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7252019 The Iron Cycle in Chronic Kidney Disease (CKD)
The iron cycle in chronic kidney disease (CKD) from geneticsand experimental models to CKD patients
Kimberly Zumbrennen-Bullough and Jodie L Babitt
Program in Anemia Signaling Research Division of Nephrology Program in Membrane Biology Center for Systems Biology Massachusetts
General Hospital Harvard Medical School Boston MA USA
Correspondence and offprint requests to Jodie L Babitt E-mail babittjodiemghharvardedu
A B S T R A C T
Iron is essential for most living organisms but iron excess canbe toxic Cellular and systemic iron balance is therefore tightly controlled Iron homeostasis is dysregulated in chronic kidney disease (CKD) and contributes to the anemia that is prevalentin this patient population Iron supplementation is one cor-nerstone of anemia management in CKD patients but has notbeen rigorously studied in large prospective randomized con-trolled trials This review highlights important advances fromgenetic studies and animal models that have provided key in-sights into the molecular mechanisms governing iron homeo-stasis and its disturbance in CKD and summarizes how these1047297ndings may yield advances in the care of this patient popu-lation
As a transition metal that can donate and accept electronsiron has a critical role in fundamental biological processes in-cluding oxygen and electron transport cellular respiration andDNA synthesis However excess iron can lead to the pro-duction of toxic free radicals and cell death Disturbances of iron homeostasis lead to many common diseases such asanemia and the iron overload disorder hemochromatosis thatin aggregate affect over 1 billion people worldwide [1] Iron istherefore tightly controlled via a network of proteins involvedin the import storage export and transport of iron at bothcellular and systemic levels
Humans have a daily requirement of sim25 mg of ironnearly 80 of which is used for erythropoiesis [1] A smallfraction of this iron is provided by dietary absorption (sim1ndash2mg) while the majority is provided by recycling iron from se-nescent erythrocytes via macrophages in liver spleen andbone marrow The circulating pool of iron contains only sim10 (sim3 mg) of the daily requirement for erythropoiesisand therefore must be turned over every 2ndash3 h Iron loss is anunregulated process that occurs primarily through cell shed-ding and blood loss (Figure 1)
D I E T A R Y I R O N A B S O R P T I O N
Dietary iron absorption occurs primarily in the duodenum(Figure 2) Dietary iron exists in both heme and non-hemeforms but the molecular mechanisms underlying heme ab-sorption are poorly understood The liberation of non-hemeiron from food and its solubilization is aided by the acidic pHof the stomach [2] Soluble iron is reduced from the ferric (Fe3+) to the ferrous form (Fe2+) in a process that is thought toinvolve ferrireductases located on the intestinal apical cell
membrane and ascorbic acid [3] One candidate ferrireductaseis DCYTB [3] but Dcytb null mice do not appear to have a sig-ni1047297cant iron phenotype suggesting that additional ferrireduc-tases may play a redundant role in iron reduction [4] Ferrousiron is then transported across the apical surface of the duode-nal enterocyte via divalent metal transporter 1 (DMT1)mutations of which lead to iron de1047297ciency anemia [5ndash7] AH+Fe2+ symporter DMT1-mediated iron uptake is also aidedby an acidic microenvironment [6] Although the mechanismof heme uptake by the enterocyte remains obscure it has beensuggested that subsequent intracellular metabolism by heme
copy The Author 2013 Published by Oxford University Press onbehalf of ERA-EDTA All rights reserved 263
7252019 The Iron Cycle in Chronic Kidney Disease (CKD)
oxygenase 1 releases iron into a common pathway shared withnon-heme iron [8]
Once taken up into duodenal enterocytes iron is either uti-lized by the cell stored or exported across the basolateralmembrane into circulation for systemic use Iron is stored inan inactive Fe3+ form in ferritin a multimeric protein com-
prising 24 light and heavy chain subunits surrounding a coreof up to 4500 iron atoms [1] In the absence of export acrossthe basolateral membrane this stored iron is lost as entero-cytes are sloughed off into the gut lumen every few days
Iron export across the enterocyte basolateral membraneinto circulation is mediated by ferroportin Importantly ferro-portin is also expressed in iron recycling macrophages andhepatocytes and is the only known mammalian iron exportprotein responsible for iron entry into the bloodstream [9ndash11]Iron export by ferroportin is coupled with ferroxidases includ-ing hephaestin in the intestine and ceruloplasmin that convert
Fe
2+
back to Fe
3+
and facilitate iron loading onto the plasmairon carrying protein transferrin (Tf [12ndash15])
T H E H E P C I D I NndashF E R R O P O R T I N A X I S
R E G U L A T E S S Y S T E M I C I R O N B A L A N C E
Ferroportin and its ligand hepcidin are key regulators of sys-temic iron balance coordinating communication betweentissues and cells that acquire store and utilize iron Discoveredin 2000ndash01 hepcidin is a 25 amino acid peptide hormone pri-marily secreted by the liver that resembles other proteins in-
volved in innate immunity [16ndash
18] Hepcidin was soonrecognized to have an important role in iron homeostasisregulation since hepcidin null mice and human patients withhepcidin mutations develop a severe juvenile-onset form of hemochromatosis [19ndash21] In contrast hepcidin transgenicmice and human patients with hepcidin-expressing adenomasdevelop profound iron de1047297ciency anemia [22 23] In 2004 itwas demonstrated that ferroportin was the receptor for hepci-din and that hepcidin binding caused ferroportin to be inter-nalized and degraded [24] The hepcidinndashferroportin axistherefore controls iron entry into circulation from dietary
F I G U R E 2 Enterocyte iron uptake Dietary iron absorption occurs via the reduction of ferric (Fe3+) iron to ferrous (Fe2+) iron by ferrire-ductases such as DCYTB Ferrous iron is then transported across theapical membrane of duodenal enterocytes by the symporter DMT1
Heme is also an important source of dietary iron although the mech-anism for heme uptake is unclear Heme oxygenase 1 (HO1) isthought to facilitate the degradation of heme into iron biliverdin andcarbon monoxide Cytoplasmic iron can be stored by the ferritincomplex utilized by various molecular enzymes or exported into thebloodstream by ferroportin (FPN) The multicopper ferroxidase he-phaestin (HEPH) works in conjunction with ferroportin to facilitateiron export coupled with oxidization of Fe2+ to Fe3+ and loading ontoTf
F I G U R E 1 Systemic iron regulation Iron is absorbed by the duode-
num where it is released into the circulation via the iron exporter fer-roportin to be loaded onto transferrin (Tf) The majority of iron isutilized by red blood cells (RBCs) for the synthesis of the hemoglobinrequiring sim25 mg of iron per day The daily requirements for intesti-nal iron uptake are only 1ndash2 mg per day due to ef 1047297cient recycling of iron from RBCs Iron recycling is performed primarily by reticuloen-dothelial macrophages which phagocytize senescent RBCs and thenexport iron via ferroportin back into the circulating pool of Tf-boundiron Excess iron is also stored within hepatocytes Hepcidin regulatessystemic iron balance by inducing ferroportin degradation to inhibitiron absorption from the duodenum and iron release from macro-phage and hepatocyte stores Hepcidin production in the liver isstimulated by iron and in1047298ammation to limit iron availability whilehepcidin production is inhibited by iron de1047297ciency anemia and
hypoxia to increase iron availability Several other growth factors andsteroid hormones have recently been demonstrated to suppress hepci-din expression in the liver including EGF HGF testosterone andestrogen
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7252019 The Iron Cycle in Chronic Kidney Disease (CKD)
sources iron recycling macrophages and hepatocyte stores(Figure 1)
Although the hepcidinndashferroportin axis has a central role inregulating body iron balance there are many additional levelsof regulation For example the enterocyte exerts local controlover iron absorption through the regulation of proteins in- volved in iron transport (DMT1 and ferroportin) and seques-tration (ferritin) via both transcriptional and post-transcriptional mechanisms involving hypoxia inducible
factors (particularly HIF-2α) and iron regulatory proteins (re- viewed in ref [25])
H E P C I D I N R E G U L A T I O N
Hepcidin expression in the liver is regulated by a number of factors (Figure 1) Iron increases hepcidin expression as ahomeostatic mechanism to limit further iron entry into thebloodstream [17 26 27] In1047298ammation also stimulates hepci-din expression [17 26ndash30] which is hypothesized to functionas a protective mechanism to sequester iron from infectiousorganisms However in chronic in1047298ammatory states this
results in macrophage iron sequestration hypoferremia andiron restricted erythropoiesis that contributes to anemia of chronic disease [31] Iron de1047297ciency hypoxia and anemiainhibit hepcidin expression to increase iron availability for ery-thropoiesis [26] Recently several growth factors steroid hor-mones and other endocrine signals have also been identi1047297ed tohave a role in hepcidin regulation [32ndash36]
Hepcidin regulation by iron
Key insights into the iron-mediated hepcidin regulatory pathways came from studying the genetic iron overload dis-order hereditary hemochromatosis This is a heterogeneousdisorder caused by mutations in any of several genes that ulti-mately result in impaired regulation of the hepcidinndashferropor-tin axis leading to increased dietary iron absorption increasediron release from macrophage stores progressive tissue irondeposition and consequent multiorgan damage and disease[37] Hereditary hemochromatosis can be caused by mutationsin hepcidin itself mutations in ferroportin that interfere withhepcidin binding or hepcidin-mediated internalization ormutations in one of three other genes that are involved in theiron-mediated regulation of hepcidin expression hemojuvelin(HJV also known as HFE2) HFE and transferrin receptor 2(TFR2) [37] Among these genes HJV has the most criticalrole in hepcidin regulation since HJV mutations lead to the
more severe juvenile onset form of hemochromatosis that issimilar to the phenotype seen with mutations in hepcidin itself [21 38]
HJV functions as a co-receptor for the bone morphogeneticprotein (BMP)-SMAD signaling pathway [39] which iscentral to hepcidin transcriptional regulation in response toiron [40 41] (Figure 3) A subfamily of the transforming growth factor beta (TGF-β) superfamily of signaling mol-ecules BMPs have an important role in a number of biologicfunctions particularly during development [42] Moreoverthere is redundancy in the system with a number of BMP
ligands and several BMP type I and type II receptors that canlead to the same intracellular SMAD signaling cascade [42]Nevertheless HJV mediates a crucial and unique function of BMP-SMAD signaling in the liver to regulate hepcidinexpression and systemic iron balance since mice and patientswith HJV mutations have hepcidin de1047297ciency and hemochro-matosis but no other obvious phenotype [38 43 44] It ishypothesized that HJV expression sensitizes hepatocytes torespond to low levels of BMP ligand which would not other-
wise generate a response in the absence of the co-receptor[39] By enhancing the af 1047297nity of the binding interaction HJVmay also help cells to selectively respond to a certain subset of BMP ligands using a certain subset of BMP type I and type IIreceptors that are required to speci1047297cally regulate hepcidin inliver cells in particular the ligand BMP6 [45ndash47] the BMPtype I receptors ALK3 and ALK2 [48 49] and the BMP typeII receptor ACTRIIA [48] (Figure 3)
HJV may also connect the BMP-SMAD signaling responseto molecules involved in iron sensing but the molecularmechanisms for this remain to be fully elucidated It has beenhypothesized that HFE and TFR2 sense circulating iron levelsin the form of iron-bound transferrin since TFR2 can bind to
transferrin [50 51] and HFE competes for transferrin binding to transferrin receptor 1 (TFR1) [52ndash55] (Figure 3) There issome evidence from in vitro overexpression systems that HFETFR2 and HJV can interact with each other [56ndash58] but it isuncertain whether this occurs in vivo HFE and TFR2 doappear to intersect with the BMP-SMAD pathway at somelevel since mice and human patients with HFE and TFR2mutations exhibit impairment in liver SMAD signaling [59ndash64] However the functions of HFE and TFR2 in regulating hepcidin are not entirely overlapping given the differential se- verity of the iron overload phenotype in mice and patientswith HFE mutations alone TFR2 mutations alone and doubleHFETFR2 mutations [61 64 65]
Hepcidin regulation by in1047298ammation
Another well-characterized hepcidin regulatory pathway isthe IL6-JAK-STAT3 pathway which mediates at least in parthepcidin transcriptional induction in response to in1047298am-mation [27 28 66ndash68] (Figure 3) Other mediators of in1047298am-mation and infection including IL-22 type I interferon tumornecrosis factor alpha and endoplasmic reticulum stress havealso been implicated in hepcidin regulation [27 29 30]Notably liver SMAD signaling is also induced in many in1047298ammatory models [69 70] and hepcidin induction by in1047298ammation is reduced when the BMP-SMAD signaling
pathway is inhibited indicating crosstalk between these regu-latory pathways [41 71ndash75] Hypothesized mechanisms forthis crosstalk are an interaction between STAT3 and SMADsat the level of the hepcidin promoter and the TGF-β super-family member activin B [70 73] (Figure 3)
Hepcidin regulation by erythropoietic activity and
hypoxia
Increased erythropoietic activity for example in response toanemia or erythropoiesis-stimulating agent (ESA) adminis-tration is a potent suppressor of hepcidin expression This
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T h e i r o n c y c l e i n C K D265
7252019 The Iron Cycle in Chronic Kidney Disease (CKD)
appears to be mediated by a secreted factor from proliferating red blood cell (RBC) precursors in the bone marrow sinceinhibition of erythropoiesis by chemotherapy irradiation oran erythropoietin blocking antibody prevents hepcidin sup-pression by anemia or ESAs [76 77] TGF-βBMP superfamily modulators GDF15 and TWSG1 have been proposed tomediate hepcidin suppression in iron loading anemias with in-effective erythropoiesis such as β-thalassemia [78 79] butmay not mediate hepcidin suppression in other contexts [8081] Recent data in genetic mouse models suggest thathypoxia-mediated hepcidin suppression occurs indirectly by
stimulating erythropoiesis [82 83] although other mechan-isms for hypoxia-mediated hepcidin suppression have alsobeen proposed [84 85]
Hepcidin regulation by growth factors steroid hormones
and other endocrine factors
Recently several growth factors and steroid hormones havebeen demonstrated to suppress hepcidin expression in theliver including hepatocyte growth factor (HGF 32) epidermalgrowth factor (EGF 32) estrogen [33 34] and testosterone[35 36] (Figure 1) HGF EGF and testosterone are proposed
to intersect with BMP-SMAD signaling in the regulation of hepcidin [32 35 36] while estrogen is suggested to act via anestrogen response element in the hepcidin promoter [33 34]The effects of steroid hormones on hepcidin regulation may help explain gender differences in iron homeostasis that havebeen observed [86] Recent data presented in abstract formsuggests that vitamin D administration may also suppress cir-culating hepcidin levels and that vitamin D inhibits hepcidintranscription in mononuclear cells [87] In contrast prolongedfasting [88] and glucose [89] have been shown to increase cir-culating hepcidin levels and the glucose-mediated hepcidin
increase was associated with a decrease in serum iron levels[89] The mechanism of hepcidin regulation by glucose andfasting is still undetermined but interestingly while glucosedid not affect hepcidin secretion in hepatoma-derived cell cul-tures it did induce hepcidin secretion by insulinoma-derivedcell cultures [89] These 1047297ndings suggest intriguing linksbetween iron metabolism and multiple endocrine systems andraise the possibility that hepcidin production in non-hepatictissues may functionally contribute to circulating hepcidinlevels and systemic iron balance in some contexts althoughthis will need to be validated by future studies
F I G U R E 3 Molecular regulation of hepcidin by iron and in1047298ammation Increased systemic iron stimulates the production of the ligand bonemorphogenetic protein 6 (BMP6) which binds to the BMP Type I (ALK2ALK3) and II (ACTRIIA) receptors and the co-receptor HJV tostimulate phosphorylation of the SMAD158 intracellular signaling molecules Phosphorylated SMAD 158 binds to SMAD4 and translocatesto the nuclease to activate hepcidin transcription The mechanism by which the hemochromatosis protein HFE andor TFR2 regulate hepcidinexpression is unknown but appears to involve an interaction with the BMP-SMAD signaling pathway It has been proposed that an interactionbetween HFE and TFR1 is reduced under high iron conditions due to competitive binding of holotransferrin to TFR1 Displaced HFE couldthen associate with TFR2 and possibly the HJV-BMP receptor complex to regulate hepcidin In1047298ammation also stimulates hepcidin productionin part via a canonical janus kinase (JAK)signal transducer and activator of transcription (STAT) pathway in which in1047298ammation increases in-terleukin 6 (IL6) binding to the IL6-receptor (IL6R) and thereby stimulating phosphorylation of JAKs and STAT3 Phosphorylated-STAT3homodimers translocate to the nuclease and bind to the hepcidin promoter to stimulate hepcidin expression Other mediators of in1047298ammationand infection can also regulate hepcidin expression in this context (not shown) A mechanism of crosstalk between in1047298ammatory signals andBMP signaling has been proposed in which in1047298ammation induces activin B which binds to BMP receptors to stimulate SMAD158 phosphoryl-ation SMADs and STAT3 may also interact at the level of the hepcidin promoter
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7252019 The Iron Cycle in Chronic Kidney Disease (CKD)
D I S O R D E R E D I R O N B A L A N C E I N C H R O N I C
K I D N E Y D I S E A S E
Iron de1047297ciency-limiting erythropoiesis is an important causeof anemia and resistance to ESAs in chronic kidney disease(CKD) patients [90ndash93] Iron administration is therefore a vital part of CKD anemia management Moreover the use of iron agents appears to be increasing [94] in the wake of recentlarge clinical trials that raised safety concerns for ESAs [95ndash97] and clinical practice guidelines that have liberalized rec-ommendations regarding iron use in CKD patients [98]However current diagnostic tests to evaluate iron status arelimited the targets of iron therapy are largely opinion basedand the safety of iron has not been rigorously evaluated inlarge prospective randomized controlled trials in this patientpopulation [98] Our increasing understanding about the mol-ecular mechanisms governing iron homeostasis regulation andits disturbance in CKD may lead to improved diagnostic andtherapeutic strategies for managing this patient population
The causes of iron de1047297ciency in CKD patients are multifac-torial (Figure 4) Some patients have true iron de1047297ciency
characterized by decreases in both circulating iron levels andtotal body iron stores Other patients have functional ironde1047297ciency characterized by a decrease in circulating iron thatlimits erythropoiesis which can occur even in the context of normal or adequate body iron stores A combination of thesefeatures may also be present Factors predisposing CKDpatients to iron de1047297ciency include increased blood loss in-creased iron utilization from ESA therapy impaired dietary iron absorption and impaired iron release from body storagesites [84] (Figure 4) Blood loss can arise from frequent
phlebotomy blood trapping in the dialysis apparatus and gas-trointestinal or other bleeding as a result of uremic plateletdysfunction Dietary iron absorption can be impaired by antacid medications or phosphate binders that block entero-cyte iron uptake It is now apparent that hepcidin excess alsocontributes to the impaired dietary iron absorption and im-paired iron release from body storage sites in CKD patients by downregulating ferroportin expression to block iron entry intothe circulation [99ndash101] Mechanisms leading to hepcidin
excess in these patients are thought to include reduced renalclearance of this small peptide hormone and increasedin1047298ammatory-mediated hepcidin transcription caused by thedialysis procedure itself andor the underlying disease process[84] Hepcidin levels in CKD patients are also in1047298uenced by iron and ESA administration (Figure 4) [84 100]
I R O N S T A T U S E VA L U A T I O N I N C K D
Current Kidney Disease Improving Global Outcomes clinicalpractice guidelines regarding the use of iron agents to manageanemia of CKD [98] revolve around two diagnostic tests
serum Tf saturation and serum ferritin levels Serum Tf satur-ation measures circulating iron that is immediately availablefor erythropoiesis while serum ferritin serves as a surrogatemeasure of body iron levels A major limitation of these diag-nostic tools is that they are not reliable for estimating body iron stores or predicting which patients will respond well toiron therapy [98 102ndash105] Indeed ferritin is also an acutephase reactant and so must be interpreted with caution in thesetting of in1047298ammation While there is general agreement thatpatients with total body iron de1047297ciency as indicated by low Tf saturation and low ferritin should be treated with iron therapythere are limited data on how to manage patients as ferritinlevels rise [98 106] There is therefore a need for new diagnos-tic tests to understand the iron status of CKD patients and tohelp determine which patients will bene1047297t from iron therapy
A L T E R N AT I V E A N D N O V E L D I A G N O S T I C
T O O L S F O R I R O N A N D A N E M I A
M A N A G E M E N T I N C K D
Reticulocyte hemoglobin content
By evaluating the hemoglobin content of reticulocyteswhich are early RBC forms reticulocyte hemoglobin content(CHr) provides an indication of iron availability for erythro-
poiesis within the last few days Several studies have suggestedthat CHr may also be helpful to predict responsiveness to ironin hemodialysis patients [107ndash112] although it is less wellstudied and may not be as widely available as Tf sat and ferri-tin
Percentage of hypochromic RBCs
Percentage of hypochromic RBCs measures the concen-tration of hemoglobin in RBCs which re1047298ects both the absol-ute amount of hemoglobin and the RBC size This test has alsoshown utility in predicting iron responsiveness in
F I G U R E 4 Disordered iron balance in CKD Chronic in1047298am-mation and reduced renal clearance in patients with CKD lead to in-creased levels of hepcidin which reduces duodenal iron uptake andiron release from cellular iron stores Intestinal iron uptake is also in-hibited by medications such as phosphate binders and antacids ESAsstimulate increased iron usage for erythropoiesis while blood loss dueto frequent phlebotomy blood trapping in the dialysis apparatus andgastrointestinal bleeding further deplete the circulating iron poolIron administration stimulates hepcidin expression which can para-doxically worsen the iron restriction while ESAs have an inhibitory effect on hepcidin expression
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7252019 The Iron Cycle in Chronic Kidney Disease (CKD)
hemodialysis patients [110 113] but can be impacted by blood storage time which leads to arti1047297cial RBC expansionthereby limiting utility in dialysis centers that use national lab-oratories [114]
Soluble transferrin receptor
Transferrin receptor 1 mediates uptake of iron into devel-oping RBCs Its expression and release into circulation assoluble transferrin receptor (sTFR) is increased in the setting
of iron de1047297ciency and increased erythroid activity Althoughthe literature on sTFR is limited a few studies have suggestedthat sTFR may be helpful to predict iron responsiveness [110113] However interpretation of this test in patients on ESAsis complicated by the fact that erythropoiesis itself increasessTFR levels [115] The use of this assay is also limited by lack of widespread availability and cost
Hepcidin
The understanding that hepcidin excess contributes to dis-ordered iron homeostasis in CKD patients has garnered inter-est in measuring hepcidin levels as a marker of iron statusiron responsiveness andor ESA responsiveness in CKD
patients There are two general types of assays now available tothe research community to measure circulating hepcidinlevels immunologic and mass spectrometry-based assaysBoth types of assays have their inherent strengths and weak-nesses and give an overall large variation in the absolute valuesof hepcidin levels but do show overall good correlation in rela-tive hepcidin levels with each other [116] Older assays thatalso recognize the precursor form of hepcidin (prohepcidin)are not useful because prohepcidin levels do not correlate withhepcidin biological activity [117 118] Using the more recentassays many studies have now con1047297rmed that circulating hep-cidin levels are increased in CKD patients with the highestlevels in patients on hemodialysis [99ndash101] Hepcidin levels inCKD patients have the strongest correlation with serum ferri-tin [100 101 119] but are also in1047298uenced at least in somestudies by in1047298ammation iron administration estimated glo-merular 1047297ltration rate dialysis clearance ESA dose and hemo-globin [100 101 119ndash121] One important limitation for theuse of hepcidin levels as a diagnostic tool in CKD patients isthe large intra-individual variability of both immunologic andmass spectrometry-based assays [122 123] Notably hepcidinlevels have not been shown to consistently predict responsive-ness or resistance to iron therapy or ESAs [ 120 124] Thus forthe time being there is no convincing evidence that hepcidinassays offer any advantage or additional information com-
pared with currently available diagnostic tests with regard toCKD iron and anemia management but this remains an areaof active investigation
Soluble HJV
Recent studies have explored the utility of measuring circu-lating levels of endogenous soluble HJV (sHJV) as a measureof iron status in human patients both without and with CKD[125ndash129] sHJV release from cells can be mediated by theproprotein convertase furin the transmembrane serine pro-tease TMPRSS6 and phospholipase C [130ndash134] and sHJV
has been detected in the conditioned media of transfected cellsand in the bloodstream of animals and humans [125ndash130135ndash137] While cell-surface GPI-anchored HJV functions asa BMP co-receptor to stimulate hepcidin expression (Figure 3)[39] sHJV can function as an inhibitor of BMP signaling andhepcidin expression presumably by sequestering BMP ligandsfrom interacting with cell surface signaling receptors [45 72135] Interestingly some studies have suggested that sHJVmay be decreased by iron treatment and increased by iron
de1047297ciency [125 130 135ndash137] suggesting that (i) sHJV couldbe useful as a diagnostic tool to indicate iron status and (ii) thegeneration sHJV could have a functional role to inhibit hepci-din expression in the context of iron de1047297ciency However oneimportant concern regarding these early human studies quan-titating sHJV levels is assay validity Indeed one commercialELISA assay used in studies focusing on CKD patients [128129] has subsequently been shown not to recognize HJV[138] Future studies will be needed using well-validated assaysand larger patient populations to determine if sHJV couldhave value as a diagnostic marker to guide iron therapy inCKD patients
Other markers
The putative role of GDF15 hepcidin regulation by erythro-poietic drive has generated interest in investigating this mol-ecule as a novel diagnostic tool for iron and anemiamanagement in CKD patients [139] However currently avail-able clinical data are very limited [139] Moreover while onestudy suggested that GDF15 may be increased by ironde1047297ciency [140] this was not robustly supported by anotherstudy [141] and GDF15 levels may also be in1047298uenced by in1047298ammation [141 142] malnutrition [142] and kidney disease [142] which may complicate its usefulness in thissetting
I R O N T H E R A P Y F O R C K D P A T I E N T S
Iron administration remains one of the cornerstones of anemia management in CKD patients to improve hemoglobinlevels and ESA responsiveness [98] Iron supplementation iscurrently given in two general forms oral or parenteral Oraliron supplementation is the easiest and cheapest Howeveroral iron agents can have gastrointestinal side effects that limitadherence due to the formation of local reactive oxygenspecies and oxidative damage in the gut mucosa [143] More-over several studies have suggested that oral iron is less effec-
tive than parenteral iron particularly in hemodialysis patientsfor improving or preventing iron de1047297ciency ameliorating anemia or reducing ESA dose [98 144ndash146] The limited ef-fectiveness of oral iron supplements in this patient populationis likely due to medications such as antacids and phosphatebinders that inhibit iron entry into duodenal enterocytes andhepcidin excess that decreases ferroportin expression to limitiron release from duodenal enterocytes into the bloodstream(Figure 4)
There are several intravenous (IV) iron preparations thatcan be used to treat iron-restricted erythropoiesis in CKD
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7252019 The Iron Cycle in Chronic Kidney Disease (CKD)
patients including iron dextrans iron sucrose ferric gluco-nate ferric carboxymaltose iron isomaltoside 1000 and feru-moxytol These preparations are generally comprising an ironcore shielded by a carbohydrate shell with different molecularweights and physiochemical properties yielding differentialdegradation kinetics and ability to release lsquofreersquo iron into thecirculation [143] This determines the maximal single dose foreach preparation with the newer higher molecular weightmore stable complexes enabling larger doses over shorter time
frames [143] Iron dextrans ( particularly high molecularweight dextrans) have been limited by dextran-induced ana-phylactic reactions in sim06ndash07 of patients [98] There issome limited data suggesting that various iron preparationsmay have different effects on markers of oxidative stress andin1047298ammation but this did not necessarily correlate with thecompoundsrsquo molecular weight stability or ability to releasefree iron into circulation [147 148] Comparative safety of these IV iron preparations in CKD patients remains largely unknown due to the lack of direct head-to-head clinical trials
Understanding the physiology of systemic iron balance andits pathophysiology in CKD and other iron disorders raisesseveral potential limitations shared by all IV iron preparations
Regardless of the iron preparation once the iron is taken upinto erythrocytes macrophages or other body storage siteshepcidin excess and ferroportin downregulation will limit theavailability of the iron for recycling and subsequent use More-over iron itself stimulates hepcidin expression and thereforecan paradoxically worsen the iron restriction (Figure 4)Additional concerns particularly with regard to repetitive ironadministration as ferritin levels rise are the potential foroxidant-mediated tissue injury from excess iron deposition asseen in iron overload disorders such as hemochromatosis Irondeposition has also been associated with the pathogenesis of many other common disorders including neurodegenerativediseases diabetes mellitus and atherosclerosis [1 149 150]Additionally withholding iron from invading pathogens is animportant function of the immune system and iron loading isassociated with worse outcomes in several infectious diseasesincluding malaria tuberculosis and HIV [151ndash153] Largeprospective randomized trials in the CKD population are long overdue to evaluate the ef 1047297cacy of repetitive IV iron adminis-tration with regard to hard clinical outcomes and long-termsafety to further characterize which patients will bene1047297t fromiron therapy and to determine treatment targets of irontherapy
N O V E L T R E A T M E N T S T R A T E G I E S F O R
I R O N - R E S T R I C T E D E R Y T H R O P O I E S I S I N
C K D P A T I E N T S
The understanding that hepcidin excess contributes to iron-re-stricted erythropoiesis in CKD patients has generated interestin developing new therapies that target the hepcidinndashferropor-tin axis to more directly address the underlying pathophysiol-ogy of this disease Such therapies would be expected toincrease iron availability from the diet and from the patients
own body iron stores and are a particularly attractive optionfor patients with higher ferritin levels
Several categories of hepcidinferroportin-based thera-peutics are currently in development (reviewed in [31]) Onecategory is direct hepcidin antagonists including anti-hepci-din antibodies other hepcidin-binding proteins (anticalins)hepcidin-binding spiegelmers and hepcidin siRNAs and anti-sense oligonucleotides [31] Dialysis itself also reduces hepci-din levels [121 154] but the levels quickly rebound [154]
potentially due in part to the induction of in1047298ammatory cyto-kines by the dialysis procedure as well as the high basal turn-over rate of hepcidin [155] Another category is agents thatinhibit hepcidin production by targeting either the BMP-SMAD signaling pathway or the IL6-STAT3 pathway [31]BMP-SMAD pathway inhibitors include anti-BMP6 anti-bodies sHJV linked to the constant region of IgG1 (HJVFc)small molecule BMP type I receptor antagonists (LDN-193189) and heparin (which has been shown to sequesterBMP ligands) [31 41 45 72 74 75 156] IL6-STAT3 pathway inhibitors include anti-IL6 antibodies (Siltuximab) anti-IL6receptor antibodies (Tocilizumab) JAK2 inhibitors (AG490)and STAT3 inhibitors (PpYLKTK) [31] ESAs and other
stimulators of ESA production such as prolyl hydroxylaseinhibitors also fall in this category since they inhibit hepcidinproduction A third category is ferroportin agonistsstabilizersincluding anti-ferroportin antibodies and thiol-reactive com-pounds that interfere with hepcidin binding to ferroportin aswell as agents that interfere with ferroportin internalization orpotentiate ferroportin synthesis [31] Notably many of theseagents have shown ef 1047297cacy for treating iron-restricted erythro-poiesis and anemia in animal models with anemia of chronicdisease [74 75 157ndash160] and several are currently in humanclinical trials [161ndash164] The safety and ef 1047297cacy of these agentsin human CKD patients compared with current treatmentstrategies remains to be determined
C O N C L U S I O N S
The last 13 years have yielded signi1047297cant advances in under-standing the molecular mechanisms underlying systemic ironbalance and its dysregulation in CKD patients These studieshold the promise for developing new rationally designed diag-nostic and therapeutic tools to improve anemia managementin CKD patients Novel therapies targeting hepcidin haveshown particular promise and several have already enteredhuman clinical trials More research is needed to better under-
stand the ef 1047297cacy long-term safety and targets of current irontherapies as well as novel hepcidin-lowering approaches inlarge prospective randomized controlled trials
A C K N O W L E D G E M E N T S
JLB was supported in part by NIH grant RO1-DK087727and a Howard Goodman Fellowship Award from the Massa-chusetts General Hospital
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C O N F L I C T O F I N T E R E S T S T A T E M E N T
JLB has ownership interest in a start-up company FerruMax Pharmaceuticals which has licensed technology from theMassachusetts General Hospital based on the work cited hereand in prior publications
R E F E R E N C E S
1 Hentze MW Muckenthaler MU Andrews NC Balancing acts molecular
control of mammalian iron metabolism Cell 2004 117 285ndash297
2 Kovac S Anderson GJ Baldwin GS Gastrins iron homeostasis and col-
orectal cancer Biochim Biophys Acta 2011 1813 889ndash895
3 McKie AT Barrow D Latunde-Dada GO et al An iron-regulated ferric
reductase associated with the absorption of dietary iron Science 2001
291 1755ndash1759
4 Gunshin H Starr CN Direnzo C et al Cybrd1(duodenal cytochrome b)
is not necessary for dietary iron absorption in mice Blood 2005 16 16
5 Fleming MD Trenor CC III Su MA et al Microcytic anaemia mice have
a mutation in Nramp2 a candidate iron transporter gene Nat Genet
1997 16 383ndash386
6 Gunshin H Mackenzie B Berger UV et al Cloning and characterization
of a mammalian proton-ion transporter Nature 1997 388 482ndash488
7 Gunshin H Fujiwara Y Custodio AO et al Slc11a2 is required for intesti-
nal iron absorption and erythropoiesis but dispensable in placenta and
liver J Clin Invest 2005 115 1258ndash1266
8 Weintraub LR Weinstein MB Huser HJ et al Absorption of hemoglobin
iron the role of a heme-splitting substance in the intestinal mucosa J
Clin Invest 1968 47 531ndash539
9 Donovan A Brownlie A Zhou Y et al Positional cloning of zebra1047297sh fer-
roportin1 identi1047297es a conserved vertebrate iron exporter Nature 2000
403 776ndash781
10 Abboud S Haile DJ A novel mammalian iron-regulated protein involved
in intracellular iron metabolism J Biol Chem 2000 275 19906ndash19912
11 McKie AT Marciani P Rolfs A et al A novel duodenal iron-regulated
transporter IREG1 implicated in the basolateral transfer of iron to the
circulation Mol Cell 2000 5 299ndash309
12 Osaki S Johnson DA Mobilization of liver iron by ferroxidase (cerulo-plasmin) J Biol Chem 1969 244 5757ndash5758
13 Osaki S Johnson DA Frieden E The possible signi1047297cance of the ferrous
oxidase activity of ceruloplasmin in normal human serum J Biol Chem
1966 241 2746ndash2751
14 Roeser HP Lee GR Nacht S et al The role of ceruloplasmin in iron
metabolism J Clin Invest 1970 49 2408ndash2417
15 Vulpe CD Kuo YM Murphy TL et al Hephaestin a ceruloplasmin
homologue implicated in intestinal iron transport is defective in the sla
mouse Nat Genet 1999 21 195ndash199
16 Krause A Neitz S Maumlgert HJ et al LEAP-1 a novel highly disul1047297de-
bonded human peptide exhibits antimicrobial activity FEBS Lett 2000
480 147ndash150
17 Pigeon C Ilyin G Courselaud B et al A new mouse liver-speci1047297c gene
encoding a protein homologous to human antimicrobial peptide hepci-
din is overexpressed during iron overload J Biol Chem 2001 2767811ndash7819
18 Park CH Valore EV Waring AJ et al Hepcidin a urinary antimicrobial
peptide synthesized in the liver J Biol Chem 2001 276 7806ndash7810
19 Nicolas G Bennoun M Devaux I et al Lack of hepcidin gene expression
and severe tissue iron overload in upstream stimulatory factor 2 (USF2)
knockout mice Proc Natl Acad Sci USA 2001 98 8780ndash8785
20 Lesbordes-Brion JC Viatte L Bennoun M et al Targeted disruption of
the hepcidin 1 gene results in severe hemochromatosis Blood 2006 108
1402ndash1405
21 Roetto A Papanikolaou G Politou M et al Mutant antimicrobial peptide
hepcidin is associated with severe juvenile hemochromatosis Nat Genet
2003 33 21ndash22
22 Nicolas G Bennoun M Porteu A et al Severe iron de1047297ciency anemia in
C o p y r i g h t o f N e p h r o l o g y D i a l y s i s T r a n s p l a n t a t i o n i s t h e p r o p e r t y o f O x f o r d U n i v e r s i t y P r e s s
U S A a n d i t s c o n t e n t m a y n o t b e c o p i e d o r e m a i l e d t o m u l t i p l e s i t e s o r p o s t e d t o a l i s t s e r v
w i t h o u t t h e c o p y r i g h t h o l d e r s e x p r e s s w r i t t e n p e r m i s s i o n H o w e v e r u s e r s m a y p r i n t
d o w n l o a d o r e m a i l a r t i c l e s f o r i n d i v i d u a l u s e
7252019 The Iron Cycle in Chronic Kidney Disease (CKD)
oxygenase 1 releases iron into a common pathway shared withnon-heme iron [8]
Once taken up into duodenal enterocytes iron is either uti-lized by the cell stored or exported across the basolateralmembrane into circulation for systemic use Iron is stored inan inactive Fe3+ form in ferritin a multimeric protein com-
prising 24 light and heavy chain subunits surrounding a coreof up to 4500 iron atoms [1] In the absence of export acrossthe basolateral membrane this stored iron is lost as entero-cytes are sloughed off into the gut lumen every few days
Iron export across the enterocyte basolateral membraneinto circulation is mediated by ferroportin Importantly ferro-portin is also expressed in iron recycling macrophages andhepatocytes and is the only known mammalian iron exportprotein responsible for iron entry into the bloodstream [9ndash11]Iron export by ferroportin is coupled with ferroxidases includ-ing hephaestin in the intestine and ceruloplasmin that convert
Fe
2+
back to Fe
3+
and facilitate iron loading onto the plasmairon carrying protein transferrin (Tf [12ndash15])
T H E H E P C I D I NndashF E R R O P O R T I N A X I S
R E G U L A T E S S Y S T E M I C I R O N B A L A N C E
Ferroportin and its ligand hepcidin are key regulators of sys-temic iron balance coordinating communication betweentissues and cells that acquire store and utilize iron Discoveredin 2000ndash01 hepcidin is a 25 amino acid peptide hormone pri-marily secreted by the liver that resembles other proteins in-
volved in innate immunity [16ndash
18] Hepcidin was soonrecognized to have an important role in iron homeostasisregulation since hepcidin null mice and human patients withhepcidin mutations develop a severe juvenile-onset form of hemochromatosis [19ndash21] In contrast hepcidin transgenicmice and human patients with hepcidin-expressing adenomasdevelop profound iron de1047297ciency anemia [22 23] In 2004 itwas demonstrated that ferroportin was the receptor for hepci-din and that hepcidin binding caused ferroportin to be inter-nalized and degraded [24] The hepcidinndashferroportin axistherefore controls iron entry into circulation from dietary
F I G U R E 2 Enterocyte iron uptake Dietary iron absorption occurs via the reduction of ferric (Fe3+) iron to ferrous (Fe2+) iron by ferrire-ductases such as DCYTB Ferrous iron is then transported across theapical membrane of duodenal enterocytes by the symporter DMT1
Heme is also an important source of dietary iron although the mech-anism for heme uptake is unclear Heme oxygenase 1 (HO1) isthought to facilitate the degradation of heme into iron biliverdin andcarbon monoxide Cytoplasmic iron can be stored by the ferritincomplex utilized by various molecular enzymes or exported into thebloodstream by ferroportin (FPN) The multicopper ferroxidase he-phaestin (HEPH) works in conjunction with ferroportin to facilitateiron export coupled with oxidization of Fe2+ to Fe3+ and loading ontoTf
F I G U R E 1 Systemic iron regulation Iron is absorbed by the duode-
num where it is released into the circulation via the iron exporter fer-roportin to be loaded onto transferrin (Tf) The majority of iron isutilized by red blood cells (RBCs) for the synthesis of the hemoglobinrequiring sim25 mg of iron per day The daily requirements for intesti-nal iron uptake are only 1ndash2 mg per day due to ef 1047297cient recycling of iron from RBCs Iron recycling is performed primarily by reticuloen-dothelial macrophages which phagocytize senescent RBCs and thenexport iron via ferroportin back into the circulating pool of Tf-boundiron Excess iron is also stored within hepatocytes Hepcidin regulatessystemic iron balance by inducing ferroportin degradation to inhibitiron absorption from the duodenum and iron release from macro-phage and hepatocyte stores Hepcidin production in the liver isstimulated by iron and in1047298ammation to limit iron availability whilehepcidin production is inhibited by iron de1047297ciency anemia and
hypoxia to increase iron availability Several other growth factors andsteroid hormones have recently been demonstrated to suppress hepci-din expression in the liver including EGF HGF testosterone andestrogen
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sources iron recycling macrophages and hepatocyte stores(Figure 1)
Although the hepcidinndashferroportin axis has a central role inregulating body iron balance there are many additional levelsof regulation For example the enterocyte exerts local controlover iron absorption through the regulation of proteins in- volved in iron transport (DMT1 and ferroportin) and seques-tration (ferritin) via both transcriptional and post-transcriptional mechanisms involving hypoxia inducible
factors (particularly HIF-2α) and iron regulatory proteins (re- viewed in ref [25])
H E P C I D I N R E G U L A T I O N
Hepcidin expression in the liver is regulated by a number of factors (Figure 1) Iron increases hepcidin expression as ahomeostatic mechanism to limit further iron entry into thebloodstream [17 26 27] In1047298ammation also stimulates hepci-din expression [17 26ndash30] which is hypothesized to functionas a protective mechanism to sequester iron from infectiousorganisms However in chronic in1047298ammatory states this
results in macrophage iron sequestration hypoferremia andiron restricted erythropoiesis that contributes to anemia of chronic disease [31] Iron de1047297ciency hypoxia and anemiainhibit hepcidin expression to increase iron availability for ery-thropoiesis [26] Recently several growth factors steroid hor-mones and other endocrine signals have also been identi1047297ed tohave a role in hepcidin regulation [32ndash36]
Hepcidin regulation by iron
Key insights into the iron-mediated hepcidin regulatory pathways came from studying the genetic iron overload dis-order hereditary hemochromatosis This is a heterogeneousdisorder caused by mutations in any of several genes that ulti-mately result in impaired regulation of the hepcidinndashferropor-tin axis leading to increased dietary iron absorption increasediron release from macrophage stores progressive tissue irondeposition and consequent multiorgan damage and disease[37] Hereditary hemochromatosis can be caused by mutationsin hepcidin itself mutations in ferroportin that interfere withhepcidin binding or hepcidin-mediated internalization ormutations in one of three other genes that are involved in theiron-mediated regulation of hepcidin expression hemojuvelin(HJV also known as HFE2) HFE and transferrin receptor 2(TFR2) [37] Among these genes HJV has the most criticalrole in hepcidin regulation since HJV mutations lead to the
more severe juvenile onset form of hemochromatosis that issimilar to the phenotype seen with mutations in hepcidin itself [21 38]
HJV functions as a co-receptor for the bone morphogeneticprotein (BMP)-SMAD signaling pathway [39] which iscentral to hepcidin transcriptional regulation in response toiron [40 41] (Figure 3) A subfamily of the transforming growth factor beta (TGF-β) superfamily of signaling mol-ecules BMPs have an important role in a number of biologicfunctions particularly during development [42] Moreoverthere is redundancy in the system with a number of BMP
ligands and several BMP type I and type II receptors that canlead to the same intracellular SMAD signaling cascade [42]Nevertheless HJV mediates a crucial and unique function of BMP-SMAD signaling in the liver to regulate hepcidinexpression and systemic iron balance since mice and patientswith HJV mutations have hepcidin de1047297ciency and hemochro-matosis but no other obvious phenotype [38 43 44] It ishypothesized that HJV expression sensitizes hepatocytes torespond to low levels of BMP ligand which would not other-
wise generate a response in the absence of the co-receptor[39] By enhancing the af 1047297nity of the binding interaction HJVmay also help cells to selectively respond to a certain subset of BMP ligands using a certain subset of BMP type I and type IIreceptors that are required to speci1047297cally regulate hepcidin inliver cells in particular the ligand BMP6 [45ndash47] the BMPtype I receptors ALK3 and ALK2 [48 49] and the BMP typeII receptor ACTRIIA [48] (Figure 3)
HJV may also connect the BMP-SMAD signaling responseto molecules involved in iron sensing but the molecularmechanisms for this remain to be fully elucidated It has beenhypothesized that HFE and TFR2 sense circulating iron levelsin the form of iron-bound transferrin since TFR2 can bind to
transferrin [50 51] and HFE competes for transferrin binding to transferrin receptor 1 (TFR1) [52ndash55] (Figure 3) There issome evidence from in vitro overexpression systems that HFETFR2 and HJV can interact with each other [56ndash58] but it isuncertain whether this occurs in vivo HFE and TFR2 doappear to intersect with the BMP-SMAD pathway at somelevel since mice and human patients with HFE and TFR2mutations exhibit impairment in liver SMAD signaling [59ndash64] However the functions of HFE and TFR2 in regulating hepcidin are not entirely overlapping given the differential se- verity of the iron overload phenotype in mice and patientswith HFE mutations alone TFR2 mutations alone and doubleHFETFR2 mutations [61 64 65]
Hepcidin regulation by in1047298ammation
Another well-characterized hepcidin regulatory pathway isthe IL6-JAK-STAT3 pathway which mediates at least in parthepcidin transcriptional induction in response to in1047298am-mation [27 28 66ndash68] (Figure 3) Other mediators of in1047298am-mation and infection including IL-22 type I interferon tumornecrosis factor alpha and endoplasmic reticulum stress havealso been implicated in hepcidin regulation [27 29 30]Notably liver SMAD signaling is also induced in many in1047298ammatory models [69 70] and hepcidin induction by in1047298ammation is reduced when the BMP-SMAD signaling
pathway is inhibited indicating crosstalk between these regu-latory pathways [41 71ndash75] Hypothesized mechanisms forthis crosstalk are an interaction between STAT3 and SMADsat the level of the hepcidin promoter and the TGF-β super-family member activin B [70 73] (Figure 3)
Hepcidin regulation by erythropoietic activity and
hypoxia
Increased erythropoietic activity for example in response toanemia or erythropoiesis-stimulating agent (ESA) adminis-tration is a potent suppressor of hepcidin expression This
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appears to be mediated by a secreted factor from proliferating red blood cell (RBC) precursors in the bone marrow sinceinhibition of erythropoiesis by chemotherapy irradiation oran erythropoietin blocking antibody prevents hepcidin sup-pression by anemia or ESAs [76 77] TGF-βBMP superfamily modulators GDF15 and TWSG1 have been proposed tomediate hepcidin suppression in iron loading anemias with in-effective erythropoiesis such as β-thalassemia [78 79] butmay not mediate hepcidin suppression in other contexts [8081] Recent data in genetic mouse models suggest thathypoxia-mediated hepcidin suppression occurs indirectly by
stimulating erythropoiesis [82 83] although other mechan-isms for hypoxia-mediated hepcidin suppression have alsobeen proposed [84 85]
Hepcidin regulation by growth factors steroid hormones
and other endocrine factors
Recently several growth factors and steroid hormones havebeen demonstrated to suppress hepcidin expression in theliver including hepatocyte growth factor (HGF 32) epidermalgrowth factor (EGF 32) estrogen [33 34] and testosterone[35 36] (Figure 1) HGF EGF and testosterone are proposed
to intersect with BMP-SMAD signaling in the regulation of hepcidin [32 35 36] while estrogen is suggested to act via anestrogen response element in the hepcidin promoter [33 34]The effects of steroid hormones on hepcidin regulation may help explain gender differences in iron homeostasis that havebeen observed [86] Recent data presented in abstract formsuggests that vitamin D administration may also suppress cir-culating hepcidin levels and that vitamin D inhibits hepcidintranscription in mononuclear cells [87] In contrast prolongedfasting [88] and glucose [89] have been shown to increase cir-culating hepcidin levels and the glucose-mediated hepcidin
increase was associated with a decrease in serum iron levels[89] The mechanism of hepcidin regulation by glucose andfasting is still undetermined but interestingly while glucosedid not affect hepcidin secretion in hepatoma-derived cell cul-tures it did induce hepcidin secretion by insulinoma-derivedcell cultures [89] These 1047297ndings suggest intriguing linksbetween iron metabolism and multiple endocrine systems andraise the possibility that hepcidin production in non-hepatictissues may functionally contribute to circulating hepcidinlevels and systemic iron balance in some contexts althoughthis will need to be validated by future studies
F I G U R E 3 Molecular regulation of hepcidin by iron and in1047298ammation Increased systemic iron stimulates the production of the ligand bonemorphogenetic protein 6 (BMP6) which binds to the BMP Type I (ALK2ALK3) and II (ACTRIIA) receptors and the co-receptor HJV tostimulate phosphorylation of the SMAD158 intracellular signaling molecules Phosphorylated SMAD 158 binds to SMAD4 and translocatesto the nuclease to activate hepcidin transcription The mechanism by which the hemochromatosis protein HFE andor TFR2 regulate hepcidinexpression is unknown but appears to involve an interaction with the BMP-SMAD signaling pathway It has been proposed that an interactionbetween HFE and TFR1 is reduced under high iron conditions due to competitive binding of holotransferrin to TFR1 Displaced HFE couldthen associate with TFR2 and possibly the HJV-BMP receptor complex to regulate hepcidin In1047298ammation also stimulates hepcidin productionin part via a canonical janus kinase (JAK)signal transducer and activator of transcription (STAT) pathway in which in1047298ammation increases in-terleukin 6 (IL6) binding to the IL6-receptor (IL6R) and thereby stimulating phosphorylation of JAKs and STAT3 Phosphorylated-STAT3homodimers translocate to the nuclease and bind to the hepcidin promoter to stimulate hepcidin expression Other mediators of in1047298ammationand infection can also regulate hepcidin expression in this context (not shown) A mechanism of crosstalk between in1047298ammatory signals andBMP signaling has been proposed in which in1047298ammation induces activin B which binds to BMP receptors to stimulate SMAD158 phosphoryl-ation SMADs and STAT3 may also interact at the level of the hepcidin promoter
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D I S O R D E R E D I R O N B A L A N C E I N C H R O N I C
K I D N E Y D I S E A S E
Iron de1047297ciency-limiting erythropoiesis is an important causeof anemia and resistance to ESAs in chronic kidney disease(CKD) patients [90ndash93] Iron administration is therefore a vital part of CKD anemia management Moreover the use of iron agents appears to be increasing [94] in the wake of recentlarge clinical trials that raised safety concerns for ESAs [95ndash97] and clinical practice guidelines that have liberalized rec-ommendations regarding iron use in CKD patients [98]However current diagnostic tests to evaluate iron status arelimited the targets of iron therapy are largely opinion basedand the safety of iron has not been rigorously evaluated inlarge prospective randomized controlled trials in this patientpopulation [98] Our increasing understanding about the mol-ecular mechanisms governing iron homeostasis regulation andits disturbance in CKD may lead to improved diagnostic andtherapeutic strategies for managing this patient population
The causes of iron de1047297ciency in CKD patients are multifac-torial (Figure 4) Some patients have true iron de1047297ciency
characterized by decreases in both circulating iron levels andtotal body iron stores Other patients have functional ironde1047297ciency characterized by a decrease in circulating iron thatlimits erythropoiesis which can occur even in the context of normal or adequate body iron stores A combination of thesefeatures may also be present Factors predisposing CKDpatients to iron de1047297ciency include increased blood loss in-creased iron utilization from ESA therapy impaired dietary iron absorption and impaired iron release from body storagesites [84] (Figure 4) Blood loss can arise from frequent
phlebotomy blood trapping in the dialysis apparatus and gas-trointestinal or other bleeding as a result of uremic plateletdysfunction Dietary iron absorption can be impaired by antacid medications or phosphate binders that block entero-cyte iron uptake It is now apparent that hepcidin excess alsocontributes to the impaired dietary iron absorption and im-paired iron release from body storage sites in CKD patients by downregulating ferroportin expression to block iron entry intothe circulation [99ndash101] Mechanisms leading to hepcidin
excess in these patients are thought to include reduced renalclearance of this small peptide hormone and increasedin1047298ammatory-mediated hepcidin transcription caused by thedialysis procedure itself andor the underlying disease process[84] Hepcidin levels in CKD patients are also in1047298uenced by iron and ESA administration (Figure 4) [84 100]
I R O N S T A T U S E VA L U A T I O N I N C K D
Current Kidney Disease Improving Global Outcomes clinicalpractice guidelines regarding the use of iron agents to manageanemia of CKD [98] revolve around two diagnostic tests
serum Tf saturation and serum ferritin levels Serum Tf satur-ation measures circulating iron that is immediately availablefor erythropoiesis while serum ferritin serves as a surrogatemeasure of body iron levels A major limitation of these diag-nostic tools is that they are not reliable for estimating body iron stores or predicting which patients will respond well toiron therapy [98 102ndash105] Indeed ferritin is also an acutephase reactant and so must be interpreted with caution in thesetting of in1047298ammation While there is general agreement thatpatients with total body iron de1047297ciency as indicated by low Tf saturation and low ferritin should be treated with iron therapythere are limited data on how to manage patients as ferritinlevels rise [98 106] There is therefore a need for new diagnos-tic tests to understand the iron status of CKD patients and tohelp determine which patients will bene1047297t from iron therapy
A L T E R N AT I V E A N D N O V E L D I A G N O S T I C
T O O L S F O R I R O N A N D A N E M I A
M A N A G E M E N T I N C K D
Reticulocyte hemoglobin content
By evaluating the hemoglobin content of reticulocyteswhich are early RBC forms reticulocyte hemoglobin content(CHr) provides an indication of iron availability for erythro-
poiesis within the last few days Several studies have suggestedthat CHr may also be helpful to predict responsiveness to ironin hemodialysis patients [107ndash112] although it is less wellstudied and may not be as widely available as Tf sat and ferri-tin
Percentage of hypochromic RBCs
Percentage of hypochromic RBCs measures the concen-tration of hemoglobin in RBCs which re1047298ects both the absol-ute amount of hemoglobin and the RBC size This test has alsoshown utility in predicting iron responsiveness in
F I G U R E 4 Disordered iron balance in CKD Chronic in1047298am-mation and reduced renal clearance in patients with CKD lead to in-creased levels of hepcidin which reduces duodenal iron uptake andiron release from cellular iron stores Intestinal iron uptake is also in-hibited by medications such as phosphate binders and antacids ESAsstimulate increased iron usage for erythropoiesis while blood loss dueto frequent phlebotomy blood trapping in the dialysis apparatus andgastrointestinal bleeding further deplete the circulating iron poolIron administration stimulates hepcidin expression which can para-doxically worsen the iron restriction while ESAs have an inhibitory effect on hepcidin expression
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hemodialysis patients [110 113] but can be impacted by blood storage time which leads to arti1047297cial RBC expansionthereby limiting utility in dialysis centers that use national lab-oratories [114]
Soluble transferrin receptor
Transferrin receptor 1 mediates uptake of iron into devel-oping RBCs Its expression and release into circulation assoluble transferrin receptor (sTFR) is increased in the setting
of iron de1047297ciency and increased erythroid activity Althoughthe literature on sTFR is limited a few studies have suggestedthat sTFR may be helpful to predict iron responsiveness [110113] However interpretation of this test in patients on ESAsis complicated by the fact that erythropoiesis itself increasessTFR levels [115] The use of this assay is also limited by lack of widespread availability and cost
Hepcidin
The understanding that hepcidin excess contributes to dis-ordered iron homeostasis in CKD patients has garnered inter-est in measuring hepcidin levels as a marker of iron statusiron responsiveness andor ESA responsiveness in CKD
patients There are two general types of assays now available tothe research community to measure circulating hepcidinlevels immunologic and mass spectrometry-based assaysBoth types of assays have their inherent strengths and weak-nesses and give an overall large variation in the absolute valuesof hepcidin levels but do show overall good correlation in rela-tive hepcidin levels with each other [116] Older assays thatalso recognize the precursor form of hepcidin (prohepcidin)are not useful because prohepcidin levels do not correlate withhepcidin biological activity [117 118] Using the more recentassays many studies have now con1047297rmed that circulating hep-cidin levels are increased in CKD patients with the highestlevels in patients on hemodialysis [99ndash101] Hepcidin levels inCKD patients have the strongest correlation with serum ferri-tin [100 101 119] but are also in1047298uenced at least in somestudies by in1047298ammation iron administration estimated glo-merular 1047297ltration rate dialysis clearance ESA dose and hemo-globin [100 101 119ndash121] One important limitation for theuse of hepcidin levels as a diagnostic tool in CKD patients isthe large intra-individual variability of both immunologic andmass spectrometry-based assays [122 123] Notably hepcidinlevels have not been shown to consistently predict responsive-ness or resistance to iron therapy or ESAs [ 120 124] Thus forthe time being there is no convincing evidence that hepcidinassays offer any advantage or additional information com-
pared with currently available diagnostic tests with regard toCKD iron and anemia management but this remains an areaof active investigation
Soluble HJV
Recent studies have explored the utility of measuring circu-lating levels of endogenous soluble HJV (sHJV) as a measureof iron status in human patients both without and with CKD[125ndash129] sHJV release from cells can be mediated by theproprotein convertase furin the transmembrane serine pro-tease TMPRSS6 and phospholipase C [130ndash134] and sHJV
has been detected in the conditioned media of transfected cellsand in the bloodstream of animals and humans [125ndash130135ndash137] While cell-surface GPI-anchored HJV functions asa BMP co-receptor to stimulate hepcidin expression (Figure 3)[39] sHJV can function as an inhibitor of BMP signaling andhepcidin expression presumably by sequestering BMP ligandsfrom interacting with cell surface signaling receptors [45 72135] Interestingly some studies have suggested that sHJVmay be decreased by iron treatment and increased by iron
de1047297ciency [125 130 135ndash137] suggesting that (i) sHJV couldbe useful as a diagnostic tool to indicate iron status and (ii) thegeneration sHJV could have a functional role to inhibit hepci-din expression in the context of iron de1047297ciency However oneimportant concern regarding these early human studies quan-titating sHJV levels is assay validity Indeed one commercialELISA assay used in studies focusing on CKD patients [128129] has subsequently been shown not to recognize HJV[138] Future studies will be needed using well-validated assaysand larger patient populations to determine if sHJV couldhave value as a diagnostic marker to guide iron therapy inCKD patients
Other markers
The putative role of GDF15 hepcidin regulation by erythro-poietic drive has generated interest in investigating this mol-ecule as a novel diagnostic tool for iron and anemiamanagement in CKD patients [139] However currently avail-able clinical data are very limited [139] Moreover while onestudy suggested that GDF15 may be increased by ironde1047297ciency [140] this was not robustly supported by anotherstudy [141] and GDF15 levels may also be in1047298uenced by in1047298ammation [141 142] malnutrition [142] and kidney disease [142] which may complicate its usefulness in thissetting
I R O N T H E R A P Y F O R C K D P A T I E N T S
Iron administration remains one of the cornerstones of anemia management in CKD patients to improve hemoglobinlevels and ESA responsiveness [98] Iron supplementation iscurrently given in two general forms oral or parenteral Oraliron supplementation is the easiest and cheapest Howeveroral iron agents can have gastrointestinal side effects that limitadherence due to the formation of local reactive oxygenspecies and oxidative damage in the gut mucosa [143] More-over several studies have suggested that oral iron is less effec-
tive than parenteral iron particularly in hemodialysis patientsfor improving or preventing iron de1047297ciency ameliorating anemia or reducing ESA dose [98 144ndash146] The limited ef-fectiveness of oral iron supplements in this patient populationis likely due to medications such as antacids and phosphatebinders that inhibit iron entry into duodenal enterocytes andhepcidin excess that decreases ferroportin expression to limitiron release from duodenal enterocytes into the bloodstream(Figure 4)
There are several intravenous (IV) iron preparations thatcan be used to treat iron-restricted erythropoiesis in CKD
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patients including iron dextrans iron sucrose ferric gluco-nate ferric carboxymaltose iron isomaltoside 1000 and feru-moxytol These preparations are generally comprising an ironcore shielded by a carbohydrate shell with different molecularweights and physiochemical properties yielding differentialdegradation kinetics and ability to release lsquofreersquo iron into thecirculation [143] This determines the maximal single dose foreach preparation with the newer higher molecular weightmore stable complexes enabling larger doses over shorter time
frames [143] Iron dextrans ( particularly high molecularweight dextrans) have been limited by dextran-induced ana-phylactic reactions in sim06ndash07 of patients [98] There issome limited data suggesting that various iron preparationsmay have different effects on markers of oxidative stress andin1047298ammation but this did not necessarily correlate with thecompoundsrsquo molecular weight stability or ability to releasefree iron into circulation [147 148] Comparative safety of these IV iron preparations in CKD patients remains largely unknown due to the lack of direct head-to-head clinical trials
Understanding the physiology of systemic iron balance andits pathophysiology in CKD and other iron disorders raisesseveral potential limitations shared by all IV iron preparations
Regardless of the iron preparation once the iron is taken upinto erythrocytes macrophages or other body storage siteshepcidin excess and ferroportin downregulation will limit theavailability of the iron for recycling and subsequent use More-over iron itself stimulates hepcidin expression and thereforecan paradoxically worsen the iron restriction (Figure 4)Additional concerns particularly with regard to repetitive ironadministration as ferritin levels rise are the potential foroxidant-mediated tissue injury from excess iron deposition asseen in iron overload disorders such as hemochromatosis Irondeposition has also been associated with the pathogenesis of many other common disorders including neurodegenerativediseases diabetes mellitus and atherosclerosis [1 149 150]Additionally withholding iron from invading pathogens is animportant function of the immune system and iron loading isassociated with worse outcomes in several infectious diseasesincluding malaria tuberculosis and HIV [151ndash153] Largeprospective randomized trials in the CKD population are long overdue to evaluate the ef 1047297cacy of repetitive IV iron adminis-tration with regard to hard clinical outcomes and long-termsafety to further characterize which patients will bene1047297t fromiron therapy and to determine treatment targets of irontherapy
N O V E L T R E A T M E N T S T R A T E G I E S F O R
I R O N - R E S T R I C T E D E R Y T H R O P O I E S I S I N
C K D P A T I E N T S
The understanding that hepcidin excess contributes to iron-re-stricted erythropoiesis in CKD patients has generated interestin developing new therapies that target the hepcidinndashferropor-tin axis to more directly address the underlying pathophysiol-ogy of this disease Such therapies would be expected toincrease iron availability from the diet and from the patients
own body iron stores and are a particularly attractive optionfor patients with higher ferritin levels
Several categories of hepcidinferroportin-based thera-peutics are currently in development (reviewed in [31]) Onecategory is direct hepcidin antagonists including anti-hepci-din antibodies other hepcidin-binding proteins (anticalins)hepcidin-binding spiegelmers and hepcidin siRNAs and anti-sense oligonucleotides [31] Dialysis itself also reduces hepci-din levels [121 154] but the levels quickly rebound [154]
potentially due in part to the induction of in1047298ammatory cyto-kines by the dialysis procedure as well as the high basal turn-over rate of hepcidin [155] Another category is agents thatinhibit hepcidin production by targeting either the BMP-SMAD signaling pathway or the IL6-STAT3 pathway [31]BMP-SMAD pathway inhibitors include anti-BMP6 anti-bodies sHJV linked to the constant region of IgG1 (HJVFc)small molecule BMP type I receptor antagonists (LDN-193189) and heparin (which has been shown to sequesterBMP ligands) [31 41 45 72 74 75 156] IL6-STAT3 pathway inhibitors include anti-IL6 antibodies (Siltuximab) anti-IL6receptor antibodies (Tocilizumab) JAK2 inhibitors (AG490)and STAT3 inhibitors (PpYLKTK) [31] ESAs and other
stimulators of ESA production such as prolyl hydroxylaseinhibitors also fall in this category since they inhibit hepcidinproduction A third category is ferroportin agonistsstabilizersincluding anti-ferroportin antibodies and thiol-reactive com-pounds that interfere with hepcidin binding to ferroportin aswell as agents that interfere with ferroportin internalization orpotentiate ferroportin synthesis [31] Notably many of theseagents have shown ef 1047297cacy for treating iron-restricted erythro-poiesis and anemia in animal models with anemia of chronicdisease [74 75 157ndash160] and several are currently in humanclinical trials [161ndash164] The safety and ef 1047297cacy of these agentsin human CKD patients compared with current treatmentstrategies remains to be determined
C O N C L U S I O N S
The last 13 years have yielded signi1047297cant advances in under-standing the molecular mechanisms underlying systemic ironbalance and its dysregulation in CKD patients These studieshold the promise for developing new rationally designed diag-nostic and therapeutic tools to improve anemia managementin CKD patients Novel therapies targeting hepcidin haveshown particular promise and several have already enteredhuman clinical trials More research is needed to better under-
stand the ef 1047297cacy long-term safety and targets of current irontherapies as well as novel hepcidin-lowering approaches inlarge prospective randomized controlled trials
A C K N O W L E D G E M E N T S
JLB was supported in part by NIH grant RO1-DK087727and a Howard Goodman Fellowship Award from the Massa-chusetts General Hospital
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C O N F L I C T O F I N T E R E S T S T A T E M E N T
JLB has ownership interest in a start-up company FerruMax Pharmaceuticals which has licensed technology from theMassachusetts General Hospital based on the work cited hereand in prior publications
R E F E R E N C E S
1 Hentze MW Muckenthaler MU Andrews NC Balancing acts molecular
control of mammalian iron metabolism Cell 2004 117 285ndash297
2 Kovac S Anderson GJ Baldwin GS Gastrins iron homeostasis and col-
orectal cancer Biochim Biophys Acta 2011 1813 889ndash895
3 McKie AT Barrow D Latunde-Dada GO et al An iron-regulated ferric
reductase associated with the absorption of dietary iron Science 2001
291 1755ndash1759
4 Gunshin H Starr CN Direnzo C et al Cybrd1(duodenal cytochrome b)
is not necessary for dietary iron absorption in mice Blood 2005 16 16
5 Fleming MD Trenor CC III Su MA et al Microcytic anaemia mice have
a mutation in Nramp2 a candidate iron transporter gene Nat Genet
1997 16 383ndash386
6 Gunshin H Mackenzie B Berger UV et al Cloning and characterization
of a mammalian proton-ion transporter Nature 1997 388 482ndash488
7 Gunshin H Fujiwara Y Custodio AO et al Slc11a2 is required for intesti-
nal iron absorption and erythropoiesis but dispensable in placenta and
liver J Clin Invest 2005 115 1258ndash1266
8 Weintraub LR Weinstein MB Huser HJ et al Absorption of hemoglobin
iron the role of a heme-splitting substance in the intestinal mucosa J
Clin Invest 1968 47 531ndash539
9 Donovan A Brownlie A Zhou Y et al Positional cloning of zebra1047297sh fer-
roportin1 identi1047297es a conserved vertebrate iron exporter Nature 2000
403 776ndash781
10 Abboud S Haile DJ A novel mammalian iron-regulated protein involved
in intracellular iron metabolism J Biol Chem 2000 275 19906ndash19912
11 McKie AT Marciani P Rolfs A et al A novel duodenal iron-regulated
transporter IREG1 implicated in the basolateral transfer of iron to the
circulation Mol Cell 2000 5 299ndash309
12 Osaki S Johnson DA Mobilization of liver iron by ferroxidase (cerulo-plasmin) J Biol Chem 1969 244 5757ndash5758
13 Osaki S Johnson DA Frieden E The possible signi1047297cance of the ferrous
oxidase activity of ceruloplasmin in normal human serum J Biol Chem
1966 241 2746ndash2751
14 Roeser HP Lee GR Nacht S et al The role of ceruloplasmin in iron
metabolism J Clin Invest 1970 49 2408ndash2417
15 Vulpe CD Kuo YM Murphy TL et al Hephaestin a ceruloplasmin
homologue implicated in intestinal iron transport is defective in the sla
mouse Nat Genet 1999 21 195ndash199
16 Krause A Neitz S Maumlgert HJ et al LEAP-1 a novel highly disul1047297de-
bonded human peptide exhibits antimicrobial activity FEBS Lett 2000
480 147ndash150
17 Pigeon C Ilyin G Courselaud B et al A new mouse liver-speci1047297c gene
encoding a protein homologous to human antimicrobial peptide hepci-
din is overexpressed during iron overload J Biol Chem 2001 2767811ndash7819
18 Park CH Valore EV Waring AJ et al Hepcidin a urinary antimicrobial
peptide synthesized in the liver J Biol Chem 2001 276 7806ndash7810
19 Nicolas G Bennoun M Devaux I et al Lack of hepcidin gene expression
and severe tissue iron overload in upstream stimulatory factor 2 (USF2)
knockout mice Proc Natl Acad Sci USA 2001 98 8780ndash8785
20 Lesbordes-Brion JC Viatte L Bennoun M et al Targeted disruption of
the hepcidin 1 gene results in severe hemochromatosis Blood 2006 108
1402ndash1405
21 Roetto A Papanikolaou G Politou M et al Mutant antimicrobial peptide
hepcidin is associated with severe juvenile hemochromatosis Nat Genet
2003 33 21ndash22
22 Nicolas G Bennoun M Porteu A et al Severe iron de1047297ciency anemia in
C o p y r i g h t o f N e p h r o l o g y D i a l y s i s T r a n s p l a n t a t i o n i s t h e p r o p e r t y o f O x f o r d U n i v e r s i t y P r e s s
U S A a n d i t s c o n t e n t m a y n o t b e c o p i e d o r e m a i l e d t o m u l t i p l e s i t e s o r p o s t e d t o a l i s t s e r v
w i t h o u t t h e c o p y r i g h t h o l d e r s e x p r e s s w r i t t e n p e r m i s s i o n H o w e v e r u s e r s m a y p r i n t
d o w n l o a d o r e m a i l a r t i c l e s f o r i n d i v i d u a l u s e
7252019 The Iron Cycle in Chronic Kidney Disease (CKD)
sources iron recycling macrophages and hepatocyte stores(Figure 1)
Although the hepcidinndashferroportin axis has a central role inregulating body iron balance there are many additional levelsof regulation For example the enterocyte exerts local controlover iron absorption through the regulation of proteins in- volved in iron transport (DMT1 and ferroportin) and seques-tration (ferritin) via both transcriptional and post-transcriptional mechanisms involving hypoxia inducible
factors (particularly HIF-2α) and iron regulatory proteins (re- viewed in ref [25])
H E P C I D I N R E G U L A T I O N
Hepcidin expression in the liver is regulated by a number of factors (Figure 1) Iron increases hepcidin expression as ahomeostatic mechanism to limit further iron entry into thebloodstream [17 26 27] In1047298ammation also stimulates hepci-din expression [17 26ndash30] which is hypothesized to functionas a protective mechanism to sequester iron from infectiousorganisms However in chronic in1047298ammatory states this
results in macrophage iron sequestration hypoferremia andiron restricted erythropoiesis that contributes to anemia of chronic disease [31] Iron de1047297ciency hypoxia and anemiainhibit hepcidin expression to increase iron availability for ery-thropoiesis [26] Recently several growth factors steroid hor-mones and other endocrine signals have also been identi1047297ed tohave a role in hepcidin regulation [32ndash36]
Hepcidin regulation by iron
Key insights into the iron-mediated hepcidin regulatory pathways came from studying the genetic iron overload dis-order hereditary hemochromatosis This is a heterogeneousdisorder caused by mutations in any of several genes that ulti-mately result in impaired regulation of the hepcidinndashferropor-tin axis leading to increased dietary iron absorption increasediron release from macrophage stores progressive tissue irondeposition and consequent multiorgan damage and disease[37] Hereditary hemochromatosis can be caused by mutationsin hepcidin itself mutations in ferroportin that interfere withhepcidin binding or hepcidin-mediated internalization ormutations in one of three other genes that are involved in theiron-mediated regulation of hepcidin expression hemojuvelin(HJV also known as HFE2) HFE and transferrin receptor 2(TFR2) [37] Among these genes HJV has the most criticalrole in hepcidin regulation since HJV mutations lead to the
more severe juvenile onset form of hemochromatosis that issimilar to the phenotype seen with mutations in hepcidin itself [21 38]
HJV functions as a co-receptor for the bone morphogeneticprotein (BMP)-SMAD signaling pathway [39] which iscentral to hepcidin transcriptional regulation in response toiron [40 41] (Figure 3) A subfamily of the transforming growth factor beta (TGF-β) superfamily of signaling mol-ecules BMPs have an important role in a number of biologicfunctions particularly during development [42] Moreoverthere is redundancy in the system with a number of BMP
ligands and several BMP type I and type II receptors that canlead to the same intracellular SMAD signaling cascade [42]Nevertheless HJV mediates a crucial and unique function of BMP-SMAD signaling in the liver to regulate hepcidinexpression and systemic iron balance since mice and patientswith HJV mutations have hepcidin de1047297ciency and hemochro-matosis but no other obvious phenotype [38 43 44] It ishypothesized that HJV expression sensitizes hepatocytes torespond to low levels of BMP ligand which would not other-
wise generate a response in the absence of the co-receptor[39] By enhancing the af 1047297nity of the binding interaction HJVmay also help cells to selectively respond to a certain subset of BMP ligands using a certain subset of BMP type I and type IIreceptors that are required to speci1047297cally regulate hepcidin inliver cells in particular the ligand BMP6 [45ndash47] the BMPtype I receptors ALK3 and ALK2 [48 49] and the BMP typeII receptor ACTRIIA [48] (Figure 3)
HJV may also connect the BMP-SMAD signaling responseto molecules involved in iron sensing but the molecularmechanisms for this remain to be fully elucidated It has beenhypothesized that HFE and TFR2 sense circulating iron levelsin the form of iron-bound transferrin since TFR2 can bind to
transferrin [50 51] and HFE competes for transferrin binding to transferrin receptor 1 (TFR1) [52ndash55] (Figure 3) There issome evidence from in vitro overexpression systems that HFETFR2 and HJV can interact with each other [56ndash58] but it isuncertain whether this occurs in vivo HFE and TFR2 doappear to intersect with the BMP-SMAD pathway at somelevel since mice and human patients with HFE and TFR2mutations exhibit impairment in liver SMAD signaling [59ndash64] However the functions of HFE and TFR2 in regulating hepcidin are not entirely overlapping given the differential se- verity of the iron overload phenotype in mice and patientswith HFE mutations alone TFR2 mutations alone and doubleHFETFR2 mutations [61 64 65]
Hepcidin regulation by in1047298ammation
Another well-characterized hepcidin regulatory pathway isthe IL6-JAK-STAT3 pathway which mediates at least in parthepcidin transcriptional induction in response to in1047298am-mation [27 28 66ndash68] (Figure 3) Other mediators of in1047298am-mation and infection including IL-22 type I interferon tumornecrosis factor alpha and endoplasmic reticulum stress havealso been implicated in hepcidin regulation [27 29 30]Notably liver SMAD signaling is also induced in many in1047298ammatory models [69 70] and hepcidin induction by in1047298ammation is reduced when the BMP-SMAD signaling
pathway is inhibited indicating crosstalk between these regu-latory pathways [41 71ndash75] Hypothesized mechanisms forthis crosstalk are an interaction between STAT3 and SMADsat the level of the hepcidin promoter and the TGF-β super-family member activin B [70 73] (Figure 3)
Hepcidin regulation by erythropoietic activity and
hypoxia
Increased erythropoietic activity for example in response toanemia or erythropoiesis-stimulating agent (ESA) adminis-tration is a potent suppressor of hepcidin expression This
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appears to be mediated by a secreted factor from proliferating red blood cell (RBC) precursors in the bone marrow sinceinhibition of erythropoiesis by chemotherapy irradiation oran erythropoietin blocking antibody prevents hepcidin sup-pression by anemia or ESAs [76 77] TGF-βBMP superfamily modulators GDF15 and TWSG1 have been proposed tomediate hepcidin suppression in iron loading anemias with in-effective erythropoiesis such as β-thalassemia [78 79] butmay not mediate hepcidin suppression in other contexts [8081] Recent data in genetic mouse models suggest thathypoxia-mediated hepcidin suppression occurs indirectly by
stimulating erythropoiesis [82 83] although other mechan-isms for hypoxia-mediated hepcidin suppression have alsobeen proposed [84 85]
Hepcidin regulation by growth factors steroid hormones
and other endocrine factors
Recently several growth factors and steroid hormones havebeen demonstrated to suppress hepcidin expression in theliver including hepatocyte growth factor (HGF 32) epidermalgrowth factor (EGF 32) estrogen [33 34] and testosterone[35 36] (Figure 1) HGF EGF and testosterone are proposed
to intersect with BMP-SMAD signaling in the regulation of hepcidin [32 35 36] while estrogen is suggested to act via anestrogen response element in the hepcidin promoter [33 34]The effects of steroid hormones on hepcidin regulation may help explain gender differences in iron homeostasis that havebeen observed [86] Recent data presented in abstract formsuggests that vitamin D administration may also suppress cir-culating hepcidin levels and that vitamin D inhibits hepcidintranscription in mononuclear cells [87] In contrast prolongedfasting [88] and glucose [89] have been shown to increase cir-culating hepcidin levels and the glucose-mediated hepcidin
increase was associated with a decrease in serum iron levels[89] The mechanism of hepcidin regulation by glucose andfasting is still undetermined but interestingly while glucosedid not affect hepcidin secretion in hepatoma-derived cell cul-tures it did induce hepcidin secretion by insulinoma-derivedcell cultures [89] These 1047297ndings suggest intriguing linksbetween iron metabolism and multiple endocrine systems andraise the possibility that hepcidin production in non-hepatictissues may functionally contribute to circulating hepcidinlevels and systemic iron balance in some contexts althoughthis will need to be validated by future studies
F I G U R E 3 Molecular regulation of hepcidin by iron and in1047298ammation Increased systemic iron stimulates the production of the ligand bonemorphogenetic protein 6 (BMP6) which binds to the BMP Type I (ALK2ALK3) and II (ACTRIIA) receptors and the co-receptor HJV tostimulate phosphorylation of the SMAD158 intracellular signaling molecules Phosphorylated SMAD 158 binds to SMAD4 and translocatesto the nuclease to activate hepcidin transcription The mechanism by which the hemochromatosis protein HFE andor TFR2 regulate hepcidinexpression is unknown but appears to involve an interaction with the BMP-SMAD signaling pathway It has been proposed that an interactionbetween HFE and TFR1 is reduced under high iron conditions due to competitive binding of holotransferrin to TFR1 Displaced HFE couldthen associate with TFR2 and possibly the HJV-BMP receptor complex to regulate hepcidin In1047298ammation also stimulates hepcidin productionin part via a canonical janus kinase (JAK)signal transducer and activator of transcription (STAT) pathway in which in1047298ammation increases in-terleukin 6 (IL6) binding to the IL6-receptor (IL6R) and thereby stimulating phosphorylation of JAKs and STAT3 Phosphorylated-STAT3homodimers translocate to the nuclease and bind to the hepcidin promoter to stimulate hepcidin expression Other mediators of in1047298ammationand infection can also regulate hepcidin expression in this context (not shown) A mechanism of crosstalk between in1047298ammatory signals andBMP signaling has been proposed in which in1047298ammation induces activin B which binds to BMP receptors to stimulate SMAD158 phosphoryl-ation SMADs and STAT3 may also interact at the level of the hepcidin promoter
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D I S O R D E R E D I R O N B A L A N C E I N C H R O N I C
K I D N E Y D I S E A S E
Iron de1047297ciency-limiting erythropoiesis is an important causeof anemia and resistance to ESAs in chronic kidney disease(CKD) patients [90ndash93] Iron administration is therefore a vital part of CKD anemia management Moreover the use of iron agents appears to be increasing [94] in the wake of recentlarge clinical trials that raised safety concerns for ESAs [95ndash97] and clinical practice guidelines that have liberalized rec-ommendations regarding iron use in CKD patients [98]However current diagnostic tests to evaluate iron status arelimited the targets of iron therapy are largely opinion basedand the safety of iron has not been rigorously evaluated inlarge prospective randomized controlled trials in this patientpopulation [98] Our increasing understanding about the mol-ecular mechanisms governing iron homeostasis regulation andits disturbance in CKD may lead to improved diagnostic andtherapeutic strategies for managing this patient population
The causes of iron de1047297ciency in CKD patients are multifac-torial (Figure 4) Some patients have true iron de1047297ciency
characterized by decreases in both circulating iron levels andtotal body iron stores Other patients have functional ironde1047297ciency characterized by a decrease in circulating iron thatlimits erythropoiesis which can occur even in the context of normal or adequate body iron stores A combination of thesefeatures may also be present Factors predisposing CKDpatients to iron de1047297ciency include increased blood loss in-creased iron utilization from ESA therapy impaired dietary iron absorption and impaired iron release from body storagesites [84] (Figure 4) Blood loss can arise from frequent
phlebotomy blood trapping in the dialysis apparatus and gas-trointestinal or other bleeding as a result of uremic plateletdysfunction Dietary iron absorption can be impaired by antacid medications or phosphate binders that block entero-cyte iron uptake It is now apparent that hepcidin excess alsocontributes to the impaired dietary iron absorption and im-paired iron release from body storage sites in CKD patients by downregulating ferroportin expression to block iron entry intothe circulation [99ndash101] Mechanisms leading to hepcidin
excess in these patients are thought to include reduced renalclearance of this small peptide hormone and increasedin1047298ammatory-mediated hepcidin transcription caused by thedialysis procedure itself andor the underlying disease process[84] Hepcidin levels in CKD patients are also in1047298uenced by iron and ESA administration (Figure 4) [84 100]
I R O N S T A T U S E VA L U A T I O N I N C K D
Current Kidney Disease Improving Global Outcomes clinicalpractice guidelines regarding the use of iron agents to manageanemia of CKD [98] revolve around two diagnostic tests
serum Tf saturation and serum ferritin levels Serum Tf satur-ation measures circulating iron that is immediately availablefor erythropoiesis while serum ferritin serves as a surrogatemeasure of body iron levels A major limitation of these diag-nostic tools is that they are not reliable for estimating body iron stores or predicting which patients will respond well toiron therapy [98 102ndash105] Indeed ferritin is also an acutephase reactant and so must be interpreted with caution in thesetting of in1047298ammation While there is general agreement thatpatients with total body iron de1047297ciency as indicated by low Tf saturation and low ferritin should be treated with iron therapythere are limited data on how to manage patients as ferritinlevels rise [98 106] There is therefore a need for new diagnos-tic tests to understand the iron status of CKD patients and tohelp determine which patients will bene1047297t from iron therapy
A L T E R N AT I V E A N D N O V E L D I A G N O S T I C
T O O L S F O R I R O N A N D A N E M I A
M A N A G E M E N T I N C K D
Reticulocyte hemoglobin content
By evaluating the hemoglobin content of reticulocyteswhich are early RBC forms reticulocyte hemoglobin content(CHr) provides an indication of iron availability for erythro-
poiesis within the last few days Several studies have suggestedthat CHr may also be helpful to predict responsiveness to ironin hemodialysis patients [107ndash112] although it is less wellstudied and may not be as widely available as Tf sat and ferri-tin
Percentage of hypochromic RBCs
Percentage of hypochromic RBCs measures the concen-tration of hemoglobin in RBCs which re1047298ects both the absol-ute amount of hemoglobin and the RBC size This test has alsoshown utility in predicting iron responsiveness in
F I G U R E 4 Disordered iron balance in CKD Chronic in1047298am-mation and reduced renal clearance in patients with CKD lead to in-creased levels of hepcidin which reduces duodenal iron uptake andiron release from cellular iron stores Intestinal iron uptake is also in-hibited by medications such as phosphate binders and antacids ESAsstimulate increased iron usage for erythropoiesis while blood loss dueto frequent phlebotomy blood trapping in the dialysis apparatus andgastrointestinal bleeding further deplete the circulating iron poolIron administration stimulates hepcidin expression which can para-doxically worsen the iron restriction while ESAs have an inhibitory effect on hepcidin expression
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hemodialysis patients [110 113] but can be impacted by blood storage time which leads to arti1047297cial RBC expansionthereby limiting utility in dialysis centers that use national lab-oratories [114]
Soluble transferrin receptor
Transferrin receptor 1 mediates uptake of iron into devel-oping RBCs Its expression and release into circulation assoluble transferrin receptor (sTFR) is increased in the setting
of iron de1047297ciency and increased erythroid activity Althoughthe literature on sTFR is limited a few studies have suggestedthat sTFR may be helpful to predict iron responsiveness [110113] However interpretation of this test in patients on ESAsis complicated by the fact that erythropoiesis itself increasessTFR levels [115] The use of this assay is also limited by lack of widespread availability and cost
Hepcidin
The understanding that hepcidin excess contributes to dis-ordered iron homeostasis in CKD patients has garnered inter-est in measuring hepcidin levels as a marker of iron statusiron responsiveness andor ESA responsiveness in CKD
patients There are two general types of assays now available tothe research community to measure circulating hepcidinlevels immunologic and mass spectrometry-based assaysBoth types of assays have their inherent strengths and weak-nesses and give an overall large variation in the absolute valuesof hepcidin levels but do show overall good correlation in rela-tive hepcidin levels with each other [116] Older assays thatalso recognize the precursor form of hepcidin (prohepcidin)are not useful because prohepcidin levels do not correlate withhepcidin biological activity [117 118] Using the more recentassays many studies have now con1047297rmed that circulating hep-cidin levels are increased in CKD patients with the highestlevels in patients on hemodialysis [99ndash101] Hepcidin levels inCKD patients have the strongest correlation with serum ferri-tin [100 101 119] but are also in1047298uenced at least in somestudies by in1047298ammation iron administration estimated glo-merular 1047297ltration rate dialysis clearance ESA dose and hemo-globin [100 101 119ndash121] One important limitation for theuse of hepcidin levels as a diagnostic tool in CKD patients isthe large intra-individual variability of both immunologic andmass spectrometry-based assays [122 123] Notably hepcidinlevels have not been shown to consistently predict responsive-ness or resistance to iron therapy or ESAs [ 120 124] Thus forthe time being there is no convincing evidence that hepcidinassays offer any advantage or additional information com-
pared with currently available diagnostic tests with regard toCKD iron and anemia management but this remains an areaof active investigation
Soluble HJV
Recent studies have explored the utility of measuring circu-lating levels of endogenous soluble HJV (sHJV) as a measureof iron status in human patients both without and with CKD[125ndash129] sHJV release from cells can be mediated by theproprotein convertase furin the transmembrane serine pro-tease TMPRSS6 and phospholipase C [130ndash134] and sHJV
has been detected in the conditioned media of transfected cellsand in the bloodstream of animals and humans [125ndash130135ndash137] While cell-surface GPI-anchored HJV functions asa BMP co-receptor to stimulate hepcidin expression (Figure 3)[39] sHJV can function as an inhibitor of BMP signaling andhepcidin expression presumably by sequestering BMP ligandsfrom interacting with cell surface signaling receptors [45 72135] Interestingly some studies have suggested that sHJVmay be decreased by iron treatment and increased by iron
de1047297ciency [125 130 135ndash137] suggesting that (i) sHJV couldbe useful as a diagnostic tool to indicate iron status and (ii) thegeneration sHJV could have a functional role to inhibit hepci-din expression in the context of iron de1047297ciency However oneimportant concern regarding these early human studies quan-titating sHJV levels is assay validity Indeed one commercialELISA assay used in studies focusing on CKD patients [128129] has subsequently been shown not to recognize HJV[138] Future studies will be needed using well-validated assaysand larger patient populations to determine if sHJV couldhave value as a diagnostic marker to guide iron therapy inCKD patients
Other markers
The putative role of GDF15 hepcidin regulation by erythro-poietic drive has generated interest in investigating this mol-ecule as a novel diagnostic tool for iron and anemiamanagement in CKD patients [139] However currently avail-able clinical data are very limited [139] Moreover while onestudy suggested that GDF15 may be increased by ironde1047297ciency [140] this was not robustly supported by anotherstudy [141] and GDF15 levels may also be in1047298uenced by in1047298ammation [141 142] malnutrition [142] and kidney disease [142] which may complicate its usefulness in thissetting
I R O N T H E R A P Y F O R C K D P A T I E N T S
Iron administration remains one of the cornerstones of anemia management in CKD patients to improve hemoglobinlevels and ESA responsiveness [98] Iron supplementation iscurrently given in two general forms oral or parenteral Oraliron supplementation is the easiest and cheapest Howeveroral iron agents can have gastrointestinal side effects that limitadherence due to the formation of local reactive oxygenspecies and oxidative damage in the gut mucosa [143] More-over several studies have suggested that oral iron is less effec-
tive than parenteral iron particularly in hemodialysis patientsfor improving or preventing iron de1047297ciency ameliorating anemia or reducing ESA dose [98 144ndash146] The limited ef-fectiveness of oral iron supplements in this patient populationis likely due to medications such as antacids and phosphatebinders that inhibit iron entry into duodenal enterocytes andhepcidin excess that decreases ferroportin expression to limitiron release from duodenal enterocytes into the bloodstream(Figure 4)
There are several intravenous (IV) iron preparations thatcan be used to treat iron-restricted erythropoiesis in CKD
F U L L R E V I E W
K Zumbrennen-Bullough and JL Babitt 268
7252019 The Iron Cycle in Chronic Kidney Disease (CKD)
patients including iron dextrans iron sucrose ferric gluco-nate ferric carboxymaltose iron isomaltoside 1000 and feru-moxytol These preparations are generally comprising an ironcore shielded by a carbohydrate shell with different molecularweights and physiochemical properties yielding differentialdegradation kinetics and ability to release lsquofreersquo iron into thecirculation [143] This determines the maximal single dose foreach preparation with the newer higher molecular weightmore stable complexes enabling larger doses over shorter time
frames [143] Iron dextrans ( particularly high molecularweight dextrans) have been limited by dextran-induced ana-phylactic reactions in sim06ndash07 of patients [98] There issome limited data suggesting that various iron preparationsmay have different effects on markers of oxidative stress andin1047298ammation but this did not necessarily correlate with thecompoundsrsquo molecular weight stability or ability to releasefree iron into circulation [147 148] Comparative safety of these IV iron preparations in CKD patients remains largely unknown due to the lack of direct head-to-head clinical trials
Understanding the physiology of systemic iron balance andits pathophysiology in CKD and other iron disorders raisesseveral potential limitations shared by all IV iron preparations
Regardless of the iron preparation once the iron is taken upinto erythrocytes macrophages or other body storage siteshepcidin excess and ferroportin downregulation will limit theavailability of the iron for recycling and subsequent use More-over iron itself stimulates hepcidin expression and thereforecan paradoxically worsen the iron restriction (Figure 4)Additional concerns particularly with regard to repetitive ironadministration as ferritin levels rise are the potential foroxidant-mediated tissue injury from excess iron deposition asseen in iron overload disorders such as hemochromatosis Irondeposition has also been associated with the pathogenesis of many other common disorders including neurodegenerativediseases diabetes mellitus and atherosclerosis [1 149 150]Additionally withholding iron from invading pathogens is animportant function of the immune system and iron loading isassociated with worse outcomes in several infectious diseasesincluding malaria tuberculosis and HIV [151ndash153] Largeprospective randomized trials in the CKD population are long overdue to evaluate the ef 1047297cacy of repetitive IV iron adminis-tration with regard to hard clinical outcomes and long-termsafety to further characterize which patients will bene1047297t fromiron therapy and to determine treatment targets of irontherapy
N O V E L T R E A T M E N T S T R A T E G I E S F O R
I R O N - R E S T R I C T E D E R Y T H R O P O I E S I S I N
C K D P A T I E N T S
The understanding that hepcidin excess contributes to iron-re-stricted erythropoiesis in CKD patients has generated interestin developing new therapies that target the hepcidinndashferropor-tin axis to more directly address the underlying pathophysiol-ogy of this disease Such therapies would be expected toincrease iron availability from the diet and from the patients
own body iron stores and are a particularly attractive optionfor patients with higher ferritin levels
Several categories of hepcidinferroportin-based thera-peutics are currently in development (reviewed in [31]) Onecategory is direct hepcidin antagonists including anti-hepci-din antibodies other hepcidin-binding proteins (anticalins)hepcidin-binding spiegelmers and hepcidin siRNAs and anti-sense oligonucleotides [31] Dialysis itself also reduces hepci-din levels [121 154] but the levels quickly rebound [154]
potentially due in part to the induction of in1047298ammatory cyto-kines by the dialysis procedure as well as the high basal turn-over rate of hepcidin [155] Another category is agents thatinhibit hepcidin production by targeting either the BMP-SMAD signaling pathway or the IL6-STAT3 pathway [31]BMP-SMAD pathway inhibitors include anti-BMP6 anti-bodies sHJV linked to the constant region of IgG1 (HJVFc)small molecule BMP type I receptor antagonists (LDN-193189) and heparin (which has been shown to sequesterBMP ligands) [31 41 45 72 74 75 156] IL6-STAT3 pathway inhibitors include anti-IL6 antibodies (Siltuximab) anti-IL6receptor antibodies (Tocilizumab) JAK2 inhibitors (AG490)and STAT3 inhibitors (PpYLKTK) [31] ESAs and other
stimulators of ESA production such as prolyl hydroxylaseinhibitors also fall in this category since they inhibit hepcidinproduction A third category is ferroportin agonistsstabilizersincluding anti-ferroportin antibodies and thiol-reactive com-pounds that interfere with hepcidin binding to ferroportin aswell as agents that interfere with ferroportin internalization orpotentiate ferroportin synthesis [31] Notably many of theseagents have shown ef 1047297cacy for treating iron-restricted erythro-poiesis and anemia in animal models with anemia of chronicdisease [74 75 157ndash160] and several are currently in humanclinical trials [161ndash164] The safety and ef 1047297cacy of these agentsin human CKD patients compared with current treatmentstrategies remains to be determined
C O N C L U S I O N S
The last 13 years have yielded signi1047297cant advances in under-standing the molecular mechanisms underlying systemic ironbalance and its dysregulation in CKD patients These studieshold the promise for developing new rationally designed diag-nostic and therapeutic tools to improve anemia managementin CKD patients Novel therapies targeting hepcidin haveshown particular promise and several have already enteredhuman clinical trials More research is needed to better under-
stand the ef 1047297cacy long-term safety and targets of current irontherapies as well as novel hepcidin-lowering approaches inlarge prospective randomized controlled trials
A C K N O W L E D G E M E N T S
JLB was supported in part by NIH grant RO1-DK087727and a Howard Goodman Fellowship Award from the Massa-chusetts General Hospital
F UL L R E V I E W
T h e i r o n c y c l e i n C K D269
7252019 The Iron Cycle in Chronic Kidney Disease (CKD)
C O N F L I C T O F I N T E R E S T S T A T E M E N T
JLB has ownership interest in a start-up company FerruMax Pharmaceuticals which has licensed technology from theMassachusetts General Hospital based on the work cited hereand in prior publications
R E F E R E N C E S
1 Hentze MW Muckenthaler MU Andrews NC Balancing acts molecular
control of mammalian iron metabolism Cell 2004 117 285ndash297
2 Kovac S Anderson GJ Baldwin GS Gastrins iron homeostasis and col-
orectal cancer Biochim Biophys Acta 2011 1813 889ndash895
3 McKie AT Barrow D Latunde-Dada GO et al An iron-regulated ferric
reductase associated with the absorption of dietary iron Science 2001
291 1755ndash1759
4 Gunshin H Starr CN Direnzo C et al Cybrd1(duodenal cytochrome b)
is not necessary for dietary iron absorption in mice Blood 2005 16 16
5 Fleming MD Trenor CC III Su MA et al Microcytic anaemia mice have
a mutation in Nramp2 a candidate iron transporter gene Nat Genet
1997 16 383ndash386
6 Gunshin H Mackenzie B Berger UV et al Cloning and characterization
of a mammalian proton-ion transporter Nature 1997 388 482ndash488
7 Gunshin H Fujiwara Y Custodio AO et al Slc11a2 is required for intesti-
nal iron absorption and erythropoiesis but dispensable in placenta and
liver J Clin Invest 2005 115 1258ndash1266
8 Weintraub LR Weinstein MB Huser HJ et al Absorption of hemoglobin
iron the role of a heme-splitting substance in the intestinal mucosa J
Clin Invest 1968 47 531ndash539
9 Donovan A Brownlie A Zhou Y et al Positional cloning of zebra1047297sh fer-
roportin1 identi1047297es a conserved vertebrate iron exporter Nature 2000
403 776ndash781
10 Abboud S Haile DJ A novel mammalian iron-regulated protein involved
in intracellular iron metabolism J Biol Chem 2000 275 19906ndash19912
11 McKie AT Marciani P Rolfs A et al A novel duodenal iron-regulated
transporter IREG1 implicated in the basolateral transfer of iron to the
circulation Mol Cell 2000 5 299ndash309
12 Osaki S Johnson DA Mobilization of liver iron by ferroxidase (cerulo-plasmin) J Biol Chem 1969 244 5757ndash5758
13 Osaki S Johnson DA Frieden E The possible signi1047297cance of the ferrous
oxidase activity of ceruloplasmin in normal human serum J Biol Chem
1966 241 2746ndash2751
14 Roeser HP Lee GR Nacht S et al The role of ceruloplasmin in iron
metabolism J Clin Invest 1970 49 2408ndash2417
15 Vulpe CD Kuo YM Murphy TL et al Hephaestin a ceruloplasmin
homologue implicated in intestinal iron transport is defective in the sla
mouse Nat Genet 1999 21 195ndash199
16 Krause A Neitz S Maumlgert HJ et al LEAP-1 a novel highly disul1047297de-
bonded human peptide exhibits antimicrobial activity FEBS Lett 2000
480 147ndash150
17 Pigeon C Ilyin G Courselaud B et al A new mouse liver-speci1047297c gene
encoding a protein homologous to human antimicrobial peptide hepci-
din is overexpressed during iron overload J Biol Chem 2001 2767811ndash7819
18 Park CH Valore EV Waring AJ et al Hepcidin a urinary antimicrobial
peptide synthesized in the liver J Biol Chem 2001 276 7806ndash7810
19 Nicolas G Bennoun M Devaux I et al Lack of hepcidin gene expression
and severe tissue iron overload in upstream stimulatory factor 2 (USF2)
knockout mice Proc Natl Acad Sci USA 2001 98 8780ndash8785
20 Lesbordes-Brion JC Viatte L Bennoun M et al Targeted disruption of
the hepcidin 1 gene results in severe hemochromatosis Blood 2006 108
1402ndash1405
21 Roetto A Papanikolaou G Politou M et al Mutant antimicrobial peptide
hepcidin is associated with severe juvenile hemochromatosis Nat Genet
2003 33 21ndash22
22 Nicolas G Bennoun M Porteu A et al Severe iron de1047297ciency anemia in
C o p y r i g h t o f N e p h r o l o g y D i a l y s i s T r a n s p l a n t a t i o n i s t h e p r o p e r t y o f O x f o r d U n i v e r s i t y P r e s s
U S A a n d i t s c o n t e n t m a y n o t b e c o p i e d o r e m a i l e d t o m u l t i p l e s i t e s o r p o s t e d t o a l i s t s e r v
w i t h o u t t h e c o p y r i g h t h o l d e r s e x p r e s s w r i t t e n p e r m i s s i o n H o w e v e r u s e r s m a y p r i n t
d o w n l o a d o r e m a i l a r t i c l e s f o r i n d i v i d u a l u s e
7252019 The Iron Cycle in Chronic Kidney Disease (CKD)
appears to be mediated by a secreted factor from proliferating red blood cell (RBC) precursors in the bone marrow sinceinhibition of erythropoiesis by chemotherapy irradiation oran erythropoietin blocking antibody prevents hepcidin sup-pression by anemia or ESAs [76 77] TGF-βBMP superfamily modulators GDF15 and TWSG1 have been proposed tomediate hepcidin suppression in iron loading anemias with in-effective erythropoiesis such as β-thalassemia [78 79] butmay not mediate hepcidin suppression in other contexts [8081] Recent data in genetic mouse models suggest thathypoxia-mediated hepcidin suppression occurs indirectly by
stimulating erythropoiesis [82 83] although other mechan-isms for hypoxia-mediated hepcidin suppression have alsobeen proposed [84 85]
Hepcidin regulation by growth factors steroid hormones
and other endocrine factors
Recently several growth factors and steroid hormones havebeen demonstrated to suppress hepcidin expression in theliver including hepatocyte growth factor (HGF 32) epidermalgrowth factor (EGF 32) estrogen [33 34] and testosterone[35 36] (Figure 1) HGF EGF and testosterone are proposed
to intersect with BMP-SMAD signaling in the regulation of hepcidin [32 35 36] while estrogen is suggested to act via anestrogen response element in the hepcidin promoter [33 34]The effects of steroid hormones on hepcidin regulation may help explain gender differences in iron homeostasis that havebeen observed [86] Recent data presented in abstract formsuggests that vitamin D administration may also suppress cir-culating hepcidin levels and that vitamin D inhibits hepcidintranscription in mononuclear cells [87] In contrast prolongedfasting [88] and glucose [89] have been shown to increase cir-culating hepcidin levels and the glucose-mediated hepcidin
increase was associated with a decrease in serum iron levels[89] The mechanism of hepcidin regulation by glucose andfasting is still undetermined but interestingly while glucosedid not affect hepcidin secretion in hepatoma-derived cell cul-tures it did induce hepcidin secretion by insulinoma-derivedcell cultures [89] These 1047297ndings suggest intriguing linksbetween iron metabolism and multiple endocrine systems andraise the possibility that hepcidin production in non-hepatictissues may functionally contribute to circulating hepcidinlevels and systemic iron balance in some contexts althoughthis will need to be validated by future studies
F I G U R E 3 Molecular regulation of hepcidin by iron and in1047298ammation Increased systemic iron stimulates the production of the ligand bonemorphogenetic protein 6 (BMP6) which binds to the BMP Type I (ALK2ALK3) and II (ACTRIIA) receptors and the co-receptor HJV tostimulate phosphorylation of the SMAD158 intracellular signaling molecules Phosphorylated SMAD 158 binds to SMAD4 and translocatesto the nuclease to activate hepcidin transcription The mechanism by which the hemochromatosis protein HFE andor TFR2 regulate hepcidinexpression is unknown but appears to involve an interaction with the BMP-SMAD signaling pathway It has been proposed that an interactionbetween HFE and TFR1 is reduced under high iron conditions due to competitive binding of holotransferrin to TFR1 Displaced HFE couldthen associate with TFR2 and possibly the HJV-BMP receptor complex to regulate hepcidin In1047298ammation also stimulates hepcidin productionin part via a canonical janus kinase (JAK)signal transducer and activator of transcription (STAT) pathway in which in1047298ammation increases in-terleukin 6 (IL6) binding to the IL6-receptor (IL6R) and thereby stimulating phosphorylation of JAKs and STAT3 Phosphorylated-STAT3homodimers translocate to the nuclease and bind to the hepcidin promoter to stimulate hepcidin expression Other mediators of in1047298ammationand infection can also regulate hepcidin expression in this context (not shown) A mechanism of crosstalk between in1047298ammatory signals andBMP signaling has been proposed in which in1047298ammation induces activin B which binds to BMP receptors to stimulate SMAD158 phosphoryl-ation SMADs and STAT3 may also interact at the level of the hepcidin promoter
F U L L R E V I E W
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7252019 The Iron Cycle in Chronic Kidney Disease (CKD)
D I S O R D E R E D I R O N B A L A N C E I N C H R O N I C
K I D N E Y D I S E A S E
Iron de1047297ciency-limiting erythropoiesis is an important causeof anemia and resistance to ESAs in chronic kidney disease(CKD) patients [90ndash93] Iron administration is therefore a vital part of CKD anemia management Moreover the use of iron agents appears to be increasing [94] in the wake of recentlarge clinical trials that raised safety concerns for ESAs [95ndash97] and clinical practice guidelines that have liberalized rec-ommendations regarding iron use in CKD patients [98]However current diagnostic tests to evaluate iron status arelimited the targets of iron therapy are largely opinion basedand the safety of iron has not been rigorously evaluated inlarge prospective randomized controlled trials in this patientpopulation [98] Our increasing understanding about the mol-ecular mechanisms governing iron homeostasis regulation andits disturbance in CKD may lead to improved diagnostic andtherapeutic strategies for managing this patient population
The causes of iron de1047297ciency in CKD patients are multifac-torial (Figure 4) Some patients have true iron de1047297ciency
characterized by decreases in both circulating iron levels andtotal body iron stores Other patients have functional ironde1047297ciency characterized by a decrease in circulating iron thatlimits erythropoiesis which can occur even in the context of normal or adequate body iron stores A combination of thesefeatures may also be present Factors predisposing CKDpatients to iron de1047297ciency include increased blood loss in-creased iron utilization from ESA therapy impaired dietary iron absorption and impaired iron release from body storagesites [84] (Figure 4) Blood loss can arise from frequent
phlebotomy blood trapping in the dialysis apparatus and gas-trointestinal or other bleeding as a result of uremic plateletdysfunction Dietary iron absorption can be impaired by antacid medications or phosphate binders that block entero-cyte iron uptake It is now apparent that hepcidin excess alsocontributes to the impaired dietary iron absorption and im-paired iron release from body storage sites in CKD patients by downregulating ferroportin expression to block iron entry intothe circulation [99ndash101] Mechanisms leading to hepcidin
excess in these patients are thought to include reduced renalclearance of this small peptide hormone and increasedin1047298ammatory-mediated hepcidin transcription caused by thedialysis procedure itself andor the underlying disease process[84] Hepcidin levels in CKD patients are also in1047298uenced by iron and ESA administration (Figure 4) [84 100]
I R O N S T A T U S E VA L U A T I O N I N C K D
Current Kidney Disease Improving Global Outcomes clinicalpractice guidelines regarding the use of iron agents to manageanemia of CKD [98] revolve around two diagnostic tests
serum Tf saturation and serum ferritin levels Serum Tf satur-ation measures circulating iron that is immediately availablefor erythropoiesis while serum ferritin serves as a surrogatemeasure of body iron levels A major limitation of these diag-nostic tools is that they are not reliable for estimating body iron stores or predicting which patients will respond well toiron therapy [98 102ndash105] Indeed ferritin is also an acutephase reactant and so must be interpreted with caution in thesetting of in1047298ammation While there is general agreement thatpatients with total body iron de1047297ciency as indicated by low Tf saturation and low ferritin should be treated with iron therapythere are limited data on how to manage patients as ferritinlevels rise [98 106] There is therefore a need for new diagnos-tic tests to understand the iron status of CKD patients and tohelp determine which patients will bene1047297t from iron therapy
A L T E R N AT I V E A N D N O V E L D I A G N O S T I C
T O O L S F O R I R O N A N D A N E M I A
M A N A G E M E N T I N C K D
Reticulocyte hemoglobin content
By evaluating the hemoglobin content of reticulocyteswhich are early RBC forms reticulocyte hemoglobin content(CHr) provides an indication of iron availability for erythro-
poiesis within the last few days Several studies have suggestedthat CHr may also be helpful to predict responsiveness to ironin hemodialysis patients [107ndash112] although it is less wellstudied and may not be as widely available as Tf sat and ferri-tin
Percentage of hypochromic RBCs
Percentage of hypochromic RBCs measures the concen-tration of hemoglobin in RBCs which re1047298ects both the absol-ute amount of hemoglobin and the RBC size This test has alsoshown utility in predicting iron responsiveness in
F I G U R E 4 Disordered iron balance in CKD Chronic in1047298am-mation and reduced renal clearance in patients with CKD lead to in-creased levels of hepcidin which reduces duodenal iron uptake andiron release from cellular iron stores Intestinal iron uptake is also in-hibited by medications such as phosphate binders and antacids ESAsstimulate increased iron usage for erythropoiesis while blood loss dueto frequent phlebotomy blood trapping in the dialysis apparatus andgastrointestinal bleeding further deplete the circulating iron poolIron administration stimulates hepcidin expression which can para-doxically worsen the iron restriction while ESAs have an inhibitory effect on hepcidin expression
F UL L R E V I E W
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7252019 The Iron Cycle in Chronic Kidney Disease (CKD)
hemodialysis patients [110 113] but can be impacted by blood storage time which leads to arti1047297cial RBC expansionthereby limiting utility in dialysis centers that use national lab-oratories [114]
Soluble transferrin receptor
Transferrin receptor 1 mediates uptake of iron into devel-oping RBCs Its expression and release into circulation assoluble transferrin receptor (sTFR) is increased in the setting
of iron de1047297ciency and increased erythroid activity Althoughthe literature on sTFR is limited a few studies have suggestedthat sTFR may be helpful to predict iron responsiveness [110113] However interpretation of this test in patients on ESAsis complicated by the fact that erythropoiesis itself increasessTFR levels [115] The use of this assay is also limited by lack of widespread availability and cost
Hepcidin
The understanding that hepcidin excess contributes to dis-ordered iron homeostasis in CKD patients has garnered inter-est in measuring hepcidin levels as a marker of iron statusiron responsiveness andor ESA responsiveness in CKD
patients There are two general types of assays now available tothe research community to measure circulating hepcidinlevels immunologic and mass spectrometry-based assaysBoth types of assays have their inherent strengths and weak-nesses and give an overall large variation in the absolute valuesof hepcidin levels but do show overall good correlation in rela-tive hepcidin levels with each other [116] Older assays thatalso recognize the precursor form of hepcidin (prohepcidin)are not useful because prohepcidin levels do not correlate withhepcidin biological activity [117 118] Using the more recentassays many studies have now con1047297rmed that circulating hep-cidin levels are increased in CKD patients with the highestlevels in patients on hemodialysis [99ndash101] Hepcidin levels inCKD patients have the strongest correlation with serum ferri-tin [100 101 119] but are also in1047298uenced at least in somestudies by in1047298ammation iron administration estimated glo-merular 1047297ltration rate dialysis clearance ESA dose and hemo-globin [100 101 119ndash121] One important limitation for theuse of hepcidin levels as a diagnostic tool in CKD patients isthe large intra-individual variability of both immunologic andmass spectrometry-based assays [122 123] Notably hepcidinlevels have not been shown to consistently predict responsive-ness or resistance to iron therapy or ESAs [ 120 124] Thus forthe time being there is no convincing evidence that hepcidinassays offer any advantage or additional information com-
pared with currently available diagnostic tests with regard toCKD iron and anemia management but this remains an areaof active investigation
Soluble HJV
Recent studies have explored the utility of measuring circu-lating levels of endogenous soluble HJV (sHJV) as a measureof iron status in human patients both without and with CKD[125ndash129] sHJV release from cells can be mediated by theproprotein convertase furin the transmembrane serine pro-tease TMPRSS6 and phospholipase C [130ndash134] and sHJV
has been detected in the conditioned media of transfected cellsand in the bloodstream of animals and humans [125ndash130135ndash137] While cell-surface GPI-anchored HJV functions asa BMP co-receptor to stimulate hepcidin expression (Figure 3)[39] sHJV can function as an inhibitor of BMP signaling andhepcidin expression presumably by sequestering BMP ligandsfrom interacting with cell surface signaling receptors [45 72135] Interestingly some studies have suggested that sHJVmay be decreased by iron treatment and increased by iron
de1047297ciency [125 130 135ndash137] suggesting that (i) sHJV couldbe useful as a diagnostic tool to indicate iron status and (ii) thegeneration sHJV could have a functional role to inhibit hepci-din expression in the context of iron de1047297ciency However oneimportant concern regarding these early human studies quan-titating sHJV levels is assay validity Indeed one commercialELISA assay used in studies focusing on CKD patients [128129] has subsequently been shown not to recognize HJV[138] Future studies will be needed using well-validated assaysand larger patient populations to determine if sHJV couldhave value as a diagnostic marker to guide iron therapy inCKD patients
Other markers
The putative role of GDF15 hepcidin regulation by erythro-poietic drive has generated interest in investigating this mol-ecule as a novel diagnostic tool for iron and anemiamanagement in CKD patients [139] However currently avail-able clinical data are very limited [139] Moreover while onestudy suggested that GDF15 may be increased by ironde1047297ciency [140] this was not robustly supported by anotherstudy [141] and GDF15 levels may also be in1047298uenced by in1047298ammation [141 142] malnutrition [142] and kidney disease [142] which may complicate its usefulness in thissetting
I R O N T H E R A P Y F O R C K D P A T I E N T S
Iron administration remains one of the cornerstones of anemia management in CKD patients to improve hemoglobinlevels and ESA responsiveness [98] Iron supplementation iscurrently given in two general forms oral or parenteral Oraliron supplementation is the easiest and cheapest Howeveroral iron agents can have gastrointestinal side effects that limitadherence due to the formation of local reactive oxygenspecies and oxidative damage in the gut mucosa [143] More-over several studies have suggested that oral iron is less effec-
tive than parenteral iron particularly in hemodialysis patientsfor improving or preventing iron de1047297ciency ameliorating anemia or reducing ESA dose [98 144ndash146] The limited ef-fectiveness of oral iron supplements in this patient populationis likely due to medications such as antacids and phosphatebinders that inhibit iron entry into duodenal enterocytes andhepcidin excess that decreases ferroportin expression to limitiron release from duodenal enterocytes into the bloodstream(Figure 4)
There are several intravenous (IV) iron preparations thatcan be used to treat iron-restricted erythropoiesis in CKD
F U L L R E V I E W
K Zumbrennen-Bullough and JL Babitt 268
7252019 The Iron Cycle in Chronic Kidney Disease (CKD)
patients including iron dextrans iron sucrose ferric gluco-nate ferric carboxymaltose iron isomaltoside 1000 and feru-moxytol These preparations are generally comprising an ironcore shielded by a carbohydrate shell with different molecularweights and physiochemical properties yielding differentialdegradation kinetics and ability to release lsquofreersquo iron into thecirculation [143] This determines the maximal single dose foreach preparation with the newer higher molecular weightmore stable complexes enabling larger doses over shorter time
frames [143] Iron dextrans ( particularly high molecularweight dextrans) have been limited by dextran-induced ana-phylactic reactions in sim06ndash07 of patients [98] There issome limited data suggesting that various iron preparationsmay have different effects on markers of oxidative stress andin1047298ammation but this did not necessarily correlate with thecompoundsrsquo molecular weight stability or ability to releasefree iron into circulation [147 148] Comparative safety of these IV iron preparations in CKD patients remains largely unknown due to the lack of direct head-to-head clinical trials
Understanding the physiology of systemic iron balance andits pathophysiology in CKD and other iron disorders raisesseveral potential limitations shared by all IV iron preparations
Regardless of the iron preparation once the iron is taken upinto erythrocytes macrophages or other body storage siteshepcidin excess and ferroportin downregulation will limit theavailability of the iron for recycling and subsequent use More-over iron itself stimulates hepcidin expression and thereforecan paradoxically worsen the iron restriction (Figure 4)Additional concerns particularly with regard to repetitive ironadministration as ferritin levels rise are the potential foroxidant-mediated tissue injury from excess iron deposition asseen in iron overload disorders such as hemochromatosis Irondeposition has also been associated with the pathogenesis of many other common disorders including neurodegenerativediseases diabetes mellitus and atherosclerosis [1 149 150]Additionally withholding iron from invading pathogens is animportant function of the immune system and iron loading isassociated with worse outcomes in several infectious diseasesincluding malaria tuberculosis and HIV [151ndash153] Largeprospective randomized trials in the CKD population are long overdue to evaluate the ef 1047297cacy of repetitive IV iron adminis-tration with regard to hard clinical outcomes and long-termsafety to further characterize which patients will bene1047297t fromiron therapy and to determine treatment targets of irontherapy
N O V E L T R E A T M E N T S T R A T E G I E S F O R
I R O N - R E S T R I C T E D E R Y T H R O P O I E S I S I N
C K D P A T I E N T S
The understanding that hepcidin excess contributes to iron-re-stricted erythropoiesis in CKD patients has generated interestin developing new therapies that target the hepcidinndashferropor-tin axis to more directly address the underlying pathophysiol-ogy of this disease Such therapies would be expected toincrease iron availability from the diet and from the patients
own body iron stores and are a particularly attractive optionfor patients with higher ferritin levels
Several categories of hepcidinferroportin-based thera-peutics are currently in development (reviewed in [31]) Onecategory is direct hepcidin antagonists including anti-hepci-din antibodies other hepcidin-binding proteins (anticalins)hepcidin-binding spiegelmers and hepcidin siRNAs and anti-sense oligonucleotides [31] Dialysis itself also reduces hepci-din levels [121 154] but the levels quickly rebound [154]
potentially due in part to the induction of in1047298ammatory cyto-kines by the dialysis procedure as well as the high basal turn-over rate of hepcidin [155] Another category is agents thatinhibit hepcidin production by targeting either the BMP-SMAD signaling pathway or the IL6-STAT3 pathway [31]BMP-SMAD pathway inhibitors include anti-BMP6 anti-bodies sHJV linked to the constant region of IgG1 (HJVFc)small molecule BMP type I receptor antagonists (LDN-193189) and heparin (which has been shown to sequesterBMP ligands) [31 41 45 72 74 75 156] IL6-STAT3 pathway inhibitors include anti-IL6 antibodies (Siltuximab) anti-IL6receptor antibodies (Tocilizumab) JAK2 inhibitors (AG490)and STAT3 inhibitors (PpYLKTK) [31] ESAs and other
stimulators of ESA production such as prolyl hydroxylaseinhibitors also fall in this category since they inhibit hepcidinproduction A third category is ferroportin agonistsstabilizersincluding anti-ferroportin antibodies and thiol-reactive com-pounds that interfere with hepcidin binding to ferroportin aswell as agents that interfere with ferroportin internalization orpotentiate ferroportin synthesis [31] Notably many of theseagents have shown ef 1047297cacy for treating iron-restricted erythro-poiesis and anemia in animal models with anemia of chronicdisease [74 75 157ndash160] and several are currently in humanclinical trials [161ndash164] The safety and ef 1047297cacy of these agentsin human CKD patients compared with current treatmentstrategies remains to be determined
C O N C L U S I O N S
The last 13 years have yielded signi1047297cant advances in under-standing the molecular mechanisms underlying systemic ironbalance and its dysregulation in CKD patients These studieshold the promise for developing new rationally designed diag-nostic and therapeutic tools to improve anemia managementin CKD patients Novel therapies targeting hepcidin haveshown particular promise and several have already enteredhuman clinical trials More research is needed to better under-
stand the ef 1047297cacy long-term safety and targets of current irontherapies as well as novel hepcidin-lowering approaches inlarge prospective randomized controlled trials
A C K N O W L E D G E M E N T S
JLB was supported in part by NIH grant RO1-DK087727and a Howard Goodman Fellowship Award from the Massa-chusetts General Hospital
F UL L R E V I E W
T h e i r o n c y c l e i n C K D269
7252019 The Iron Cycle in Chronic Kidney Disease (CKD)
C O N F L I C T O F I N T E R E S T S T A T E M E N T
JLB has ownership interest in a start-up company FerruMax Pharmaceuticals which has licensed technology from theMassachusetts General Hospital based on the work cited hereand in prior publications
R E F E R E N C E S
1 Hentze MW Muckenthaler MU Andrews NC Balancing acts molecular
control of mammalian iron metabolism Cell 2004 117 285ndash297
2 Kovac S Anderson GJ Baldwin GS Gastrins iron homeostasis and col-
orectal cancer Biochim Biophys Acta 2011 1813 889ndash895
3 McKie AT Barrow D Latunde-Dada GO et al An iron-regulated ferric
reductase associated with the absorption of dietary iron Science 2001
291 1755ndash1759
4 Gunshin H Starr CN Direnzo C et al Cybrd1(duodenal cytochrome b)
is not necessary for dietary iron absorption in mice Blood 2005 16 16
5 Fleming MD Trenor CC III Su MA et al Microcytic anaemia mice have
a mutation in Nramp2 a candidate iron transporter gene Nat Genet
1997 16 383ndash386
6 Gunshin H Mackenzie B Berger UV et al Cloning and characterization
of a mammalian proton-ion transporter Nature 1997 388 482ndash488
7 Gunshin H Fujiwara Y Custodio AO et al Slc11a2 is required for intesti-
nal iron absorption and erythropoiesis but dispensable in placenta and
liver J Clin Invest 2005 115 1258ndash1266
8 Weintraub LR Weinstein MB Huser HJ et al Absorption of hemoglobin
iron the role of a heme-splitting substance in the intestinal mucosa J
Clin Invest 1968 47 531ndash539
9 Donovan A Brownlie A Zhou Y et al Positional cloning of zebra1047297sh fer-
roportin1 identi1047297es a conserved vertebrate iron exporter Nature 2000
403 776ndash781
10 Abboud S Haile DJ A novel mammalian iron-regulated protein involved
in intracellular iron metabolism J Biol Chem 2000 275 19906ndash19912
11 McKie AT Marciani P Rolfs A et al A novel duodenal iron-regulated
transporter IREG1 implicated in the basolateral transfer of iron to the
circulation Mol Cell 2000 5 299ndash309
12 Osaki S Johnson DA Mobilization of liver iron by ferroxidase (cerulo-plasmin) J Biol Chem 1969 244 5757ndash5758
13 Osaki S Johnson DA Frieden E The possible signi1047297cance of the ferrous
oxidase activity of ceruloplasmin in normal human serum J Biol Chem
1966 241 2746ndash2751
14 Roeser HP Lee GR Nacht S et al The role of ceruloplasmin in iron
metabolism J Clin Invest 1970 49 2408ndash2417
15 Vulpe CD Kuo YM Murphy TL et al Hephaestin a ceruloplasmin
homologue implicated in intestinal iron transport is defective in the sla
mouse Nat Genet 1999 21 195ndash199
16 Krause A Neitz S Maumlgert HJ et al LEAP-1 a novel highly disul1047297de-
bonded human peptide exhibits antimicrobial activity FEBS Lett 2000
480 147ndash150
17 Pigeon C Ilyin G Courselaud B et al A new mouse liver-speci1047297c gene
encoding a protein homologous to human antimicrobial peptide hepci-
din is overexpressed during iron overload J Biol Chem 2001 2767811ndash7819
18 Park CH Valore EV Waring AJ et al Hepcidin a urinary antimicrobial
peptide synthesized in the liver J Biol Chem 2001 276 7806ndash7810
19 Nicolas G Bennoun M Devaux I et al Lack of hepcidin gene expression
and severe tissue iron overload in upstream stimulatory factor 2 (USF2)
knockout mice Proc Natl Acad Sci USA 2001 98 8780ndash8785
20 Lesbordes-Brion JC Viatte L Bennoun M et al Targeted disruption of
the hepcidin 1 gene results in severe hemochromatosis Blood 2006 108
1402ndash1405
21 Roetto A Papanikolaou G Politou M et al Mutant antimicrobial peptide
hepcidin is associated with severe juvenile hemochromatosis Nat Genet
2003 33 21ndash22
22 Nicolas G Bennoun M Porteu A et al Severe iron de1047297ciency anemia in
C o p y r i g h t o f N e p h r o l o g y D i a l y s i s T r a n s p l a n t a t i o n i s t h e p r o p e r t y o f O x f o r d U n i v e r s i t y P r e s s
U S A a n d i t s c o n t e n t m a y n o t b e c o p i e d o r e m a i l e d t o m u l t i p l e s i t e s o r p o s t e d t o a l i s t s e r v
w i t h o u t t h e c o p y r i g h t h o l d e r s e x p r e s s w r i t t e n p e r m i s s i o n H o w e v e r u s e r s m a y p r i n t
d o w n l o a d o r e m a i l a r t i c l e s f o r i n d i v i d u a l u s e
7252019 The Iron Cycle in Chronic Kidney Disease (CKD)
D I S O R D E R E D I R O N B A L A N C E I N C H R O N I C
K I D N E Y D I S E A S E
Iron de1047297ciency-limiting erythropoiesis is an important causeof anemia and resistance to ESAs in chronic kidney disease(CKD) patients [90ndash93] Iron administration is therefore a vital part of CKD anemia management Moreover the use of iron agents appears to be increasing [94] in the wake of recentlarge clinical trials that raised safety concerns for ESAs [95ndash97] and clinical practice guidelines that have liberalized rec-ommendations regarding iron use in CKD patients [98]However current diagnostic tests to evaluate iron status arelimited the targets of iron therapy are largely opinion basedand the safety of iron has not been rigorously evaluated inlarge prospective randomized controlled trials in this patientpopulation [98] Our increasing understanding about the mol-ecular mechanisms governing iron homeostasis regulation andits disturbance in CKD may lead to improved diagnostic andtherapeutic strategies for managing this patient population
The causes of iron de1047297ciency in CKD patients are multifac-torial (Figure 4) Some patients have true iron de1047297ciency
characterized by decreases in both circulating iron levels andtotal body iron stores Other patients have functional ironde1047297ciency characterized by a decrease in circulating iron thatlimits erythropoiesis which can occur even in the context of normal or adequate body iron stores A combination of thesefeatures may also be present Factors predisposing CKDpatients to iron de1047297ciency include increased blood loss in-creased iron utilization from ESA therapy impaired dietary iron absorption and impaired iron release from body storagesites [84] (Figure 4) Blood loss can arise from frequent
phlebotomy blood trapping in the dialysis apparatus and gas-trointestinal or other bleeding as a result of uremic plateletdysfunction Dietary iron absorption can be impaired by antacid medications or phosphate binders that block entero-cyte iron uptake It is now apparent that hepcidin excess alsocontributes to the impaired dietary iron absorption and im-paired iron release from body storage sites in CKD patients by downregulating ferroportin expression to block iron entry intothe circulation [99ndash101] Mechanisms leading to hepcidin
excess in these patients are thought to include reduced renalclearance of this small peptide hormone and increasedin1047298ammatory-mediated hepcidin transcription caused by thedialysis procedure itself andor the underlying disease process[84] Hepcidin levels in CKD patients are also in1047298uenced by iron and ESA administration (Figure 4) [84 100]
I R O N S T A T U S E VA L U A T I O N I N C K D
Current Kidney Disease Improving Global Outcomes clinicalpractice guidelines regarding the use of iron agents to manageanemia of CKD [98] revolve around two diagnostic tests
serum Tf saturation and serum ferritin levels Serum Tf satur-ation measures circulating iron that is immediately availablefor erythropoiesis while serum ferritin serves as a surrogatemeasure of body iron levels A major limitation of these diag-nostic tools is that they are not reliable for estimating body iron stores or predicting which patients will respond well toiron therapy [98 102ndash105] Indeed ferritin is also an acutephase reactant and so must be interpreted with caution in thesetting of in1047298ammation While there is general agreement thatpatients with total body iron de1047297ciency as indicated by low Tf saturation and low ferritin should be treated with iron therapythere are limited data on how to manage patients as ferritinlevels rise [98 106] There is therefore a need for new diagnos-tic tests to understand the iron status of CKD patients and tohelp determine which patients will bene1047297t from iron therapy
A L T E R N AT I V E A N D N O V E L D I A G N O S T I C
T O O L S F O R I R O N A N D A N E M I A
M A N A G E M E N T I N C K D
Reticulocyte hemoglobin content
By evaluating the hemoglobin content of reticulocyteswhich are early RBC forms reticulocyte hemoglobin content(CHr) provides an indication of iron availability for erythro-
poiesis within the last few days Several studies have suggestedthat CHr may also be helpful to predict responsiveness to ironin hemodialysis patients [107ndash112] although it is less wellstudied and may not be as widely available as Tf sat and ferri-tin
Percentage of hypochromic RBCs
Percentage of hypochromic RBCs measures the concen-tration of hemoglobin in RBCs which re1047298ects both the absol-ute amount of hemoglobin and the RBC size This test has alsoshown utility in predicting iron responsiveness in
F I G U R E 4 Disordered iron balance in CKD Chronic in1047298am-mation and reduced renal clearance in patients with CKD lead to in-creased levels of hepcidin which reduces duodenal iron uptake andiron release from cellular iron stores Intestinal iron uptake is also in-hibited by medications such as phosphate binders and antacids ESAsstimulate increased iron usage for erythropoiesis while blood loss dueto frequent phlebotomy blood trapping in the dialysis apparatus andgastrointestinal bleeding further deplete the circulating iron poolIron administration stimulates hepcidin expression which can para-doxically worsen the iron restriction while ESAs have an inhibitory effect on hepcidin expression
F UL L R E V I E W
T h e i r o n c y c l e i n C K D267
7252019 The Iron Cycle in Chronic Kidney Disease (CKD)
hemodialysis patients [110 113] but can be impacted by blood storage time which leads to arti1047297cial RBC expansionthereby limiting utility in dialysis centers that use national lab-oratories [114]
Soluble transferrin receptor
Transferrin receptor 1 mediates uptake of iron into devel-oping RBCs Its expression and release into circulation assoluble transferrin receptor (sTFR) is increased in the setting
of iron de1047297ciency and increased erythroid activity Althoughthe literature on sTFR is limited a few studies have suggestedthat sTFR may be helpful to predict iron responsiveness [110113] However interpretation of this test in patients on ESAsis complicated by the fact that erythropoiesis itself increasessTFR levels [115] The use of this assay is also limited by lack of widespread availability and cost
Hepcidin
The understanding that hepcidin excess contributes to dis-ordered iron homeostasis in CKD patients has garnered inter-est in measuring hepcidin levels as a marker of iron statusiron responsiveness andor ESA responsiveness in CKD
patients There are two general types of assays now available tothe research community to measure circulating hepcidinlevels immunologic and mass spectrometry-based assaysBoth types of assays have their inherent strengths and weak-nesses and give an overall large variation in the absolute valuesof hepcidin levels but do show overall good correlation in rela-tive hepcidin levels with each other [116] Older assays thatalso recognize the precursor form of hepcidin (prohepcidin)are not useful because prohepcidin levels do not correlate withhepcidin biological activity [117 118] Using the more recentassays many studies have now con1047297rmed that circulating hep-cidin levels are increased in CKD patients with the highestlevels in patients on hemodialysis [99ndash101] Hepcidin levels inCKD patients have the strongest correlation with serum ferri-tin [100 101 119] but are also in1047298uenced at least in somestudies by in1047298ammation iron administration estimated glo-merular 1047297ltration rate dialysis clearance ESA dose and hemo-globin [100 101 119ndash121] One important limitation for theuse of hepcidin levels as a diagnostic tool in CKD patients isthe large intra-individual variability of both immunologic andmass spectrometry-based assays [122 123] Notably hepcidinlevels have not been shown to consistently predict responsive-ness or resistance to iron therapy or ESAs [ 120 124] Thus forthe time being there is no convincing evidence that hepcidinassays offer any advantage or additional information com-
pared with currently available diagnostic tests with regard toCKD iron and anemia management but this remains an areaof active investigation
Soluble HJV
Recent studies have explored the utility of measuring circu-lating levels of endogenous soluble HJV (sHJV) as a measureof iron status in human patients both without and with CKD[125ndash129] sHJV release from cells can be mediated by theproprotein convertase furin the transmembrane serine pro-tease TMPRSS6 and phospholipase C [130ndash134] and sHJV
has been detected in the conditioned media of transfected cellsand in the bloodstream of animals and humans [125ndash130135ndash137] While cell-surface GPI-anchored HJV functions asa BMP co-receptor to stimulate hepcidin expression (Figure 3)[39] sHJV can function as an inhibitor of BMP signaling andhepcidin expression presumably by sequestering BMP ligandsfrom interacting with cell surface signaling receptors [45 72135] Interestingly some studies have suggested that sHJVmay be decreased by iron treatment and increased by iron
de1047297ciency [125 130 135ndash137] suggesting that (i) sHJV couldbe useful as a diagnostic tool to indicate iron status and (ii) thegeneration sHJV could have a functional role to inhibit hepci-din expression in the context of iron de1047297ciency However oneimportant concern regarding these early human studies quan-titating sHJV levels is assay validity Indeed one commercialELISA assay used in studies focusing on CKD patients [128129] has subsequently been shown not to recognize HJV[138] Future studies will be needed using well-validated assaysand larger patient populations to determine if sHJV couldhave value as a diagnostic marker to guide iron therapy inCKD patients
Other markers
The putative role of GDF15 hepcidin regulation by erythro-poietic drive has generated interest in investigating this mol-ecule as a novel diagnostic tool for iron and anemiamanagement in CKD patients [139] However currently avail-able clinical data are very limited [139] Moreover while onestudy suggested that GDF15 may be increased by ironde1047297ciency [140] this was not robustly supported by anotherstudy [141] and GDF15 levels may also be in1047298uenced by in1047298ammation [141 142] malnutrition [142] and kidney disease [142] which may complicate its usefulness in thissetting
I R O N T H E R A P Y F O R C K D P A T I E N T S
Iron administration remains one of the cornerstones of anemia management in CKD patients to improve hemoglobinlevels and ESA responsiveness [98] Iron supplementation iscurrently given in two general forms oral or parenteral Oraliron supplementation is the easiest and cheapest Howeveroral iron agents can have gastrointestinal side effects that limitadherence due to the formation of local reactive oxygenspecies and oxidative damage in the gut mucosa [143] More-over several studies have suggested that oral iron is less effec-
tive than parenteral iron particularly in hemodialysis patientsfor improving or preventing iron de1047297ciency ameliorating anemia or reducing ESA dose [98 144ndash146] The limited ef-fectiveness of oral iron supplements in this patient populationis likely due to medications such as antacids and phosphatebinders that inhibit iron entry into duodenal enterocytes andhepcidin excess that decreases ferroportin expression to limitiron release from duodenal enterocytes into the bloodstream(Figure 4)
There are several intravenous (IV) iron preparations thatcan be used to treat iron-restricted erythropoiesis in CKD
F U L L R E V I E W
K Zumbrennen-Bullough and JL Babitt 268
7252019 The Iron Cycle in Chronic Kidney Disease (CKD)
patients including iron dextrans iron sucrose ferric gluco-nate ferric carboxymaltose iron isomaltoside 1000 and feru-moxytol These preparations are generally comprising an ironcore shielded by a carbohydrate shell with different molecularweights and physiochemical properties yielding differentialdegradation kinetics and ability to release lsquofreersquo iron into thecirculation [143] This determines the maximal single dose foreach preparation with the newer higher molecular weightmore stable complexes enabling larger doses over shorter time
frames [143] Iron dextrans ( particularly high molecularweight dextrans) have been limited by dextran-induced ana-phylactic reactions in sim06ndash07 of patients [98] There issome limited data suggesting that various iron preparationsmay have different effects on markers of oxidative stress andin1047298ammation but this did not necessarily correlate with thecompoundsrsquo molecular weight stability or ability to releasefree iron into circulation [147 148] Comparative safety of these IV iron preparations in CKD patients remains largely unknown due to the lack of direct head-to-head clinical trials
Understanding the physiology of systemic iron balance andits pathophysiology in CKD and other iron disorders raisesseveral potential limitations shared by all IV iron preparations
Regardless of the iron preparation once the iron is taken upinto erythrocytes macrophages or other body storage siteshepcidin excess and ferroportin downregulation will limit theavailability of the iron for recycling and subsequent use More-over iron itself stimulates hepcidin expression and thereforecan paradoxically worsen the iron restriction (Figure 4)Additional concerns particularly with regard to repetitive ironadministration as ferritin levels rise are the potential foroxidant-mediated tissue injury from excess iron deposition asseen in iron overload disorders such as hemochromatosis Irondeposition has also been associated with the pathogenesis of many other common disorders including neurodegenerativediseases diabetes mellitus and atherosclerosis [1 149 150]Additionally withholding iron from invading pathogens is animportant function of the immune system and iron loading isassociated with worse outcomes in several infectious diseasesincluding malaria tuberculosis and HIV [151ndash153] Largeprospective randomized trials in the CKD population are long overdue to evaluate the ef 1047297cacy of repetitive IV iron adminis-tration with regard to hard clinical outcomes and long-termsafety to further characterize which patients will bene1047297t fromiron therapy and to determine treatment targets of irontherapy
N O V E L T R E A T M E N T S T R A T E G I E S F O R
I R O N - R E S T R I C T E D E R Y T H R O P O I E S I S I N
C K D P A T I E N T S
The understanding that hepcidin excess contributes to iron-re-stricted erythropoiesis in CKD patients has generated interestin developing new therapies that target the hepcidinndashferropor-tin axis to more directly address the underlying pathophysiol-ogy of this disease Such therapies would be expected toincrease iron availability from the diet and from the patients
own body iron stores and are a particularly attractive optionfor patients with higher ferritin levels
Several categories of hepcidinferroportin-based thera-peutics are currently in development (reviewed in [31]) Onecategory is direct hepcidin antagonists including anti-hepci-din antibodies other hepcidin-binding proteins (anticalins)hepcidin-binding spiegelmers and hepcidin siRNAs and anti-sense oligonucleotides [31] Dialysis itself also reduces hepci-din levels [121 154] but the levels quickly rebound [154]
potentially due in part to the induction of in1047298ammatory cyto-kines by the dialysis procedure as well as the high basal turn-over rate of hepcidin [155] Another category is agents thatinhibit hepcidin production by targeting either the BMP-SMAD signaling pathway or the IL6-STAT3 pathway [31]BMP-SMAD pathway inhibitors include anti-BMP6 anti-bodies sHJV linked to the constant region of IgG1 (HJVFc)small molecule BMP type I receptor antagonists (LDN-193189) and heparin (which has been shown to sequesterBMP ligands) [31 41 45 72 74 75 156] IL6-STAT3 pathway inhibitors include anti-IL6 antibodies (Siltuximab) anti-IL6receptor antibodies (Tocilizumab) JAK2 inhibitors (AG490)and STAT3 inhibitors (PpYLKTK) [31] ESAs and other
stimulators of ESA production such as prolyl hydroxylaseinhibitors also fall in this category since they inhibit hepcidinproduction A third category is ferroportin agonistsstabilizersincluding anti-ferroportin antibodies and thiol-reactive com-pounds that interfere with hepcidin binding to ferroportin aswell as agents that interfere with ferroportin internalization orpotentiate ferroportin synthesis [31] Notably many of theseagents have shown ef 1047297cacy for treating iron-restricted erythro-poiesis and anemia in animal models with anemia of chronicdisease [74 75 157ndash160] and several are currently in humanclinical trials [161ndash164] The safety and ef 1047297cacy of these agentsin human CKD patients compared with current treatmentstrategies remains to be determined
C O N C L U S I O N S
The last 13 years have yielded signi1047297cant advances in under-standing the molecular mechanisms underlying systemic ironbalance and its dysregulation in CKD patients These studieshold the promise for developing new rationally designed diag-nostic and therapeutic tools to improve anemia managementin CKD patients Novel therapies targeting hepcidin haveshown particular promise and several have already enteredhuman clinical trials More research is needed to better under-
stand the ef 1047297cacy long-term safety and targets of current irontherapies as well as novel hepcidin-lowering approaches inlarge prospective randomized controlled trials
A C K N O W L E D G E M E N T S
JLB was supported in part by NIH grant RO1-DK087727and a Howard Goodman Fellowship Award from the Massa-chusetts General Hospital
F UL L R E V I E W
T h e i r o n c y c l e i n C K D269
7252019 The Iron Cycle in Chronic Kidney Disease (CKD)
C O N F L I C T O F I N T E R E S T S T A T E M E N T
JLB has ownership interest in a start-up company FerruMax Pharmaceuticals which has licensed technology from theMassachusetts General Hospital based on the work cited hereand in prior publications
R E F E R E N C E S
1 Hentze MW Muckenthaler MU Andrews NC Balancing acts molecular
control of mammalian iron metabolism Cell 2004 117 285ndash297
2 Kovac S Anderson GJ Baldwin GS Gastrins iron homeostasis and col-
orectal cancer Biochim Biophys Acta 2011 1813 889ndash895
3 McKie AT Barrow D Latunde-Dada GO et al An iron-regulated ferric
reductase associated with the absorption of dietary iron Science 2001
291 1755ndash1759
4 Gunshin H Starr CN Direnzo C et al Cybrd1(duodenal cytochrome b)
is not necessary for dietary iron absorption in mice Blood 2005 16 16
5 Fleming MD Trenor CC III Su MA et al Microcytic anaemia mice have
a mutation in Nramp2 a candidate iron transporter gene Nat Genet
1997 16 383ndash386
6 Gunshin H Mackenzie B Berger UV et al Cloning and characterization
of a mammalian proton-ion transporter Nature 1997 388 482ndash488
7 Gunshin H Fujiwara Y Custodio AO et al Slc11a2 is required for intesti-
nal iron absorption and erythropoiesis but dispensable in placenta and
liver J Clin Invest 2005 115 1258ndash1266
8 Weintraub LR Weinstein MB Huser HJ et al Absorption of hemoglobin
iron the role of a heme-splitting substance in the intestinal mucosa J
Clin Invest 1968 47 531ndash539
9 Donovan A Brownlie A Zhou Y et al Positional cloning of zebra1047297sh fer-
roportin1 identi1047297es a conserved vertebrate iron exporter Nature 2000
403 776ndash781
10 Abboud S Haile DJ A novel mammalian iron-regulated protein involved
in intracellular iron metabolism J Biol Chem 2000 275 19906ndash19912
11 McKie AT Marciani P Rolfs A et al A novel duodenal iron-regulated
transporter IREG1 implicated in the basolateral transfer of iron to the
circulation Mol Cell 2000 5 299ndash309
12 Osaki S Johnson DA Mobilization of liver iron by ferroxidase (cerulo-plasmin) J Biol Chem 1969 244 5757ndash5758
13 Osaki S Johnson DA Frieden E The possible signi1047297cance of the ferrous
oxidase activity of ceruloplasmin in normal human serum J Biol Chem
1966 241 2746ndash2751
14 Roeser HP Lee GR Nacht S et al The role of ceruloplasmin in iron
metabolism J Clin Invest 1970 49 2408ndash2417
15 Vulpe CD Kuo YM Murphy TL et al Hephaestin a ceruloplasmin
homologue implicated in intestinal iron transport is defective in the sla
mouse Nat Genet 1999 21 195ndash199
16 Krause A Neitz S Maumlgert HJ et al LEAP-1 a novel highly disul1047297de-
bonded human peptide exhibits antimicrobial activity FEBS Lett 2000
480 147ndash150
17 Pigeon C Ilyin G Courselaud B et al A new mouse liver-speci1047297c gene
encoding a protein homologous to human antimicrobial peptide hepci-
din is overexpressed during iron overload J Biol Chem 2001 2767811ndash7819
18 Park CH Valore EV Waring AJ et al Hepcidin a urinary antimicrobial
peptide synthesized in the liver J Biol Chem 2001 276 7806ndash7810
19 Nicolas G Bennoun M Devaux I et al Lack of hepcidin gene expression
and severe tissue iron overload in upstream stimulatory factor 2 (USF2)
knockout mice Proc Natl Acad Sci USA 2001 98 8780ndash8785
20 Lesbordes-Brion JC Viatte L Bennoun M et al Targeted disruption of
the hepcidin 1 gene results in severe hemochromatosis Blood 2006 108
1402ndash1405
21 Roetto A Papanikolaou G Politou M et al Mutant antimicrobial peptide
hepcidin is associated with severe juvenile hemochromatosis Nat Genet
2003 33 21ndash22
22 Nicolas G Bennoun M Porteu A et al Severe iron de1047297ciency anemia in
C o p y r i g h t o f N e p h r o l o g y D i a l y s i s T r a n s p l a n t a t i o n i s t h e p r o p e r t y o f O x f o r d U n i v e r s i t y P r e s s
U S A a n d i t s c o n t e n t m a y n o t b e c o p i e d o r e m a i l e d t o m u l t i p l e s i t e s o r p o s t e d t o a l i s t s e r v
w i t h o u t t h e c o p y r i g h t h o l d e r s e x p r e s s w r i t t e n p e r m i s s i o n H o w e v e r u s e r s m a y p r i n t
d o w n l o a d o r e m a i l a r t i c l e s f o r i n d i v i d u a l u s e
7252019 The Iron Cycle in Chronic Kidney Disease (CKD)
hemodialysis patients [110 113] but can be impacted by blood storage time which leads to arti1047297cial RBC expansionthereby limiting utility in dialysis centers that use national lab-oratories [114]
Soluble transferrin receptor
Transferrin receptor 1 mediates uptake of iron into devel-oping RBCs Its expression and release into circulation assoluble transferrin receptor (sTFR) is increased in the setting
of iron de1047297ciency and increased erythroid activity Althoughthe literature on sTFR is limited a few studies have suggestedthat sTFR may be helpful to predict iron responsiveness [110113] However interpretation of this test in patients on ESAsis complicated by the fact that erythropoiesis itself increasessTFR levels [115] The use of this assay is also limited by lack of widespread availability and cost
Hepcidin
The understanding that hepcidin excess contributes to dis-ordered iron homeostasis in CKD patients has garnered inter-est in measuring hepcidin levels as a marker of iron statusiron responsiveness andor ESA responsiveness in CKD
patients There are two general types of assays now available tothe research community to measure circulating hepcidinlevels immunologic and mass spectrometry-based assaysBoth types of assays have their inherent strengths and weak-nesses and give an overall large variation in the absolute valuesof hepcidin levels but do show overall good correlation in rela-tive hepcidin levels with each other [116] Older assays thatalso recognize the precursor form of hepcidin (prohepcidin)are not useful because prohepcidin levels do not correlate withhepcidin biological activity [117 118] Using the more recentassays many studies have now con1047297rmed that circulating hep-cidin levels are increased in CKD patients with the highestlevels in patients on hemodialysis [99ndash101] Hepcidin levels inCKD patients have the strongest correlation with serum ferri-tin [100 101 119] but are also in1047298uenced at least in somestudies by in1047298ammation iron administration estimated glo-merular 1047297ltration rate dialysis clearance ESA dose and hemo-globin [100 101 119ndash121] One important limitation for theuse of hepcidin levels as a diagnostic tool in CKD patients isthe large intra-individual variability of both immunologic andmass spectrometry-based assays [122 123] Notably hepcidinlevels have not been shown to consistently predict responsive-ness or resistance to iron therapy or ESAs [ 120 124] Thus forthe time being there is no convincing evidence that hepcidinassays offer any advantage or additional information com-
pared with currently available diagnostic tests with regard toCKD iron and anemia management but this remains an areaof active investigation
Soluble HJV
Recent studies have explored the utility of measuring circu-lating levels of endogenous soluble HJV (sHJV) as a measureof iron status in human patients both without and with CKD[125ndash129] sHJV release from cells can be mediated by theproprotein convertase furin the transmembrane serine pro-tease TMPRSS6 and phospholipase C [130ndash134] and sHJV
has been detected in the conditioned media of transfected cellsand in the bloodstream of animals and humans [125ndash130135ndash137] While cell-surface GPI-anchored HJV functions asa BMP co-receptor to stimulate hepcidin expression (Figure 3)[39] sHJV can function as an inhibitor of BMP signaling andhepcidin expression presumably by sequestering BMP ligandsfrom interacting with cell surface signaling receptors [45 72135] Interestingly some studies have suggested that sHJVmay be decreased by iron treatment and increased by iron
de1047297ciency [125 130 135ndash137] suggesting that (i) sHJV couldbe useful as a diagnostic tool to indicate iron status and (ii) thegeneration sHJV could have a functional role to inhibit hepci-din expression in the context of iron de1047297ciency However oneimportant concern regarding these early human studies quan-titating sHJV levels is assay validity Indeed one commercialELISA assay used in studies focusing on CKD patients [128129] has subsequently been shown not to recognize HJV[138] Future studies will be needed using well-validated assaysand larger patient populations to determine if sHJV couldhave value as a diagnostic marker to guide iron therapy inCKD patients
Other markers
The putative role of GDF15 hepcidin regulation by erythro-poietic drive has generated interest in investigating this mol-ecule as a novel diagnostic tool for iron and anemiamanagement in CKD patients [139] However currently avail-able clinical data are very limited [139] Moreover while onestudy suggested that GDF15 may be increased by ironde1047297ciency [140] this was not robustly supported by anotherstudy [141] and GDF15 levels may also be in1047298uenced by in1047298ammation [141 142] malnutrition [142] and kidney disease [142] which may complicate its usefulness in thissetting
I R O N T H E R A P Y F O R C K D P A T I E N T S
Iron administration remains one of the cornerstones of anemia management in CKD patients to improve hemoglobinlevels and ESA responsiveness [98] Iron supplementation iscurrently given in two general forms oral or parenteral Oraliron supplementation is the easiest and cheapest Howeveroral iron agents can have gastrointestinal side effects that limitadherence due to the formation of local reactive oxygenspecies and oxidative damage in the gut mucosa [143] More-over several studies have suggested that oral iron is less effec-
tive than parenteral iron particularly in hemodialysis patientsfor improving or preventing iron de1047297ciency ameliorating anemia or reducing ESA dose [98 144ndash146] The limited ef-fectiveness of oral iron supplements in this patient populationis likely due to medications such as antacids and phosphatebinders that inhibit iron entry into duodenal enterocytes andhepcidin excess that decreases ferroportin expression to limitiron release from duodenal enterocytes into the bloodstream(Figure 4)
There are several intravenous (IV) iron preparations thatcan be used to treat iron-restricted erythropoiesis in CKD
F U L L R E V I E W
K Zumbrennen-Bullough and JL Babitt 268
7252019 The Iron Cycle in Chronic Kidney Disease (CKD)
patients including iron dextrans iron sucrose ferric gluco-nate ferric carboxymaltose iron isomaltoside 1000 and feru-moxytol These preparations are generally comprising an ironcore shielded by a carbohydrate shell with different molecularweights and physiochemical properties yielding differentialdegradation kinetics and ability to release lsquofreersquo iron into thecirculation [143] This determines the maximal single dose foreach preparation with the newer higher molecular weightmore stable complexes enabling larger doses over shorter time
frames [143] Iron dextrans ( particularly high molecularweight dextrans) have been limited by dextran-induced ana-phylactic reactions in sim06ndash07 of patients [98] There issome limited data suggesting that various iron preparationsmay have different effects on markers of oxidative stress andin1047298ammation but this did not necessarily correlate with thecompoundsrsquo molecular weight stability or ability to releasefree iron into circulation [147 148] Comparative safety of these IV iron preparations in CKD patients remains largely unknown due to the lack of direct head-to-head clinical trials
Understanding the physiology of systemic iron balance andits pathophysiology in CKD and other iron disorders raisesseveral potential limitations shared by all IV iron preparations
Regardless of the iron preparation once the iron is taken upinto erythrocytes macrophages or other body storage siteshepcidin excess and ferroportin downregulation will limit theavailability of the iron for recycling and subsequent use More-over iron itself stimulates hepcidin expression and thereforecan paradoxically worsen the iron restriction (Figure 4)Additional concerns particularly with regard to repetitive ironadministration as ferritin levels rise are the potential foroxidant-mediated tissue injury from excess iron deposition asseen in iron overload disorders such as hemochromatosis Irondeposition has also been associated with the pathogenesis of many other common disorders including neurodegenerativediseases diabetes mellitus and atherosclerosis [1 149 150]Additionally withholding iron from invading pathogens is animportant function of the immune system and iron loading isassociated with worse outcomes in several infectious diseasesincluding malaria tuberculosis and HIV [151ndash153] Largeprospective randomized trials in the CKD population are long overdue to evaluate the ef 1047297cacy of repetitive IV iron adminis-tration with regard to hard clinical outcomes and long-termsafety to further characterize which patients will bene1047297t fromiron therapy and to determine treatment targets of irontherapy
N O V E L T R E A T M E N T S T R A T E G I E S F O R
I R O N - R E S T R I C T E D E R Y T H R O P O I E S I S I N
C K D P A T I E N T S
The understanding that hepcidin excess contributes to iron-re-stricted erythropoiesis in CKD patients has generated interestin developing new therapies that target the hepcidinndashferropor-tin axis to more directly address the underlying pathophysiol-ogy of this disease Such therapies would be expected toincrease iron availability from the diet and from the patients
own body iron stores and are a particularly attractive optionfor patients with higher ferritin levels
Several categories of hepcidinferroportin-based thera-peutics are currently in development (reviewed in [31]) Onecategory is direct hepcidin antagonists including anti-hepci-din antibodies other hepcidin-binding proteins (anticalins)hepcidin-binding spiegelmers and hepcidin siRNAs and anti-sense oligonucleotides [31] Dialysis itself also reduces hepci-din levels [121 154] but the levels quickly rebound [154]
potentially due in part to the induction of in1047298ammatory cyto-kines by the dialysis procedure as well as the high basal turn-over rate of hepcidin [155] Another category is agents thatinhibit hepcidin production by targeting either the BMP-SMAD signaling pathway or the IL6-STAT3 pathway [31]BMP-SMAD pathway inhibitors include anti-BMP6 anti-bodies sHJV linked to the constant region of IgG1 (HJVFc)small molecule BMP type I receptor antagonists (LDN-193189) and heparin (which has been shown to sequesterBMP ligands) [31 41 45 72 74 75 156] IL6-STAT3 pathway inhibitors include anti-IL6 antibodies (Siltuximab) anti-IL6receptor antibodies (Tocilizumab) JAK2 inhibitors (AG490)and STAT3 inhibitors (PpYLKTK) [31] ESAs and other
stimulators of ESA production such as prolyl hydroxylaseinhibitors also fall in this category since they inhibit hepcidinproduction A third category is ferroportin agonistsstabilizersincluding anti-ferroportin antibodies and thiol-reactive com-pounds that interfere with hepcidin binding to ferroportin aswell as agents that interfere with ferroportin internalization orpotentiate ferroportin synthesis [31] Notably many of theseagents have shown ef 1047297cacy for treating iron-restricted erythro-poiesis and anemia in animal models with anemia of chronicdisease [74 75 157ndash160] and several are currently in humanclinical trials [161ndash164] The safety and ef 1047297cacy of these agentsin human CKD patients compared with current treatmentstrategies remains to be determined
C O N C L U S I O N S
The last 13 years have yielded signi1047297cant advances in under-standing the molecular mechanisms underlying systemic ironbalance and its dysregulation in CKD patients These studieshold the promise for developing new rationally designed diag-nostic and therapeutic tools to improve anemia managementin CKD patients Novel therapies targeting hepcidin haveshown particular promise and several have already enteredhuman clinical trials More research is needed to better under-
stand the ef 1047297cacy long-term safety and targets of current irontherapies as well as novel hepcidin-lowering approaches inlarge prospective randomized controlled trials
A C K N O W L E D G E M E N T S
JLB was supported in part by NIH grant RO1-DK087727and a Howard Goodman Fellowship Award from the Massa-chusetts General Hospital
F UL L R E V I E W
T h e i r o n c y c l e i n C K D269
7252019 The Iron Cycle in Chronic Kidney Disease (CKD)
C O N F L I C T O F I N T E R E S T S T A T E M E N T
JLB has ownership interest in a start-up company FerruMax Pharmaceuticals which has licensed technology from theMassachusetts General Hospital based on the work cited hereand in prior publications
R E F E R E N C E S
1 Hentze MW Muckenthaler MU Andrews NC Balancing acts molecular
control of mammalian iron metabolism Cell 2004 117 285ndash297
2 Kovac S Anderson GJ Baldwin GS Gastrins iron homeostasis and col-
orectal cancer Biochim Biophys Acta 2011 1813 889ndash895
3 McKie AT Barrow D Latunde-Dada GO et al An iron-regulated ferric
reductase associated with the absorption of dietary iron Science 2001
291 1755ndash1759
4 Gunshin H Starr CN Direnzo C et al Cybrd1(duodenal cytochrome b)
is not necessary for dietary iron absorption in mice Blood 2005 16 16
5 Fleming MD Trenor CC III Su MA et al Microcytic anaemia mice have
a mutation in Nramp2 a candidate iron transporter gene Nat Genet
1997 16 383ndash386
6 Gunshin H Mackenzie B Berger UV et al Cloning and characterization
of a mammalian proton-ion transporter Nature 1997 388 482ndash488
7 Gunshin H Fujiwara Y Custodio AO et al Slc11a2 is required for intesti-
nal iron absorption and erythropoiesis but dispensable in placenta and
liver J Clin Invest 2005 115 1258ndash1266
8 Weintraub LR Weinstein MB Huser HJ et al Absorption of hemoglobin
iron the role of a heme-splitting substance in the intestinal mucosa J
Clin Invest 1968 47 531ndash539
9 Donovan A Brownlie A Zhou Y et al Positional cloning of zebra1047297sh fer-
roportin1 identi1047297es a conserved vertebrate iron exporter Nature 2000
403 776ndash781
10 Abboud S Haile DJ A novel mammalian iron-regulated protein involved
in intracellular iron metabolism J Biol Chem 2000 275 19906ndash19912
11 McKie AT Marciani P Rolfs A et al A novel duodenal iron-regulated
transporter IREG1 implicated in the basolateral transfer of iron to the
circulation Mol Cell 2000 5 299ndash309
12 Osaki S Johnson DA Mobilization of liver iron by ferroxidase (cerulo-plasmin) J Biol Chem 1969 244 5757ndash5758
13 Osaki S Johnson DA Frieden E The possible signi1047297cance of the ferrous
oxidase activity of ceruloplasmin in normal human serum J Biol Chem
1966 241 2746ndash2751
14 Roeser HP Lee GR Nacht S et al The role of ceruloplasmin in iron
metabolism J Clin Invest 1970 49 2408ndash2417
15 Vulpe CD Kuo YM Murphy TL et al Hephaestin a ceruloplasmin
homologue implicated in intestinal iron transport is defective in the sla
mouse Nat Genet 1999 21 195ndash199
16 Krause A Neitz S Maumlgert HJ et al LEAP-1 a novel highly disul1047297de-
bonded human peptide exhibits antimicrobial activity FEBS Lett 2000
480 147ndash150
17 Pigeon C Ilyin G Courselaud B et al A new mouse liver-speci1047297c gene
encoding a protein homologous to human antimicrobial peptide hepci-
din is overexpressed during iron overload J Biol Chem 2001 2767811ndash7819
18 Park CH Valore EV Waring AJ et al Hepcidin a urinary antimicrobial
peptide synthesized in the liver J Biol Chem 2001 276 7806ndash7810
19 Nicolas G Bennoun M Devaux I et al Lack of hepcidin gene expression
and severe tissue iron overload in upstream stimulatory factor 2 (USF2)
knockout mice Proc Natl Acad Sci USA 2001 98 8780ndash8785
20 Lesbordes-Brion JC Viatte L Bennoun M et al Targeted disruption of
the hepcidin 1 gene results in severe hemochromatosis Blood 2006 108
1402ndash1405
21 Roetto A Papanikolaou G Politou M et al Mutant antimicrobial peptide
hepcidin is associated with severe juvenile hemochromatosis Nat Genet
2003 33 21ndash22
22 Nicolas G Bennoun M Porteu A et al Severe iron de1047297ciency anemia in
C o p y r i g h t o f N e p h r o l o g y D i a l y s i s T r a n s p l a n t a t i o n i s t h e p r o p e r t y o f O x f o r d U n i v e r s i t y P r e s s
U S A a n d i t s c o n t e n t m a y n o t b e c o p i e d o r e m a i l e d t o m u l t i p l e s i t e s o r p o s t e d t o a l i s t s e r v
w i t h o u t t h e c o p y r i g h t h o l d e r s e x p r e s s w r i t t e n p e r m i s s i o n H o w e v e r u s e r s m a y p r i n t
d o w n l o a d o r e m a i l a r t i c l e s f o r i n d i v i d u a l u s e
7252019 The Iron Cycle in Chronic Kidney Disease (CKD)
patients including iron dextrans iron sucrose ferric gluco-nate ferric carboxymaltose iron isomaltoside 1000 and feru-moxytol These preparations are generally comprising an ironcore shielded by a carbohydrate shell with different molecularweights and physiochemical properties yielding differentialdegradation kinetics and ability to release lsquofreersquo iron into thecirculation [143] This determines the maximal single dose foreach preparation with the newer higher molecular weightmore stable complexes enabling larger doses over shorter time
frames [143] Iron dextrans ( particularly high molecularweight dextrans) have been limited by dextran-induced ana-phylactic reactions in sim06ndash07 of patients [98] There issome limited data suggesting that various iron preparationsmay have different effects on markers of oxidative stress andin1047298ammation but this did not necessarily correlate with thecompoundsrsquo molecular weight stability or ability to releasefree iron into circulation [147 148] Comparative safety of these IV iron preparations in CKD patients remains largely unknown due to the lack of direct head-to-head clinical trials
Understanding the physiology of systemic iron balance andits pathophysiology in CKD and other iron disorders raisesseveral potential limitations shared by all IV iron preparations
Regardless of the iron preparation once the iron is taken upinto erythrocytes macrophages or other body storage siteshepcidin excess and ferroportin downregulation will limit theavailability of the iron for recycling and subsequent use More-over iron itself stimulates hepcidin expression and thereforecan paradoxically worsen the iron restriction (Figure 4)Additional concerns particularly with regard to repetitive ironadministration as ferritin levels rise are the potential foroxidant-mediated tissue injury from excess iron deposition asseen in iron overload disorders such as hemochromatosis Irondeposition has also been associated with the pathogenesis of many other common disorders including neurodegenerativediseases diabetes mellitus and atherosclerosis [1 149 150]Additionally withholding iron from invading pathogens is animportant function of the immune system and iron loading isassociated with worse outcomes in several infectious diseasesincluding malaria tuberculosis and HIV [151ndash153] Largeprospective randomized trials in the CKD population are long overdue to evaluate the ef 1047297cacy of repetitive IV iron adminis-tration with regard to hard clinical outcomes and long-termsafety to further characterize which patients will bene1047297t fromiron therapy and to determine treatment targets of irontherapy
N O V E L T R E A T M E N T S T R A T E G I E S F O R
I R O N - R E S T R I C T E D E R Y T H R O P O I E S I S I N
C K D P A T I E N T S
The understanding that hepcidin excess contributes to iron-re-stricted erythropoiesis in CKD patients has generated interestin developing new therapies that target the hepcidinndashferropor-tin axis to more directly address the underlying pathophysiol-ogy of this disease Such therapies would be expected toincrease iron availability from the diet and from the patients
own body iron stores and are a particularly attractive optionfor patients with higher ferritin levels
Several categories of hepcidinferroportin-based thera-peutics are currently in development (reviewed in [31]) Onecategory is direct hepcidin antagonists including anti-hepci-din antibodies other hepcidin-binding proteins (anticalins)hepcidin-binding spiegelmers and hepcidin siRNAs and anti-sense oligonucleotides [31] Dialysis itself also reduces hepci-din levels [121 154] but the levels quickly rebound [154]
potentially due in part to the induction of in1047298ammatory cyto-kines by the dialysis procedure as well as the high basal turn-over rate of hepcidin [155] Another category is agents thatinhibit hepcidin production by targeting either the BMP-SMAD signaling pathway or the IL6-STAT3 pathway [31]BMP-SMAD pathway inhibitors include anti-BMP6 anti-bodies sHJV linked to the constant region of IgG1 (HJVFc)small molecule BMP type I receptor antagonists (LDN-193189) and heparin (which has been shown to sequesterBMP ligands) [31 41 45 72 74 75 156] IL6-STAT3 pathway inhibitors include anti-IL6 antibodies (Siltuximab) anti-IL6receptor antibodies (Tocilizumab) JAK2 inhibitors (AG490)and STAT3 inhibitors (PpYLKTK) [31] ESAs and other
stimulators of ESA production such as prolyl hydroxylaseinhibitors also fall in this category since they inhibit hepcidinproduction A third category is ferroportin agonistsstabilizersincluding anti-ferroportin antibodies and thiol-reactive com-pounds that interfere with hepcidin binding to ferroportin aswell as agents that interfere with ferroportin internalization orpotentiate ferroportin synthesis [31] Notably many of theseagents have shown ef 1047297cacy for treating iron-restricted erythro-poiesis and anemia in animal models with anemia of chronicdisease [74 75 157ndash160] and several are currently in humanclinical trials [161ndash164] The safety and ef 1047297cacy of these agentsin human CKD patients compared with current treatmentstrategies remains to be determined
C O N C L U S I O N S
The last 13 years have yielded signi1047297cant advances in under-standing the molecular mechanisms underlying systemic ironbalance and its dysregulation in CKD patients These studieshold the promise for developing new rationally designed diag-nostic and therapeutic tools to improve anemia managementin CKD patients Novel therapies targeting hepcidin haveshown particular promise and several have already enteredhuman clinical trials More research is needed to better under-
stand the ef 1047297cacy long-term safety and targets of current irontherapies as well as novel hepcidin-lowering approaches inlarge prospective randomized controlled trials
A C K N O W L E D G E M E N T S
JLB was supported in part by NIH grant RO1-DK087727and a Howard Goodman Fellowship Award from the Massa-chusetts General Hospital
F UL L R E V I E W
T h e i r o n c y c l e i n C K D269
7252019 The Iron Cycle in Chronic Kidney Disease (CKD)
C O N F L I C T O F I N T E R E S T S T A T E M E N T
JLB has ownership interest in a start-up company FerruMax Pharmaceuticals which has licensed technology from theMassachusetts General Hospital based on the work cited hereand in prior publications
R E F E R E N C E S
1 Hentze MW Muckenthaler MU Andrews NC Balancing acts molecular
control of mammalian iron metabolism Cell 2004 117 285ndash297
2 Kovac S Anderson GJ Baldwin GS Gastrins iron homeostasis and col-
orectal cancer Biochim Biophys Acta 2011 1813 889ndash895
3 McKie AT Barrow D Latunde-Dada GO et al An iron-regulated ferric
reductase associated with the absorption of dietary iron Science 2001
291 1755ndash1759
4 Gunshin H Starr CN Direnzo C et al Cybrd1(duodenal cytochrome b)
is not necessary for dietary iron absorption in mice Blood 2005 16 16
5 Fleming MD Trenor CC III Su MA et al Microcytic anaemia mice have
a mutation in Nramp2 a candidate iron transporter gene Nat Genet
1997 16 383ndash386
6 Gunshin H Mackenzie B Berger UV et al Cloning and characterization
of a mammalian proton-ion transporter Nature 1997 388 482ndash488
7 Gunshin H Fujiwara Y Custodio AO et al Slc11a2 is required for intesti-
nal iron absorption and erythropoiesis but dispensable in placenta and
liver J Clin Invest 2005 115 1258ndash1266
8 Weintraub LR Weinstein MB Huser HJ et al Absorption of hemoglobin
iron the role of a heme-splitting substance in the intestinal mucosa J
Clin Invest 1968 47 531ndash539
9 Donovan A Brownlie A Zhou Y et al Positional cloning of zebra1047297sh fer-
roportin1 identi1047297es a conserved vertebrate iron exporter Nature 2000
403 776ndash781
10 Abboud S Haile DJ A novel mammalian iron-regulated protein involved
in intracellular iron metabolism J Biol Chem 2000 275 19906ndash19912
11 McKie AT Marciani P Rolfs A et al A novel duodenal iron-regulated
transporter IREG1 implicated in the basolateral transfer of iron to the
circulation Mol Cell 2000 5 299ndash309
12 Osaki S Johnson DA Mobilization of liver iron by ferroxidase (cerulo-plasmin) J Biol Chem 1969 244 5757ndash5758
13 Osaki S Johnson DA Frieden E The possible signi1047297cance of the ferrous
oxidase activity of ceruloplasmin in normal human serum J Biol Chem
1966 241 2746ndash2751
14 Roeser HP Lee GR Nacht S et al The role of ceruloplasmin in iron
metabolism J Clin Invest 1970 49 2408ndash2417
15 Vulpe CD Kuo YM Murphy TL et al Hephaestin a ceruloplasmin
homologue implicated in intestinal iron transport is defective in the sla
mouse Nat Genet 1999 21 195ndash199
16 Krause A Neitz S Maumlgert HJ et al LEAP-1 a novel highly disul1047297de-
bonded human peptide exhibits antimicrobial activity FEBS Lett 2000
480 147ndash150
17 Pigeon C Ilyin G Courselaud B et al A new mouse liver-speci1047297c gene
encoding a protein homologous to human antimicrobial peptide hepci-
din is overexpressed during iron overload J Biol Chem 2001 2767811ndash7819
18 Park CH Valore EV Waring AJ et al Hepcidin a urinary antimicrobial
peptide synthesized in the liver J Biol Chem 2001 276 7806ndash7810
19 Nicolas G Bennoun M Devaux I et al Lack of hepcidin gene expression
and severe tissue iron overload in upstream stimulatory factor 2 (USF2)
knockout mice Proc Natl Acad Sci USA 2001 98 8780ndash8785
20 Lesbordes-Brion JC Viatte L Bennoun M et al Targeted disruption of
the hepcidin 1 gene results in severe hemochromatosis Blood 2006 108
1402ndash1405
21 Roetto A Papanikolaou G Politou M et al Mutant antimicrobial peptide
hepcidin is associated with severe juvenile hemochromatosis Nat Genet
2003 33 21ndash22
22 Nicolas G Bennoun M Porteu A et al Severe iron de1047297ciency anemia in
C o p y r i g h t o f N e p h r o l o g y D i a l y s i s T r a n s p l a n t a t i o n i s t h e p r o p e r t y o f O x f o r d U n i v e r s i t y P r e s s
U S A a n d i t s c o n t e n t m a y n o t b e c o p i e d o r e m a i l e d t o m u l t i p l e s i t e s o r p o s t e d t o a l i s t s e r v
w i t h o u t t h e c o p y r i g h t h o l d e r s e x p r e s s w r i t t e n p e r m i s s i o n H o w e v e r u s e r s m a y p r i n t
d o w n l o a d o r e m a i l a r t i c l e s f o r i n d i v i d u a l u s e
7252019 The Iron Cycle in Chronic Kidney Disease (CKD)
C O N F L I C T O F I N T E R E S T S T A T E M E N T
JLB has ownership interest in a start-up company FerruMax Pharmaceuticals which has licensed technology from theMassachusetts General Hospital based on the work cited hereand in prior publications
R E F E R E N C E S
1 Hentze MW Muckenthaler MU Andrews NC Balancing acts molecular
control of mammalian iron metabolism Cell 2004 117 285ndash297
2 Kovac S Anderson GJ Baldwin GS Gastrins iron homeostasis and col-
orectal cancer Biochim Biophys Acta 2011 1813 889ndash895
3 McKie AT Barrow D Latunde-Dada GO et al An iron-regulated ferric
reductase associated with the absorption of dietary iron Science 2001
291 1755ndash1759
4 Gunshin H Starr CN Direnzo C et al Cybrd1(duodenal cytochrome b)
is not necessary for dietary iron absorption in mice Blood 2005 16 16
5 Fleming MD Trenor CC III Su MA et al Microcytic anaemia mice have
a mutation in Nramp2 a candidate iron transporter gene Nat Genet
1997 16 383ndash386
6 Gunshin H Mackenzie B Berger UV et al Cloning and characterization
of a mammalian proton-ion transporter Nature 1997 388 482ndash488
7 Gunshin H Fujiwara Y Custodio AO et al Slc11a2 is required for intesti-
nal iron absorption and erythropoiesis but dispensable in placenta and
liver J Clin Invest 2005 115 1258ndash1266
8 Weintraub LR Weinstein MB Huser HJ et al Absorption of hemoglobin
iron the role of a heme-splitting substance in the intestinal mucosa J
Clin Invest 1968 47 531ndash539
9 Donovan A Brownlie A Zhou Y et al Positional cloning of zebra1047297sh fer-
roportin1 identi1047297es a conserved vertebrate iron exporter Nature 2000
403 776ndash781
10 Abboud S Haile DJ A novel mammalian iron-regulated protein involved
in intracellular iron metabolism J Biol Chem 2000 275 19906ndash19912
11 McKie AT Marciani P Rolfs A et al A novel duodenal iron-regulated
transporter IREG1 implicated in the basolateral transfer of iron to the
circulation Mol Cell 2000 5 299ndash309
12 Osaki S Johnson DA Mobilization of liver iron by ferroxidase (cerulo-plasmin) J Biol Chem 1969 244 5757ndash5758
13 Osaki S Johnson DA Frieden E The possible signi1047297cance of the ferrous
oxidase activity of ceruloplasmin in normal human serum J Biol Chem
1966 241 2746ndash2751
14 Roeser HP Lee GR Nacht S et al The role of ceruloplasmin in iron
metabolism J Clin Invest 1970 49 2408ndash2417
15 Vulpe CD Kuo YM Murphy TL et al Hephaestin a ceruloplasmin
homologue implicated in intestinal iron transport is defective in the sla
mouse Nat Genet 1999 21 195ndash199
16 Krause A Neitz S Maumlgert HJ et al LEAP-1 a novel highly disul1047297de-
bonded human peptide exhibits antimicrobial activity FEBS Lett 2000
480 147ndash150
17 Pigeon C Ilyin G Courselaud B et al A new mouse liver-speci1047297c gene
encoding a protein homologous to human antimicrobial peptide hepci-
din is overexpressed during iron overload J Biol Chem 2001 2767811ndash7819
18 Park CH Valore EV Waring AJ et al Hepcidin a urinary antimicrobial
peptide synthesized in the liver J Biol Chem 2001 276 7806ndash7810
19 Nicolas G Bennoun M Devaux I et al Lack of hepcidin gene expression
and severe tissue iron overload in upstream stimulatory factor 2 (USF2)
knockout mice Proc Natl Acad Sci USA 2001 98 8780ndash8785
20 Lesbordes-Brion JC Viatte L Bennoun M et al Targeted disruption of
the hepcidin 1 gene results in severe hemochromatosis Blood 2006 108
1402ndash1405
21 Roetto A Papanikolaou G Politou M et al Mutant antimicrobial peptide
hepcidin is associated with severe juvenile hemochromatosis Nat Genet
2003 33 21ndash22
22 Nicolas G Bennoun M Porteu A et al Severe iron de1047297ciency anemia in
C o p y r i g h t o f N e p h r o l o g y D i a l y s i s T r a n s p l a n t a t i o n i s t h e p r o p e r t y o f O x f o r d U n i v e r s i t y P r e s s
U S A a n d i t s c o n t e n t m a y n o t b e c o p i e d o r e m a i l e d t o m u l t i p l e s i t e s o r p o s t e d t o a l i s t s e r v
w i t h o u t t h e c o p y r i g h t h o l d e r s e x p r e s s w r i t t e n p e r m i s s i o n H o w e v e r u s e r s m a y p r i n t
d o w n l o a d o r e m a i l a r t i c l e s f o r i n d i v i d u a l u s e
7252019 The Iron Cycle in Chronic Kidney Disease (CKD)
C o p y r i g h t o f N e p h r o l o g y D i a l y s i s T r a n s p l a n t a t i o n i s t h e p r o p e r t y o f O x f o r d U n i v e r s i t y P r e s s
U S A a n d i t s c o n t e n t m a y n o t b e c o p i e d o r e m a i l e d t o m u l t i p l e s i t e s o r p o s t e d t o a l i s t s e r v
w i t h o u t t h e c o p y r i g h t h o l d e r s e x p r e s s w r i t t e n p e r m i s s i o n H o w e v e r u s e r s m a y p r i n t
d o w n l o a d o r e m a i l a r t i c l e s f o r i n d i v i d u a l u s e
7252019 The Iron Cycle in Chronic Kidney Disease (CKD)
C o p y r i g h t o f N e p h r o l o g y D i a l y s i s T r a n s p l a n t a t i o n i s t h e p r o p e r t y o f O x f o r d U n i v e r s i t y P r e s s
U S A a n d i t s c o n t e n t m a y n o t b e c o p i e d o r e m a i l e d t o m u l t i p l e s i t e s o r p o s t e d t o a l i s t s e r v
w i t h o u t t h e c o p y r i g h t h o l d e r s e x p r e s s w r i t t e n p e r m i s s i o n H o w e v e r u s e r s m a y p r i n t
d o w n l o a d o r e m a i l a r t i c l e s f o r i n d i v i d u a l u s e
7252019 The Iron Cycle in Chronic Kidney Disease (CKD)
C o p y r i g h t o f N e p h r o l o g y D i a l y s i s T r a n s p l a n t a t i o n i s t h e p r o p e r t y o f O x f o r d U n i v e r s i t y P r e s s
U S A a n d i t s c o n t e n t m a y n o t b e c o p i e d o r e m a i l e d t o m u l t i p l e s i t e s o r p o s t e d t o a l i s t s e r v
w i t h o u t t h e c o p y r i g h t h o l d e r s e x p r e s s w r i t t e n p e r m i s s i o n H o w e v e r u s e r s m a y p r i n t
d o w n l o a d o r e m a i l a r t i c l e s f o r i n d i v i d u a l u s e
7252019 The Iron Cycle in Chronic Kidney Disease (CKD)
C o p y r i g h t o f N e p h r o l o g y D i a l y s i s T r a n s p l a n t a t i o n i s t h e p r o p e r t y o f O x f o r d U n i v e r s i t y P r e s s
U S A a n d i t s c o n t e n t m a y n o t b e c o p i e d o r e m a i l e d t o m u l t i p l e s i t e s o r p o s t e d t o a l i s t s e r v
w i t h o u t t h e c o p y r i g h t h o l d e r s e x p r e s s w r i t t e n p e r m i s s i o n H o w e v e r u s e r s m a y p r i n t
d o w n l o a d o r e m a i l a r t i c l e s f o r i n d i v i d u a l u s e