocyte maturity. They are no longer found in the cell membrane of reticulocytes and erythrocytes [1,2]. Conversely, the expression of this receptor in neoplastic cells appears to be a disturbing phenomenon. This hampers the administration of recombinant erythropoietin in patients with malignant neoplasm, which in this situation can promote tumor cell growth [3]. The situation triggering the release of erythropoietin is hypoxia of tissues of various origins (heart and lung diseases, smoking or being at high altitudes).

Therefore, EPO-R receptors occurs in tissues with high metabolism and high sensitivity to hypoxia, i.e., brain, heart muscle, skeletal muscle and kidneys [2]. In turn, the secretion of erythropoietin is disturbed in chronic kidney disease, where the production of this hormone is gradually reduced. Deficiency of erythropoietin or a lack of sensitivity to its target tissue is one of the development mechanisms of chronic diseases anemia. In addition to erythropoietin, other hematopoietic factors are necessary for the proper conduct of the erythropoiesis process. These include iron, vitamin B₁₂, folic acid, vitamin C, vitamin B₆, proteins and hormones. The absence of any hematopoietic factor or the appearance of factors directly damaging the bone marrow or mature erythrocytes will also result in the occurrence of anemia.

2. Iron Metabolism—General Remarks

Iron is one of the most important microelements of the body necessary for the synthesis of hemoglobin. In addition to its main building function, it also has a regulatory role. As a component of heme and cytochrome enzymes, it enables cellular respiration through electrons transfer in the electron transport chain, supports antioxidative processes and DNA synthesis (RNA reductase is an iron-dependent enzyme needed for DNA replication). The iron content in the human body oscillates around 3–4 g, of which approx. 2.5 g (65%) is in hemoglobin, approx. 400 mg (10%) in myoglobin, catalase and cytochrome, from 3 to 7 mg (0.1%) is bound by transferrin, while the rest is backup (25%). The stored iron can be divided into active (ferritin) and inactive (hemosiderin) pools [4].

The daily diet provides an average of 10–15 mg of iron, of which only 5–10% (i.e., 1 mg) is absorbed into the body. The human body uses 20–25 mg of iron daily for hemoglobin synthesis. Most of this element comes from the natural degradation of erythrocytes due to their damage or aging. However, there are situations in which its excessive accumulation in the body occurs. It may result from the excessive absorption of this element from the gastrointestinal tract (hemochromatosis), its excessive supply with food or from improper iron metabolism (shortage for erythropoiesis or excessive release from the liver or red blood cells). Currently, the ability of free iron to initiate oxidative stress is widely discussed, consisting mainly of the production of free radicals, including reactive oxygen species, which damage nucleic acids, lipids and proteins contributing to carcinogenesis [5,6].

3. Iron Absorption and Characterization of Proteins Involved in the Metabolism

Absorption of iron in the gastrointestinal tract is a complicated process involving many proteins (DMT1 protein, ferroportin, ferritin, hepcidin, hephaestin, transferrin, lactoferrin). At the cellular level, deficiency of this element increases the synthesis of the transferrin receptor and increases the absorption of iron in the gastrointestinal tract. While at the systemic level, its deficiency significantly reduces the synthesis of ferritin (unless the deficiency is accompanied by inflammation that is present at the same time, because ferritin is an acute phase protein). The so-called active iron, i.e., rapidly exchanging, is associated with ferritin and is found primarily in hepatocytes and liver macrophages, spleen, bone marrow and muscles. The role of ferritin is primarily intracellular iron storage, which prevents the formation of reactive oxygen species (ROS) generated by the Fenton reaction. The ferritin molecule consists of soluble protein—apoferritin and the internal part containing iron ions. The protein part of ferritin is constructed from 24 chains consisting of light L and heavy H subunits forming a quadruple helix. The H subunit has oxidoreductive properties, which are attributed to ferroxidase center. Fe 2+ ions after penetrating through the apoferritin coating into ferroxidase centers are oxidized to Fe 3+ trivalent form. Inflammation increases the expression of the H subunit of ferritin, which, thanks to ferroxidase centers, has the ability to sequestrate iron and protect against oxidative stress. The consequence of this phenomenon is a deficit of available iron and anemia observed in chronic inflammation. Under physiological conditions, only the glycosylated form of the L ferritin subunit is present. The quantitative proportions of light and heavy chains forming ferritin differ depending on the state of iron homeostasis and the type of tissue. The H subunit predominates in cells requiring high iron availability, such as erythrocytes and myocardial cells. In iron storage organs (liver, spleen), ferritin is mainly built of the L subunit, which does not contain ferroxidase centers, and its main role is to store bioavailable iron atoms [7,8]. The role of ferritin as a carrier protein has recently been confirmed. It is assumed that ferritin can transport 100 to 1000 times more iron than transferrin. To serve as an iron-providing protein, ferritin must be released from the cells in which it is produced. Some studies suggest that intracellular ferritin may be transported to the lysosome for its degradation and release of iron, which is then recycled. This process is called ferritinophagy. The iron exported to the cytosol is then used in several physiological processes that include the synthesis of mitochondrial heme and erythrocyte differentiation. In the transport of cytosolic ferritin to the lysosome, a nuclear receptor co-activator 4 (NCOA4) is required, which binds to ferritin through the C-terminal domain and delivers it to the forming autophagosome. NCOA4 activity and severity of ferritinophagy are regulated by intracellular iron levels. Under the conditions of full availability of this element, ubiquitin E3 ligase (HERC2) binds NCOA4 and leads to its proteasomal degradation. The reduced level of NCOA4 promotes stabilization of ferritin by reducing the level of intracellular iron and inhibiting ferritinophagy, which, by producing reactive oxygen species (ROS), causes ferroptosis. NCOA4 deficiency in animal models is manifested in the inability to degrade ferritin and leads to a decrease in the intracellular iron bioavailability. Ferritinophagy is involved in NCOA4-mediated erythropoiesis, which enables the release of iron from ferritin, which is necessary for the synthesis of mitochondrial heme. Therefore, NCOA4 deficiency may be one of the mechanisms responsible for the development of anemia of chronic diseases [9,10]. The iron in foods is present in the form of the Fe 3+ ion, which under the influence of gastric juice and Fe reductase (DCYTB) is reduced to the Fe 2+ ion [11]. Its absorption depends on the body’s demand for this element and takes place mostly in the proximal part of the small intestine, where optimal conditions prevail. Bile secreted by the liver enters the duodenum, where high-affinity bonds form between iron and bile salts. As a result, well-soluble iron compounds (cation-bile salt complexes) are formed, which reach the intestinal mucosa more easily. Bile, by binding iron ions, thus prevents the precipitation of sparingly soluble iron oxides (Fe₂O₃). Bile salts are therefore important buffers of iron ions, increasing their absorption in the digestive tract [12,13]. At the time of iron deficiency, the expression of DMT1 (divalent metal transporter 1) increases, which is responsible for its increased absorption. At the same time, the ferroportin-controlled release of iron into the blood increases. Hepcidin—a 25-amino acid peptide predominantly synthesized by liver cells that antagonizes ferroportin, plays a key role in regulating iron metabolism [14,15,16]. It binds to ferroportin, which limits the release of iron from enterocytes, monocytes, macrophages and hepatocytes into the blood. The amount of secreted hepcidin correlates with the intensity of erythropoiesis and iron reserves in the human body, and most importantly, its synthesis and release are stimulated by inflammatory cytokines, which are produced in excess in the course of chronic diseases [17]. The iron already released into the circulation is oxidized with the participation of hephaestin (HEPH) to the Fe 3+ ion and combined with transport proteins, i.e., transferrin and lactoferrin. Transferrin is a glycoprotein synthesized in the liver by hepatocytes, glial cells, lymphocytes, Sertoli cells and mammary gland cells. The rate of synthesis of this protein, like hepcidin, is strictly conditioned by the body’s demand for iron and the concentration of this element in the blood serum. Under normal conditions, 30 to 40% of transferrin is saturated with iron, the remaining 60% is in the form of apotransferrin [18]. The mechanism that secures the loss of these proteins in the urine during glomerular filtration is their high molecular weight of about 80 kDa. The mature transferrin molecule is formed by a long 679 amino acid polypeptide chain, which is made up of two similar N- and C-terminal domains connected by a short peptide. Each of these domains, by adopting an alternating β and α structure stabilized by 19 disulfide bridges, has one hydrophilic iron ion binding site, which ultimately allows the transport of two iron atoms by one transferrin molecule [19]. The iron carriers reach the precursor cells of the erythropoietic line (proerytroblasts), which express on their surface transferrin receptors, thanks to which fusion with iron transporting proteins is possible. The iron released by endocytosis is used for hemoglobin production and partly stored by ferritin. The iron-depleted transferrin (apotransferin) leaves the precursor cell and goes back to the bloodstream, where it functions as described above [20,21]. The presence of transferrin receptors is mainly represented by tissues with high metabolism and cells that are actively dividing. This partly may explain the increase in iron demand in people suffering from certain types of neoplasm which results not only from the constant use of this element in the production of hemoglobin, but also DNA synthesis by malignant cells [18]. Despite the fact that in the case of malignant neoplasms this is not one of the main causes determining the occurrence of anemia of chronic diseases (more often caused by chemotherapy-induced myelosuppression, blood loss, compromised iron distribution, etc.), attempts are being made to use drugs targeting the transferrin receptor, which blockade would interfere with the metabolism of actively dividing cells [22]. Nevertheless, transferrin itself as a transporter protein can be used to bind oxidative stress-generating free iron or to transfer a molecularly targeted drug to malignant cells [23].

4. Regulation of Iron Metabolism at the Cellular and Systemic Level

The expression of key proteins involved in iron absorption is controlled at many levels: transcriptional, post-transcriptional and post-translational. It turns out that the individual stages of the iron absorption process are subject to separate regulations. In the last decade, particular attention has been paid to the effects of hypoxia and molecular mechanisms of iron homeostasis. The transcriptional regulation of genes involved in the absorption and transport of iron across the intestinal mucosa was largely unknown until the HIF-2 transcription factor was discovered. HIF transcription factors are central mediators of cellular adaptation to hypoxia. They form heterodimers containing a regulatory α subunit, responsive to iron deficiency and hypoxia, and a β subunit with constitutive expression known as the aryl hydrocarbon receptor nuclear translocator (ARNT). There are three α subunit isoforms (HIF-1α, HIF-2α and HIF-3α) that are regulated at post-translational level [24]. HIF-1 has been the most widely studied subunit to date. HIF-1 has been shown to be involved in angiogenesis, glycolytic metabolism, apoptosis and cellular stress [25]. HIF-1 has also been shown to regulate expression of transferrin 1 receptor (TfR1) and expression of heme oxygenase 1 (HO-1) [26,27]. HIF-2 plays a key role in adult erythropoiesis by increasing the hepatic production of erythropoietin and the absorption of iron from the gastrointestinal tract. In enterocyte, HIF-2 regulates iron absorption by directly activating transcription of the divalent metal transporter 1 (DMT1), iron reductase (DCTYB) and ferroportin (FPN). In addition, HIF-2 inhibits hepcidin production in the liver, which molecularly binds to the iron exporter, i.e., ferroportin [28]. In patients with chronic kidney disease (CKD), the lack of EPO production by the interstitial tissue of the kidneys results in defective erythropoiesis, which consequently leads to anemia. Under physiological conditions, exposure to hypoxia stabilizes HIF-2 in EPO-producing kidney cells, leading to increased production of this hormone. At normal oxygen partial pressure (21%), the α subunits are hydroxylated and quickly degraded by iron and oxygen dependent enzymes (prolyl 4-hydroxylase, PHD). When the iron level is low, the prolyl hydroxylase activity decreases, thus the α subunits are destroyed much more slowly. During hypoxia, the hydroxylation (stabilization) of the α subunit is inhibited, which enables its translocation to the nucleus and interaction (dimerization) with the β subunit. The functional heterodimer moves to the nucleus to regulate the transcription of target genes (DCYTB, DMT1, FPN) by binding to specific sequences called hypoxia-responsive elements (HRE). Direct binding of the HIF heterodimer to HRE elements in the promoter regulatory regions has been demonstrated for DCYTB and DMT1. Their induction probably mediates the increase in iron absorption observed in conditions with low iron and oxygen content [29]. The increase in the number of CKD cases underlines the need for novel therapeutic approaches for treating anemia. Since the discovery of HIF, a concept of HIF stabilization has been suggested to support EPO production in CKD anemia. This is now possible thanks to a new class of drugs—PHD inhibitors or HIF stabilizers that prevent degradation of the HIF α subunit via the proteasome. This can improve the quality of life of patients with CKD by avoiding multiple transfusions, iron supplementation, or reducing doses of erythropoiesis stimulating agents [30].

The absorption and metabolism of iron have been presented in Figure 1 .