Hepcidin

Dysregulation of metabolism and utilization of iron can lead to the development and maintenance of anemia of CKD. Anemia is prevalent among patients with CKD. The markers of iron sufficiency or availability of iron are far from perfect which results in inaccurate diagnosis and treatment of anemia with poor outcomes. Hepcidin, a 25 amino acid peptide produced by the hepatocytes, has emerged as the key regulator of uptake and release of iron in the tissues to maintain a steady supply of iron to erythron and other tissues while avoiding higher levels of iron that could be detrimental to the organs. Hepcidin itself is regulated by the supply of iron, the need for erythropoiesis, and the state of inflammation. Alterations in hepcidin levels are associated with restricted erythropoiesis, anemia, and iron overload. Discovery of hepcidin and elucidation of its mechanism of action and consequences of its upregulation and suppression have unraveled important insight into many hematologic disorders including anemia of CKD. This knowledge has also unlocked unique opportunities to modulate hepcidin via agonists and antagonists of hepcidin and its feedback pathways to treat clinical conditions. Many such agents are being developed and have potential therapeutic utility in future. Dysregulation of metabolism and utilization of iron can lead to the development and maintenance of anemia of CKD. Anemia is prevalent among patients with CKD. The markers of iron sufficiency or availability of iron are far from perfect which results in inaccurate diagnosis and treatment of anemia with poor outcomes. Hepcidin, a 25 amino acid peptide produced by the hepatocytes, has emerged as the key regulator of uptake and release of iron in the tissues to maintain a steady supply of iron to erythron and other tissues while avoiding higher levels of iron that could be detrimental to the organs. Hepcidin itself is regulated by the supply of iron, the need for erythropoiesis, and the state of inflammation. Alterations in hepcidin levels are associated with restricted erythropoiesis, anemia, and iron overload. Discovery of hepcidin and elucidation of its mechanism of action and consequences of its upregulation and suppression have unraveled important insight into many hematologic disorders including anemia of CKD. This knowledge has also unlocked unique opportunities to modulate hepcidin via agonists and antagonists of hepcidin and its feedback pathways to treat clinical conditions. Many such agents are being developed and have potential therapeutic utility in future. Clinical Summary•Hepcidin, produced by hepatocytes, is the key regulator of uptake and release of iron in tissues; hepcidin is regulated by iron supply, erythropoietic requirements, and inflammatory status.•Changes in hepcidin concentrations are associated with iron-restricted erythropoiesis, anemia, and iron overload.•Hepcidin levels exhibit biological variation and subject to alterations in renal excretion and inflammation, thereby rendering hepcidin an unsuitable biomarker of iron status or predictor of ESA-responsiveness.•Modulation of the hepcidin-ferroportin axis by agonists/antagonists represents an attractive future target for novel preventive or therapeutic strategies for disorders of iron metabolism and erythropoiesis. •Hepcidin, produced by hepatocytes, is the key regulator of uptake and release of iron in tissues; hepcidin is regulated by iron supply, erythropoietic requirements, and inflammatory status.•Changes in hepcidin concentrations are associated with iron-restricted erythropoiesis, anemia, and iron overload.•Hepcidin levels exhibit biological variation and subject to alterations in renal excretion and inflammation, thereby rendering hepcidin an unsuitable biomarker of iron status or predictor of ESA-responsiveness.•Modulation of the hepcidin-ferroportin axis by agonists/antagonists represents an attractive future target for novel preventive or therapeutic strategies for disorders of iron metabolism and erythropoiesis. An adequate amount of iron is essential for many molecular processes including energy production as well as heme formation to facilitate oxygen delivery to the tissues. An excessive amount of iron, however, can be detrimental through the formation of reactive oxygen species with resultant oxidative stress, damage to DNA, and potentiation of lipid peroxidation and ferroptosis of cells. Even in the presence of adequate iron stores, a maldistribution of iron can result in anemia as in inflammation and chronic disease such as CKD. As iron losses are relatively constant and not regulated, the absorption and release of iron from cells is regulated through a multitude of proteins to carefully maintain intracellular and systemic levels of iron that are both adequate and safe. Hepcidin was discovered as the liver-expressed antimicrobial peptide in 2000.1Krause A. Neitz S. Magert H.J. et al.LEAP-1, a novel highly disulfide-bonded human peptide, exhibits antimicrobial activity.FEBS Lett. 2000; 480: 147-150Crossref PubMed Scopus (1070) Google Scholar, 2Park C.H. Valore E.V. Waring A.J. Ganz T. Hepcidin, a urinary antimicrobial peptide synthesized in the liver.J Biol Chem. 2001; 276: 7806-7810Crossref PubMed Scopus (1747) Google Scholar Hepcidin is produced primarily by the hepatocytes which are strategically located in the vicinity of portal veins (carrying dietary iron) as well as the Kupffer cells (sensing microbes and recycling erythrocytes).3Ganz T. Nemeth E. Hepcidin and iron homeostasis.Biochim Biophys Acta. 2012; 1823: 1434-1443Crossref PubMed Scopus (856) Google Scholar Hepcidin is also produced by macrophages and adipocytes in a small quantity.4Liu X.B. Nguyen N.B. Marquess K.D. Yang F. Haile D.J. Regulation of hepcidin and ferroportin expression by lipopolysaccharide in splenic macrophages.Blood Cell Mol Dis. 2005; 35: 47-56Crossref PubMed Scopus (124) Google Scholar, 5Bekri S. Gual P. Anty R. et al.Increased adipose tissue expression of hepcidin in severe obesity is independent from diabetes and NASH.Gastroenterology. 2006; 131: 788-796Abstract Full Text Full Text PDF PubMed Scopus (384) Google Scholar Hepcidin is encoded by the hepcidin antimicrobial peptide (HAMP) gene. It is initially synthesized as an 84 amino acid pre-pro-hepcidin. It is then processed to 60 amino acid pro-hepcidin and ultimately sliced to a mature C-terminal 25 amino acid active peptide.6Rochette R. Gudjoncik A. Guenancia C. Zeller M. Cottin Y. Vergely C. The iron-regulator hormone hepcidin: a possible therapeutic target?.Pharmacol Ther. 2015; 146: 35-52Google Scholar Hepcidin is a tightly folded peptide hormone that forms a simple hairpin structure stabilized by 4 disulfide bonds (Fig 1).7Jordan J.B. Poppe L. Haniu M. et al.Hepcidin revisited, disulfide connectivity, dynamics, and structure.J Biol Chem. 2009; 284: 24155-24167Crossref Scopus (175) Google Scholar Mutations of the HAMP gene are associated with severe iron overload and hemochromatosis. Hepcidin expression is increased in iron overload and inflammation and is diminished in states of iron deficiency and hypoxia.8Nicolas G. Chauvet C. Viatte L. et al.The gene encoding the iron regulatory peptide hepcidin is regulated by anemia, hypoxia, and inflammation.J Clin Invest. 2002; 110: 1037-1044Crossref PubMed Scopus (1348) Google Scholar Hepcidin transcription is regulated by bone morphogenic protein (BMP) and its coreceptor hemojuvelin (HJV) in the liver. A number of BMPs, specifically BMP-6, can induce HAMP expression and upregulate hepatocyte hepcidin expression, a process enhanced by HJV and blunted in hemojuvelin knockout (Hfe2−/−) hepatocytes.9Babitt J.L. Huang F.W. Wrighting D.M. et al.Bone morphogenetic protein signaling by hemojuvelin regulates hepcidin expression.Nat Genet. 2006; 38: 531-539Crossref PubMed Scopus (830) Google Scholar BMPs bind to type I and type II receptors leading to signal transduction. BMP and transforming growth factor-beta induce hepcidin expression via “small” worm phenotype and Mothers Against Decapentaplegic (SMAD) signaling.10Wang R.H. Li C. Xu X. et al.A role of SMAD4 in iron metabolism through the positive regulation of hepcidin expression.Cell Metab. 2005; 2: 399-409Abstract Full Text Full Text PDF PubMed Scopus (502) Google Scholar HJV is a member of the repulsive guidance molecule family and acts as a BMP coreceptor. HJV gene (also known as HFE2) mutations can result in hepcidin deficiency and juvenile hemochromatosis.11Ramey G. Deschemin J.C. Vaulont S. Cross-talk between the mitogen activated protein kinase and bone morphogenetic protein/hemojuvelin pathways is required for the induction of hepcidin by holotransferrin in primary mouse hepatocytes.Haematologica. 2009; 94: 765-772Crossref PubMed Scopus (99) Google Scholar A number of other pathways and compounds partake in hepcidin regulation in a complex manner.3Ganz T. Nemeth E. Hepcidin and iron homeostasis.Biochim Biophys Acta. 2012; 1823: 1434-1443Crossref PubMed Scopus (856) Google Scholar Hepcidin gene expression is upregulated by inflammation and iron through the Janus kinase/signal transducers and activators of transcription (JAK/STAT) and BMP/SMAD pathways, respectively.6Rochette R. Gudjoncik A. Guenancia C. Zeller M. Cottin Y. Vergely C. The iron-regulator hormone hepcidin: a possible therapeutic target?.Pharmacol Ther. 2015; 146: 35-52Google Scholar The BMP-6 signal acts through its receptor (BMPR), and is modulated by HJV. The TMPRSS6 (transmembrane serine protease-2) gene encodes matriptase-2 (MT2) which cleaves membrane-bound HJV. BMPs can also signal through SMAD-independent pathways, notably via mitogen-activated protein (MAP) kinases. Dorsomorphin inhibits BMP signaling through the SMAD pathway. SMAD complexes bind to BMP-responsive elements. Tumor necrosis factor (TNF), pathogens, and interleukin-6 (IL-6) stimulate hepcidin synthesis via signal transducer and activator of transcription 3 (STAT-3) activation. The P38 MAP-kinase and extracellular signal-regulated kinases (ERK) 1-2 pathways are activated in response to iron signals. Diferric transferrin (Tf) binds to TF-receptor 1 (TfR-1) on the cell surface and the complex undergoes endocytosis. HFE is a protein that competes with Tf for binding to TfR-1. Hepcidin expression in macrophages is regulated mainly through TLR4 receptors associated with adaptor proteins. Hepcidin mRNA expression in macrophages induced by lipopolysaccharide (LPS) or high mobility group protein B1 (HMGB1) depends on nuclear factor kappa-light-chain-enhancer of activated B cells (NFkB). Vitamin D is a potent negative regulator of hepcidin transcription in cultured monocytes and hepatocytes and also induces antibacterial proteins such as cathelicidin.12Bacchetta J. Zaritsky J.J. Sea J.L. et al.Suppression of iron-regulatory hepcidin by vitamin D.J Am Soc Nephrol. 2014; 25: 564-572Crossref PubMed Scopus (207) Google Scholar In an in vivo and in vitro study, seven healthy volunteers received a single oral dose of vitamin D (100,000 IU vitamin D2) which increased serum levels of 25-hydroxyvitamin D from 27 ± 2 ng/mL before supplementation to 44 ± 3 ng/mL after supplementation (P < .001). This response was associated with a 34% decrease in circulating levels of hepcidin within 24 hours of vitamin D supplementation. The high levels of hepcidin in patients with CKD may possibly be due not only to reduced renal excretion of hepcidin but also to vitamin D deficiency, which is widely prevalent in this population. Hepcidin 25 has both iron regulatory and antimicrobial activities, but hepcidin 22 and 20 have only antimicrobial activity. Hepcidin 25 binds to ferroportin, a transmembrane protein, and leads to its degradation and consequent inhibition of iron release from enterocytes, macrophages, and hepatocytes to regulate iron absorption and utilization. Hepcidin is cleared via its internalization and degradation with ferroportin as well as through its excretion by the kidneys. Although hepcidin controls iron transport, its production is regulated by the systemic availability of iron, erythropoietic requirement, as well as by the state of inflammation (Fig 2).3Ganz T. Nemeth E. Hepcidin and iron homeostasis.Biochim Biophys Acta. 2012; 1823: 1434-1443Crossref PubMed Scopus (856) Google Scholar Hepcidin production is impacted by the feedback loop of systemic availability of iron. Hepcidin is produced in the iron-rich environment (whether in plasma or in tissues) and its production is reduced or stopped when iron is deficient or in high demand. In the presence of active erythropoiesis, production of hepcidin is inhibited, at least partially via an erythroid factor (erythroferrone, ERFE) produced by the erythroblasts that suppresses the BMP/SMAD pathway in the liver. In vitro, ERFE decrease SMAD1, SMAD5, and SMAD8 phosphorylation and inhibits expression of BMP target genes, suppressing the induction of hepcidin by BMP5, BMP6, and BMP7 without affecting hepcidin induction by BMP2, BMP4, BMP9, or activin B.13Arezes J. Foy N. McHugh K. et al.Erythroferrone inhibits the induction of hepcidin by BMP6.Blood. 2018; 132: 1473-1477Crossref PubMed Scopus (146) Google Scholar, 14Pak M. Lopez M.A. Gabayan V. Ganz T. Rivera S. Suppression of hepcidin during anemia requires erythropoietic activity.Blood. 2006; 108: 3730-3735Crossref PubMed Scopus (385) Google Scholar Hepcidin levels decrease in hypoxia, although the exact mechanism is unclear. Hypoxia inducible factor may suppress hepcidin transcription directly or through activation of furin to release soluble HJV which interferes with BMP signaling and leads to inhibition of hepcidin activation.15Silvestri L. Pagani A. Camaschella C. Furin-mediated release of soluble hemojuvelin: a new link between hypoxia and iron homeostasis.Blood. 2008; 111: 924-931Crossref Scopus (253) Google Scholar, 16Palaneeswari M.S. Ganesh M. Karthikeyan T. Devi A.J. Mythili S.V. Hepcidin—minireview.J Clin Diagn Res. 2013; 7: 1767-1771Google Scholar Hepcidin synthesis is increased during periods of infection or inflammation. The hepatocyte production of hepcidin in such states is regulated by IL-6 through the STAT-3 signaling pathway. It is important to understand iron metabolism in order to understand therapeutic modulation of hepcidin. Total body iron stores are approximately 4 g with daily iron losses of 1-2 mg, which must be replenished via the absorption of dietary iron. Erythropoiesis requires 20-25 mg of iron daily, most of it coming from recycling by erythrophagocytosis of senescent red cells. Daily requirement of dietary iron differs among individuals according to their age and gender, especially in young females due to a much larger iron loss during menstruation. Iron absorption and availability in the plasma are regulated by hepcidin. Iron uptake is mediated by the H+ coupled Fe2+ transporter known as divalent cation transporter 1/divalent metal ion transporter 1 (DMT1) at the brush border of the duodenal enterocytes.17Gunshin H. Allerson C.R. Polycarpou-Schwarz M. et al.Iron-dependent regulation of the divalent metal ion transporter.FEBS Lett. 2001; 509: 309-316Crossref PubMed Scopus (267) Google Scholar This is facilitated by the membrane ferrireductase, duodenal cytochrome B. Iron enters via enterocytes, is stored in the hepatocyte cytoplasm and macrophages, and must exit the cell on demand. The exit of the reduced iron from the cell requires the presence of ferroportin, a transmembrane protein, which is the only way for iron to exit into the blood. Hepcidin-ferroportin axis is an integral mechanism regulating the export of iron from the cells. The iron is again oxidized by the membrane bound ferroxidase hephaestin and ceruloplasmin and is then taken up by Tf. Tf releases iron to the cells by binding primarily to the TfR-1 on the cell membrane which stimulates its endocytosis and transport to mitochondria for heme synthesis or to cytoplasm to be stored as ferritin. Lack of ferroportin would render intracytoplasmic iron to be trapped within the cell. Thus, ferroportin degradation by hepcidin leads to inhibition of the transport of iron out of the duodenal enterocytes and a blockade of iron release from the macrophages and hepatocytes resulting in iron sequestration and depletion of the blood pool of iron. Cells that have intracellular iron but lack ferroportin have to be recycled upon cell death by macrophages. Coordination and balance between iron uptake at the brush border and its exit from the basolateral membrane is tightly regulated. This involves a complex relationship between cellular iron accumulation, leading to activation of hepcidin with its effect on iron-binding proteins, and decreased formation of DMT1, degradation of hypoxia inducible factor, and activation of transubiquitination of DMT1.17Gunshin H. Allerson C.R. Polycarpou-Schwarz M. et al.Iron-dependent regulation of the divalent metal ion transporter.FEBS Lett. 2001; 509: 309-316Crossref PubMed Scopus (267) Google Scholar, 18Shah Y.M. Matsubara T. Ito S. Yim S.H. Gonzalez F.J. Intestinal hypoxia-inducible transcription factors are essential for iron absorption following iron deficiency.Cell Metab. 2009; 9: 152-164Abstract Full Text Full Text PDF PubMed Scopus (308) Google Scholar, 19Brasse-Lagnel C. Karim Z. Letteron P. Bekri S. Bado A. Beaumont C. Intestinal DMT1 cotransporter is down-regulated by hepcidin via proteasome internalization and degradation.Gastroenterology. 2011; 140: 1261-1271Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar Iron overload induces the formation of BMPs by the hepatic sinusoidal endothelial cells. BMPs activate phosphorylation of Smad1/5/8 phosphorylation, which forms a transcriptional activator complex with Smad4 to stimulate hepcidin transcription.20Kautz L. Meynard D. Monnier A. et al.Iron regulates phosphorylation of Smad1/5/8 and gene expression of Bmp6, Smad7, Id1, and Atoh8 in the mouse liver.Blood. 2008; 112: 1503-1509Crossref PubMed Scopus (350) Google Scholar HJV or HFE2, a membrane bound GPI-anchored protein, acts as a BMP coreceptor and promotes hepcidin transcription.9Babitt J.L. Huang F.W. Wrighting D.M. et al.Bone morphogenetic protein signaling by hemojuvelin regulates hepcidin expression.Nat Genet. 2006; 38: 531-539Crossref PubMed Scopus (830) Google Scholar A soluble form of HJV (sHJV) blocks BMP-6 and inhibits hepcidin expression.21Papanikolaou G. Samuels M.E. Ludwig E.H. et al.Mutations in HFE2 cause iron overload in chromosome 1q-linked juvenile hemochromatosis.Nat Genet. 2004; 36: 77-82Crossref PubMed Scopus (829) Google Scholar HJV is cleaved by matriptase 2 (MT2), and BMP receptor, HJV, and MT2 are stabilized by neogenin.22Enns C.A. Ahmed R. Zhang A.-S. Neogenin interacts with matriptase-2 to facilitate hemojuvelin cleavage.J Biol Chem. 2012; 287: 35104-35117Scopus (54) Google Scholar Degradation of ferroportin by hepcidin requires direct binding of hepcidin to its receptor ferroportin.23Nemeth E. Tuttle M.S. Powelson J. et al.Hepcidin regulates cellular iron efflux by binding to ferroportin and inducing its internalization.Science. 2004; 306: 2090-2093Crossref PubMed Scopus (3607) Google Scholar The binding depends upon the presence of amino acid cysteine in position 326 on the extracellular loop of ferroportin. Absence of this amino acid in this position, as in the variant ferroportin, renders hepcidin unable to bind to ferroportin and can result in iron overload. The hepcidin-ferroportin complex then undergoes a conformational change leading to its endocytosis and lysosomal degradation. Studies of hepcidin structure also reveal that the first 9 N-terminal amino acids can internalize ferroportin—a conformational concept known as mini-hepcidin.24Preza G.C. Ruchala P. Pinon R. et al.Minihepcidins are rationally designed small peptides that mimic hepcidin activity in mice and may be useful for the treatment of iron overload.J Clin Invest. 2011; 121: 4880-4888Crossref PubMed Scopus (171) Google Scholar Growth and pathogenicity of many microbes require presence of iron.25Weinberg E.D. Iron availability and infection.Biochim Biophys Acta. 2009; 1790: 600-605Google Scholar Reduction in extracellular iron concentration has evolved over time as a host defense mechanism against infection. Protein-bound iron, as in ferritin, Tf, lactoferrin, or ovotransferrin, is not readily available for uptake by the microbes. Through ferroportin degradation, hepcidin decreases blood iron level to defend against infection. Inflammation and infection result in increased production of IL-6 and TNF-alpha. IL-6 directly regulates hepcidin production through induction and subsequent promoter binding of STAT-3.26Wrighting D.M. Andrews N.C. Interleukin-6 induces hepcidin expression through STAT3.Blood. 2006; 108: 3204-3209Crossref PubMed Scopus (695) Google Scholar STAT-3 is not only necessary but also sufficient for the IL-6 responsiveness of the hepcidin promoter. STAT-3 signaling is influenced by BMP-dependent Smad activation.10Wang R.H. Li C. Xu X. et al.A role of SMAD4 in iron metabolism through the positive regulation of hepcidin expression.Cell Metab. 2005; 2: 399-409Abstract Full Text Full Text PDF PubMed Scopus (502) Google Scholar An increase in hepcidin reduces iron concentrations in the blood which might be helpful in defense against iron-dependent microbes. However, this very action of hepcidin forms the basis of iron sequestration and anemia of inflammation. Inflammatory conditions may be associated with other hepcidin independent mechanisms such as decreased red cell survival and bone marrow suppression that contribute to anemia. Dysregulation of hepcidin (HAMP) or related genes such as hemochromatosis (HFE), transferrin receptor 2 (TFR2), and HJV and conditions such as beta-thalassemia intermedia are known to result in states of dysregulated ferroportin. These conditions are often associated with increased iron absorption as well as export, resulting in excessive amounts of free systemic iron and excessive iron uptake by the tissues with consequent tissue damage. Insufficient production of hepcidin mediated by the mutations in hepcidin or HJV gene is associated with iron accumulation as in hereditary hemochromatosis. Beta-thalassemia, characterized by defective beta globin production and ineffective erythropoiesis, is also associated with hyperabsorption of dietary iron, which is linked to the presence of hepcidin suppressors such as ERFE. Hepcidin suppression is also mediated by the growth differentiation factor 15 (GDF15), a member of the transforming growth factor beta superfamily.27Tanno T. Bhanu N.V. Oneal P.A. et al.High levels of GDF15 in thalassemia suppress expression of the iron regulatory protein hepcidin.Nat Med. 2007; 13: 1096-1101Crossref PubMed Scopus (659) Google Scholar The twisted gastrulation protein homolog 1 TWSG1 also interferes with BMP 2 and 4-mediated hepcidin expression and may act with GDF15 to dysregulate iron homeostasis in thalassemia syndromes.28Tanno T. Porayette P. Sripichai O. et al.Identification of TWSG1 as a second novel erythroid regulator of hepcidin expression in murine and human cells.Blood. 2009; 114: 181-186Crossref PubMed Scopus (284) Google Scholar Therapy with hepcidin agonists may in future become a potential therapy for iron overload disorders. Hepcidin circulates in blood in a protein-bound form, but with low affinity, leading to the presence of free hepcidin which is filtered by the kidney and degraded in proximal tubule. Hepcidin levels in plasma are significantly elevated in patients on hemodialysis and can aggravate iron sequestration. Hepcidin levels decrease significantly after hemodialysis.29Zaritsky J. Young B. Gales B. et al.Reduction of serum hepcidin by hemodialysis in pediatric and adult patients.Clin J Am Soc Nephrol. 2010; 5: 1010-1014Crossref PubMed Scopus (75) Google Scholar The improvement in anemia management in patients on prolonged dialysis may be partly related to hepcidin clearance.30Schwartz D.I. Pierratos A. Richardson R.M. Fenton S.S. Chan C.T. Impact of nocturnal home hemodialysis on anemia management in patients with end-stage renal disease.Clin Nephrol. 2005; 63: 202-208Google Scholar On the other end of the spectrum, hepcidin overexpression can result in iron restrictive anemia as in the case of hepcidin-expressing hepatic adenomas or in familial iron refractory iron deficiency anemia due to the mutations in MT2 which is an inhibitory regulator of hepcidin. Ferroportin deficiency due to missense mutations can also lead to impaired export of iron from cells and lead to iron restricted erythropoiesis. Anemia of some malignancies can also be related to hepcidin overproduction. Local levels of BMP are more important than the systemic level in these circumstances. Anemia of CKD is characterized by inappropriately decreased production of erythropoietin by the kidneys where erythropoietin levels are indeed elevated but not commensurate with the severity of anemia. Anemia of CKD is truly multifactorial, resulting from a relative erythropoietin deficiency compounded by the deficiency, loss, sequestration, and poor utilization of iron with characteristics of anemia of inflammation. Interestingly, patients with anemia of CKD do respond to the supraphysiologic doses of erythropoiesis-stimulating agent (ESA) as well as high-dose intravenous iron even in the presence of adequate levels of iron.31Coyne D.W. Kapoian T. Suki W. et al.the DRIVE Study GroupFerric gluconate is highly efficacious in anemic hemodialysis patients with high serum ferritin and low transferrin saturation: results of the Dialysis Patients’ Response to IV Iron with Elevated Ferritin (DRIVE) Study.J Am Soc Nephrol. 2007; 18: 975-984Crossref PubMed Scopus (339) Google Scholar High serum levels of hepcidin have been demonstrated in patients with kidney disease. In a study of CKD patients, bioactive serum hepcidin using a novel competitive ELISA was used to accurately measure hepcidin.32Zaritsky J. Young B. Wang H.J. et al.Hepcidin—a potential novel biomarker for iron status in chronic kidney disease.Clin J Am Soc Nephrol. 2009; 4: 1051-1056Crossref PubMed Scopus (254) Google Scholar Hepcidin levels were significantly elevated compared with respective age controls as tested by analysis of variance. Additionally, each quartile of CKD patients had significantly different hepcidin levels. In this study, hepcidin levels were shown to be predicted by ferritin, C-reactive protein, and stage of kidney disease. A variety of laboratory tests have been used to assess iron deficiency in patients with anemia. These include ferritin, Tf saturation, percent of hypochromic red cells, and soluble TfR. However, current methods of assessing adequacy of iron stores and iron availability to the erythron are grossly inadequate, especially in the presence of inflammation, and are unable to guide the clinician to formulate a strategy to treat anemia. Tf saturation, soluble TfR, and ferritin levels do not provide accurate insight. The association of hepcidin levels with the stage of CKD and ferritin and its inverse relationship with ESA therapy support the possibility that hepcidin could be a biomarker of iron status.33Ashby D.R. Gale D.P. Busbridge M. et al.Plasma hepcidin levels are elevated but responsive to erythropoietin therapy in renal disease.Kidney Int. 2009; 75: 976-981Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar However, there is significant intrapatient variability in hepcidin level measurements. Additionally, hepcidin is dependent on kidney function for its excretion, and the levels increase with the presence of inflammation and are not necessarily different in patients sensitive or resistant to ESA.34Kato A. Tsuji T. Luo J. et al.Association of prohepcidin and hepcidin-25 with erythropoietin response and ferritin in hemodialysis patients.Am J Nephrol. 2008; 28: 115-121Crossref Scopus (90) Google Scholar Considering these factors along with nonharmonization of different hepcidin assays, the hepcidin levels cannot be considered a biomarker of iron status or a predictor of ESA response at this time. The availability, accuracy, interpretation, and clinical implications of hepcidin assays have been controversial. One study tested 11 different assays (5 mass spectrometry-based and 6 immunochemical-based) to quantify native individual plasma samples (n = 32) and native plasma pools (n = 8) to assess analytical performance and current and achievable equivalence.35van der Vorm L.N. Hendriks J.C.M. Laarakkers C.M. et al.Toward worldwide hepcidin assay harmonization: identification of a commutable secondary reference material.Clin Chem. 2016; 62: 993-1001Google Scholar Absolute hepcidin values and reproducibility (intrameasurement procedure coefficient of variation 2.9%–8.7%) differed substantially between measurement procedures, but all were linear and correlated well. The current equivalence (intermeasurement procedure coefficient of variation 28.6%) between the methods was mainly attributable to differences in calibration and thus could be improved by harmonization with a common calibrator. Antibody-based immunoassays and mass spectrometric assays have been available to measure hepcidin, and measurements of bioactive serum hepcidin using a novel competitive ELISA are now available and have shown clinical correlation. Due to the decreased hepcidin clearance in the presence of kidney disease, serum measurements of hepcidin might be more accurate than urinary hepcidin measurements. It is important to remember that hepcidin has a biological variation (women have a lower level of hepcidin than men) and a diurnal variation (levels are lower in the morning and higher in the afternoon) (Table 1).Table 1Hepcidin Levels in Health and DiseaseSerum hepcidin levelLowHighGenderWomenMenDiurnal variationMorningAfternoonDisease statesIron defi