Hepcidin (hepatic bactericidal protein) was initially identified as a urinary antimicrobial peptide rich in cysteine. Further studies showed that hepcidin is overexpressed in mice with iron overload and that it plays a significant role in iron homeostasis in knockout animals with iron storage disease. The complete abrogation of hepcidin entails excessive intestinal absorption of iron and increased iron release by macrophages, a condition that leads to iron overload [1-3]. Hepcidin performs its different functions via a single biochemical mechanism: hepcidin-ferroportin interaction [4]. Intestinal epithelial cells and reticuloendothelial macrophages use the same transporter, ferroportin, to transport iron in the plasma. Moreover, macrophages and enterocytes exhibit strong upregulated ferroportin expression in the erythropoietic response in an iron-restricted state.

Iron regulates hepcidin homeostasis. Increases in iron levels in the plasma and iron storage stimulate the production of hepcidin, which blocks iron absorption from the diet and its further storage [5]. Hepcidin production is suppressed in the case of iron deficiency; the feedback loop between iron and hepcidin should ensure the appropriate physiological concentration of iron in the plasma. Hepcidin production is also regulated by the erythropoietic process, whose core activity is characterized by high iron consumption. In this case, hepcidin suppression causes the stored iron to be released by hepatocytes and macrophages while the intestinal absorption of iron increases. This condition involves iron supplementation from hemoglobin synthesis activity [6]. Hepcidin is elevated during inflammation and/or infection. This can cause iron dysregulation with hypoferremia and anemia related to inflammatory disease [7]. Hypoferremia can also represent a strategic host defense to limit iron availability to microorganisms.

Bone morphogenetic proteins (BMPs) are a group of growth factors that activate the transduction signal by interacting with specific receptors. They use intracellular and extracellular iron detection mechanisms to alter hepcidin expression. BMPs increase hepcidin both in vivo and in vitro. BMP6 is the primary regulator of endogenous hepcidin. Hemojuvelin (HJV) is a BMP co-receptor specialized for iron regulation [8]. The hepcidin adjustment process in the iron response occurs in the liver on the membranes of hepatocytes. Once BMP6 binds the co-receptor HJV, the SMAD transcriptional system (SMAD1/5/8) is activated [9-11]. The activated SMAD complexes bind directly to BMP-responsive elements on the hepcidin promoter, thus inducing hepcidin transcription [12].

The TFR2 and HFE genes, which are altered in adults with hereditary hemochromatosis, appear to adjust to the detection of iron-transferrin concentration. TFR2, a TfR1 homolog, is mainly expressed in the liver. HFE is similar to major histocompatibility complex type I (MHC I) molecules. Regarding the iron-transferrin concentration complex, HFE appears to function as a shuttle between TfR1 and TFR2.

Increased erythropoietic activity significantly reduces hepcidin levels. A single administration of erythropoietin (EPO) over a period of 24 hours significantly reduces hepcidin levels in humans [13]. In cases of ineffective erythropoiesis, 2 proteins are produced by erythroblasts, growth differentiation factor 15 (GDF I5) and twisted gastrulation I (TWSGI), which appear to be responsible for mediating hepcidin suppression [14, 15]. EPO indirectly influences iron homeostasis. EPO production as a normal response to hypoxic stimulation is responsible for normal erythron expansion without excessive erythropoiesis. GDF15 and TWSG1 are released as a result, ultimately suppressing hepcidin synthesis as mentioned before. EPO activation is the main event that occurs in acute hypoxia; this causes the expansion of erythropoiesis, which requires adequate iron for the hemoglobinization of red cells. The production and hemoglobinization of the erythroid lineage can still occur, if hepcidin is downregulated [16, 17].

Inflammation and infection increase hepcidin synthesis. Patients with sepsis, inflammatory bowel disease, myeloma, burns, and C reactive protein (CRP) levels >10 mg/dL exhibit significantly elevated hepcidin levels [3, 5, 7, 18, 19]. Macrophages are stimulated during the inflammatory process; the stimulation depends on the severity of inflammation. Activated macrophages release a network of cytokines. Among them is interleukin-6 (IL-6) is one of the primary inducers of hepcidin expression; an increase in hepcidin levels finally results in hypoferremia (Fig. 1). Hepcidin inhibits iron release from macrophages as well as intestinal iron absorption. In inflammatory states, hepcidin production is no longer regulated by iron burden (i.e., if the iron level is low, hepcidin synthesis should be downregulated) but is rather increased through IL-6 stimulation. Serum iron was demonstrated to affect hepcidin synthesis in healthy volunteers, in whom the early presence of hepcidin in the urine was measured after an oral iron administration dose that did not affect iron storage. Serum iron is an induction signal for hepcidin production and affects serum transferrin saturation percentage. In the case of inflammation, hepcidin can also be produced by myeloid cells via the activation of TRL4, a receptor located on the membranes of neutrophils and macrophages [20].

Understanding the physiological processes of hepcidin has made it possible to redefine the pathogenetic mechanisms of anemia.

Specific methods were recently developed to measure hepcidin and evaluate its diagnostic potential. The competitive ELISA test uses biotinylated or radioiodinated hepcidin as a tracer [5, 32]. However, mass spectrometry is used for the hepcidin assay; this involves standards labeled with hepcidin isotopically or standards that bind the hepcidin-truncated molecule [33, 34]. The use of different methods for the determination of hepcidin levels mainly depends on the employment of different hepcidin standards as well as the varying abilities of different methods to detect the hepcidin-20 and hepcidin-22 isoforms in addition to the hepcidin-25 bioactive isoform. Despite the varying abilities of the methods mentioned above, there is no difference between the samples and analytical methods used. This indicates that all methods are appropriate for detecting hepcidin isoforms in samples. The results of the first international round-robin research on the quantification of plasma and urinary hepcidin assays led to the conclusion that the differences in the levels of hepcidin isoforms between methods could be due to the use of different calibrators with assigned levels obtained by different techniques, hepcidin aggregation of either the standard solution or sample, binding of hepcidin to α2-macroglobulin or albumin, or existence of 3 hepcidin isoforms (hepcidin-25, hepcidin-22, and hepcidin-20) [35].

Since approximately 90% of circulating hepcidin is linked to α2-macroglobulin, it is important to consider whether it is necessary to measure total or unbound hepcidin and which current valuation methods can be used. The difference between immunochemical (IC) methods and mass spectrometry is another important issue. IC methods cannot selectively distinguish hepcidin-22 and hepcidin-25 from hepcidin-20. However, the reasons for including hepcidin-20 and hepcidin-22 in the total hepcidin level for determining different iron-related diseases remain unclear. The coefficient of variation among samples is lower for the plasma hepcidin assay than that for urinary hepcidin. This suggests that the difference between measured levels in the urine and plasma is not due to the method used, but due to biological mechanisms such as excretion. Hepcidin levels measured using different methods vary considerably, but the analytical variance is low and similar for other methods in general.

In order to harmonize the various methods used to measure hepcidin, it is recommended to introduce internal standards for all basic mass spectrometry methods used for clinical trials, reach assigned consensus levels, adjust the levels of calibrators used in each procedure, produce a calibrator that mimics a patient's serum, periodically analyze shared samples and/or interchangeable calibrators, and have a value assigned for quality control.

In addition to measuring hepcidin, it is possible to measure prohepcidin. However, the measurement of the latter does not appear to be biologically relevant with respect to hepcidin.

Another problem is the fluctuation of diurnal hepcidin values. Hepcidin levels are lower in the morning and increase in the afternoon. In addition, assay sensitivity is related to the amount of iron introduced by the diet [5, 36]. Various types of anemia exhibit different iron-related parameters including hepcidin, transferrin saturation, ferritin, and soluble transferrin receptor (Table 1).

Having established the central role of hepcidin in iron homeostasis, it is possible to consider its use as a novel treatment for anemia. Under particular conditions of chronic inflammation, despite low plasma iron levels, iron treatment can be harmful because it can trigger the growth of pathogens and/or contribute to neoplastic transformation because of its ability to unbalance immune functions, particularly the immune-mediated activities of T-cells [37, 38].

Although hepcidin is not used for therapeutic purposes at present, there are some ongoing studies on selected patients. Hepcidin agonists could be used to prevent or improve the accumulation of iron in both transfused and non-transfused β-thalassemic patients and even in anemia with iron storage. Hepcidin antagonists could be used in patients with diseases that cause hepcidin excess and occur with a framework of IDA or systemic IDA.

Other drugs targeting the regulation of the hepcidin system, such as dorsomorphin, a small molecule that inhibits the BMP signal, have been tested. Even soluble HJV acts as a BMP signal antagonist, decreasing the basal concentration of hepcidin in mice while increasing the liver iron concentration [39]. The use of anticytokines such as anti-IL-6 has been shown to suppress hepcidin production activity and improve anemia in some cases [40, 41]. Hydroxyproline inhibitors may be effective for inducing hepcidin inhibition; such inhibition could be caused not only by erythropoiesis stimulation but also by the ability of hydroxyproline inhibitors to interfere with the transcriptional complex, hypoxia-inducible factor [42].

Iron-related diseases are common and clinically important. Recent research on both hepcidin and the key role of the hepcidin-ferroportin axis has clarified the pathogenesis of IDA, ACD, and anemia with iron overload. This knowledge will allow the development of new tests and possibly new treatments as well.