HEPCIDINHEPCIDIN--25 25 - A USEFUL CLINICAL - A USEFUL CLINICAL GUIDE GUIDE FOR IRON-RESTRICTED FOR IRON-RESTRICTED

ERYTHROPOIESIS DETECTION IN ERYTHROPOIESIS DETECTION IN HAEMODIALYSIS PATIENTSHAEMODIALYSIS PATIENTS

Lavinia-Oltița BrătescuLavinia-Oltița Brătescu11, Liliana Bârsan, Liliana Bârsan33, Liliana , Liliana GGâârneațărneață2,32,3, Ana Stanciu, Ana Stanciu33, Mariana Lipan, Mariana Lipan11,Simona ,Simona StancuStancu2,3 2,3 and Gabriel Mircescuand Gabriel Mircescu2,32,3

11 ““Sf PantelimonSf Pantelimon““ International HealthcareInternational Healthcare SystemsSystems Nephrology Nephrology and Dialysis Medical Center, Bucharest, Romaniaand Dialysis Medical Center, Bucharest, Romania

2.2. Internal Medicine and Nephrology Dept. "Carol Davila" Internal Medicine and Nephrology Dept. "Carol Davila" University of Medicine and Pharmacy, Bucharest, University of Medicine and Pharmacy, Bucharest, Romania;Romania;

33 - Nephrology Dept. "Dr Carol Davila" Teaching Hospital of - Nephrology Dept. "Dr Carol Davila" Teaching Hospital of Nephrology, Bucharest, RomaniaNephrology, Bucharest, Romania

HepcidinHepcidin

• Low molecular weight polypeptide (2-3kDa)• Contains 8 cysteine residues which form 4 disulphide bonds,

stability of the molecule being ensured by the clip shape• The last 5 amino acids are important for binding to

ferroportin• Hepcidin seems to be the “missing link” in the

regulation of iron metabolism

Hepcidin syntesis and actions Hepcidin syntesis and actions Hepcidin isHepcidin is produced by the liver as produced by the liver as prohepcidinprohepcidin ( (8484 aa) and aa) and

thereafter converted to the active peptide thereafter converted to the active peptide hepcidin hepcidin (2(255 aa) aa)

Hepcidin accelerates the degradation of Hepcidin accelerates the degradation of ferroportin - ferroportin - the main the main cellular iron transporter in enterocyte, hepatocyte and cellular iron transporter in enterocyte, hepatocyte and macrophage - and macrophage - and induces hypoferrinemiainduces hypoferrinemia by inhibiting by inhibiting intestinal iron absorbtion and iron recyclingintestinal iron absorbtion and iron recycling

Yujie Cui, Qingyu Wu and Yiqing Zhou . Kidney Int. 23 Sept 2009; doi:10.1038/ki. 2009.357

Hepcidin actions Hepcidin actions are thought toare thought to prpreventevent iron overloadiron overload by inhibiting intestinal iron by inhibiting intestinal iron

absorbtionabsorbtion pprotectrotect against infectionagainst infectionss, by hypoferrinemia which , by hypoferrinemia which

limits the limits the iron-dependent multiplication of invading iron-dependent multiplication of invading bacteria.bacteria.

Hepcidin regulationHepcidin regulation reflects this dual role reflects this dual role llow hepcidin levelsow hepcidin levels were associated with a were associated with anemia, nemia,

dedepletepletedd iron stores, increaseiron stores, increasedd iron needs and iron needs and active active erythropoiesiserythropoiesis, while , while high hepcidin levelshigh hepcidin levels were found in were found in case of increased iron scase of increased iron sttoresores

hepcidin hepcidin is an acute phase reactantis an acute phase reactant, modulated by IL-6, modulated by IL-6.. Hepcidin excretionHepcidin excretion is unknown, but is unknown, but renal renal oror dialysis dialysis

depurationdepuration could be important could be important in defining in defining hepcidin levelhepcidin levels.s. Hepcidin integrates iron absorbtion, storage and Hepcidin integrates iron absorbtion, storage and

mobilisation from storage with mobilisation from storage with the the requirements of requirements of erythropoiesiserythropoiesis

Hepcidin in CKDHepcidin in CKD

High hepcidin levels were reported in CKD High hepcidin levels were reported in CKD patients andpatients andcould result fromcould result from

InflammationInflammation Increased iron stores resulting either from reduced (low Increased iron stores resulting either from reduced (low

epoetin driven) or ineffective erythropoiesis as well as epoetin driven) or ineffective erythropoiesis as well as overzealous iron therapyoverzealous iron therapy

Decreased hepcidin depuration by the failing kidneyDecreased hepcidin depuration by the failing kidney

could be involved in could be involved in iron restricted erythropoiesisiron restricted erythropoiesis

could be a markercould be a marker of iron status, useful to guide of iron status, useful to guide therapytherapy

Absolute iron deficiency

Absolute iron deficiency

Functional iron

deficiency

Blocked iron stores

Iron restricted erythropoiesis

Anaemia ESA responsivenessAnaemia

Iron

ther

apy

Iron therapy

Iron therapy

HypothesisHypothesis…c…controversisontroversis

Iron metabolism aIron metabolism abnormalities bnormalities are are common common inin renal anemia renal anemia Iron restricted erythropoiesis is determined in almost half of Iron restricted erythropoiesis is determined in almost half of

cases by cases by the the association between iron deficiency and iron association between iron deficiency and iron immobilization in immobilization in macrophagesmacrophages;;

Peripheral iron indices have limited diagnostics utility:Peripheral iron indices have limited diagnostics utility: Hepcidin could be a diagnostic tool and a therapeutic target in iron Hepcidin could be a diagnostic tool and a therapeutic target in iron

deficiencydeficiency The therapeutic tThe therapeutic test (est (parenteral iron supplementationparenteral iron supplementation) ) allows iron allows iron

therapy tailoringtherapy tailoring Iron deficiency is the most common manageable cause of low Iron deficiency is the most common manageable cause of low

ESA responsiveness; ESA responsiveness; The correction of iron-restricted erythropoiesis allows to avoid The correction of iron-restricted erythropoiesis allows to avoid

(30-40%) or to reduce ESA doses by 20-40%.(30-40%) or to reduce ESA doses by 20-40%.

Iron metabolism aIron metabolism abnormalities bnormalities are are common common inin renal anemia renal anemia Iron restricted erythropoiesis is determined in almost half of Iron restricted erythropoiesis is determined in almost half of

cases by cases by the the association between iron deficiency and iron association between iron deficiency and iron immobilization in immobilization in macrophagesmacrophages;;

Peripheral iron indices have limited diagnostics utility:Peripheral iron indices have limited diagnostics utility: Hepcidin could be a diagnostic tool and a therapeutic target in iron Hepcidin could be a diagnostic tool and a therapeutic target in iron

deficiencydeficiency The therapeutic tThe therapeutic test (est (parenteral iron supplementationparenteral iron supplementation) ) allows iron allows iron

therapy tailoringtherapy tailoring Iron deficiency is the most common manageable cause of low Iron deficiency is the most common manageable cause of low

ESA responsiveness; ESA responsiveness; The correction of iron-restricted erythropoiesis allows to avoid The correction of iron-restricted erythropoiesis allows to avoid

(30-40%) or to reduce ESA doses by 20-40%.(30-40%) or to reduce ESA doses by 20-40%.

FIRST STUDYFIRST STUDY: :

IIS HEPCIDIN AN IMPORTANT PLAYER IN IRON S HEPCIDIN AN IMPORTANT PLAYER IN IRON

METABOLISM IN HAEMODIALYSIS PATIENTSMETABOLISM IN HAEMODIALYSIS PATIENTS??

The hypothesisThe hypothesisHep-25 Hep-25 assessement could be useful in identifying assessement could be useful in identifying iron-restricted erythropoiesis iron-restricted erythropoiesis hemodialysis patientshemodialysis patients

Study objectiveStudy objectiveTo investigate in hemodialysis the relationships To investigate in hemodialysis the relationships between hepcidinbetween hepcidin-25-25 levels and: levels and:Peripheral iron indicesPeripheral iron indices (TSAT, fer (TSAT, ferritinritin))Parameters of inflammation (CRP, IL-6)Parameters of inflammation (CRP, IL-6)Iron supplements and darbepoietin dosesIron supplements and darbepoietin dosesPParametarameters of dialysis efficiencyers of dialysis efficiency (spKt/V) (spKt/V)

FIRST STUDYFIRST STUDY: :

IIS HEPCIDIN AN IMPORTANT PLAYER IN IRON S HEPCIDIN AN IMPORTANT PLAYER IN IRON

METABOLISM IN HAEMODIALYSIS PATIENTSMETABOLISM IN HAEMODIALYSIS PATIENTS??

The hypothesisThe hypothesisHep-25 Hep-25 assessement could be useful in identifying assessement could be useful in identifying iron-restricted erythropoiesis iron-restricted erythropoiesis hemodialysis patientshemodialysis patients

Study objectiveStudy objectiveTo investigate in hemodialysis the relationships To investigate in hemodialysis the relationships between hepcidinbetween hepcidin-25-25 levels and: levels and:Peripheral iron indicesPeripheral iron indices (TSAT, fer (TSAT, ferritinritin))Parameters of inflammation (CRP, IL-6)Parameters of inflammation (CRP, IL-6)Iron supplements and darbepoietin dosesIron supplements and darbepoietin dosesPParametarameters of dialysis efficiencyers of dialysis efficiency (spKt/V) (spKt/V)

Study designStudy design

A cross-sectional study on patients treated by HD in a A cross-sectional study on patients treated by HD in a single International Healthcare Systems dialysis unitsingle International Healthcare Systems dialysis unit

PatientsPatients:: 80 HD patients80 HD patients Median age 54 (range 21-80) yearsMedian age 54 (range 21-80) years Gender 59%MGender 59%M Median HD vintage 3.16 (range 0.3-28) yearsMedian HD vintage 3.16 (range 0.3-28) years

Primary kidney diseasePrimary kidney disease GN – 45%; IN – 21%; ADPKD 8%; VN 9%; Diabetic GN – 45%; IN – 21%; ADPKD 8%; VN 9%; Diabetic

nephropathy – 4%; Other 13%nephropathy – 4%; Other 13% Residual kidney function - 28%Residual kidney function - 28%

Inclusion criteriaInclusion criteria: : Stable HD patients for at least 3 months, with age Stable HD patients for at least 3 months, with age >18>18

yearsyears, , regardless of degree of anaemia, regardless of degree of anaemia, iinflammation, nflammation, iron status or anaemia therapy (IV iron, dabepoietin-iron status or anaemia therapy (IV iron, dabepoietin-αα))

A cross-sectional study on patients treated by HD in a A cross-sectional study on patients treated by HD in a single International Healthcare Systems dialysis unitsingle International Healthcare Systems dialysis unit

PatientsPatients:: 80 HD patients80 HD patients Median age 54 (range 21-80) yearsMedian age 54 (range 21-80) years Gender 59%MGender 59%M Median HD vintage 3.16 (range 0.3-28) yearsMedian HD vintage 3.16 (range 0.3-28) years

Primary kidney diseasePrimary kidney disease GN – 45%; IN – 21%; ADPKD 8%; VN 9%; Diabetic GN – 45%; IN – 21%; ADPKD 8%; VN 9%; Diabetic

nephropathy – 4%; Other 13%nephropathy – 4%; Other 13% Residual kidney function - 28%Residual kidney function - 28%

Inclusion criteriaInclusion criteria: : Stable HD patients for at least 3 months, with age Stable HD patients for at least 3 months, with age >18>18

yearsyears, , regardless of degree of anaemia, regardless of degree of anaemia, iinflammation, nflammation, iron status or anaemia therapy (IV iron, dabepoietin-iron status or anaemia therapy (IV iron, dabepoietin-αα))

ParametersParameters Hepcidin [competitive ELISA assay, with anti-Hepcidin-25 Hepcidin [competitive ELISA assay, with anti-Hepcidin-25

antibody, using a commercial kit produced by Peninsula antibody, using a commercial kit produced by Peninsula Laboratories, LLC (Bachem, UK) Laboratories, LLC (Bachem, UK) – – for the first time in a for the first time in a clinical studyclinical study

Anemia Anemia HemoglobinHemoglobin Darbepoetin alpha doseDarbepoetin alpha dose Iron sucrose doseIron sucrose dose

Peripheral iron indices Peripheral iron indices Transferrin saturation index (TSAT)Transferrin saturation index (TSAT) serum Ferritinserum Ferritin

Acute phase reactantsAcute phase reactants High sensitivity serum C reactive protein (CRP)High sensitivity serum C reactive protein (CRP) Interleukin-6 (IL-6)Interleukin-6 (IL-6) Serum albuminSerum albumin

HD therapyHD therapy spKt/VspKt/V Ultrafiltration volume per sessionUltrafiltration volume per session Residual diuresisResidual diuresis

General dataGeneral data

ParametersHemoglobin (g/dl)TSAT (%)Ferritin (ng/ml)Hepcidin (ng/ml)CRP (mg/l)IL-6 (pg/ml) )Albumin (g/dl)UF (l)spKt/VResidual diuresis (l)Darbepoetin doses (mcg/kg/wk)Iron doses (mg/wk)Darbepoetin resistance index (mcg/kg/wk/gHb)

All (n=80)11.56±1.33

40.89 (26.28-49.87)799 (703-886)

112.95 (107.10-122.15)5.0 (5.0-7.0)

4.52 (3.27-6.34)4.23±0.35

31.11 (23.33-38.64)0.023 (0.019-0.031)

2.45 (2.10-2.80)1.53±0.39

0.10 (0,00-0,30)0.29 (0.21-0.37)

Hepcidin levles in 80 stage 5CKD Hepcidin levles in 80 stage 5CKD HD patientsHD patients

Authors Patients Assay Hepcidin

Ashby et al2009

Stage 5HD BCR 94 pts.

RIA Anti Hep-25Bachem

58,5 ng/ml[96,7% CI 27,6-158]

Zaritsky et al2009

Stage 5PD BCR 26 pts.

ELISA Anti Hep-25 Bachem

652,4 ng/ml

Bratescu et al2009

Stage 5HD BCR 78 pts.

ELISA Anti Hep-25 Bachem

112,95 ng/ml [96,5% CI 107,10-122,15]

0

5

10

15

20

25

30

20 40 60 80 100 120 140 160

Freq

uenc

y

HEP

Histogram

ng/mL

Hepcidin (ng/ml)median112.95

[96.7% CI 107.10-122.15]

Hepcidin, anemia and peripheral Hepcidin, anemia and peripheral iron indicesiron indices

ParametriHemoglobin (g/dl)TSAT (%)Ferritin (ng/ml)

11.58±1.61 11.86±1.23 p=0.50p q1 (n=20) q4 (n=20)

36.38 (27.20-50.25) 44.75 (37.04-67.47) p=0.10p=0.002676.5 (283.0-881.0) 887.5 (716.0-1090.0)

Only ferritin was significantly higher in q4 than in q1 of hepcidin Only ferritin was significantly higher in q4 than in q1 of hepcidin

HepcidinHepcidin – – iron stores relationshipiron stores relationshipScatter Plot with Fit

0

20

40

60

80

100

120

140

160

180

200

0 500 1000 1500

Hep

cidi

n (n

g/m

l)

Ferritin (ng/ml)

Strong positive correlation was found between ferritin and hepcidin

•Iron stores (ferritin) role in defining hepcidin levels, even in HD patients, iron and epoetin treated!•Hep-25 can provide protection against iron overload

Strong positive correlation was found between ferritin and hepcidin

•Iron stores (ferritin) role in defining hepcidin levels, even in HD patients, iron and epoetin treated!•Hep-25 can provide protection against iron overload

HepcidinHepcidin – –iron available for erythropoiesis iron available for erythropoiesis

1

2

3

4

5

6

7

2.5 3 3.5 4 4.5 5

LnTSAT

Ln

HE

P

A similar, but weaker correlation was observed in case of TSAT, which suggest that•Hep-25 could be an indicator of iron available for erythropoiesis, even in HD patients

A similar, but weaker correlation was observed in case of TSAT, which suggest that•Hep-25 could be an indicator of iron available for erythropoiesis, even in HD patients

Parametri

CRP (mg/l)IL-6 (pg/ml)Albumin (g/dl)

p q 1 (n=20) q 4 (n=20)

5.0 (4.0-9.0) 5.5 (5.0-11.0) p=0.1584.50 (2.54-6.88) 4.82 (3.00-7.80) p=0.448

4.25±0.35 4.19±0.35 p=0.158

Hepcidin-25 - inflammationHepcidin-25 - inflammation, , anaemia and HD anaemia and HD therapy relationshiptherapy relationship

Hep-25 levels are little influenced by:•Inflammation parameters•Renal anaemia therapy (iron, darbepoietin) •Depuration by dialysis or by residual diuresis

Hep-25 levels are little influenced by:•Inflammation parameters•Renal anaemia therapy (iron, darbepoietin) •Depuration by dialysis or by residual diuresis

Parametersdarbepoetin doses (mcg/kg/wk)iron doses (mg/wk)darbepoietin resistance index. (mcg/kg/wk/gHb)0.028, 0.000-0.047

0.33, 0.19-0.58p q 1 (n=20) q 4 (n=20)

p=0.270.29, 0.00-0.4631.11, 30.60-69.49 p=0.13

p=0.6030.73, 15.55-38.640.027, 0.016-0.049

ParametersUltrafiltration (l)spKt/Vresidual diuresis (l)

2.10 (1.40-2.80)

p=0.6501.49±0.31

pq 1 (n=20) q 4 (n=20)

0.20 (0.10-0.50) 0.10 (0.00-0.30)

p=0.258p=0.2131.62±0.55

2.80 (2.10-3.50)

PredictorsPredictors of H of Hepcidinepcidin variation variation

Model fitting Chi2=71.45 <0.0001Pseudo R-square 0.60

Parameters Odds ratio [medie (95% CI)] Sig.Intercept q1/q4 0.12Erythropoietic activity

Hemoglobin 0.378 (0.149-0.159) 0.04Darbepoietin doses 0.000 (0.001-0.003 0.03Darbepoietin resistance index 400 (100-600) 0.03IL6 1.139 (0.909-1.428) 0.26Albumin 128.148 (3.206-5122.171) 0.01

Iron available for erythropoiesisTSAT 1.010 (0.962-1.061) 0.69

Iron storesFerritin 0.995 (0.991-0.999) 0.03Iron doses 1.058 (1.006-1.113) 0.03

Hepcidin excretionUltrafiltration volume 2.903 (1.039-8.111) 0.04spKTV 13.112 (0.227-755.826) 0.21Residual diuresis(+ vs. -) 20.330 (1.397-295.786 0.03

Other factorsGender(M vs F) 0.227 (0.020-3.866) 0.34Duration of HD session 1.289 (1.000-1.663) 0.05

First study conclusionsFirst study conclusions

1)1) Iron-restricted erythropoiesis – low Iron-restricted erythropoiesis – low Hb Hb and ferritin, high and ferritin, high EPO doses and EPO doses and ERI ERI – – werewere associated with low Hep-25 associated with low Hep-25 levelslevels

Hepcidin25 could be Hepcidin25 could be a useful marker of iron restricted a useful marker of iron restricted erythropoiesiserythropoiesis

2)2) Inflammation Inflammation (CRP and IL-6) (CRP and IL-6) appears to appears to have less have less influenceinfluence oon n Hep-25Hep-25 levels levels. .

3)3) Depuration (renal or extraDepuration (renal or extra--renal) could influence Hep-25 renal) could influence Hep-25 level, because higher level of Hep-25 level, because higher level of Hep-25 were seen in were seen in patients without patients without residual diuresis and residual diuresis and in those with in those with lower lower volumes of volumes of ultrafiltrationultrafiltration..

1)1) Iron-restricted erythropoiesis – low Iron-restricted erythropoiesis – low Hb Hb and ferritin, high and ferritin, high EPO doses and EPO doses and ERI ERI – – werewere associated with low Hep-25 associated with low Hep-25 levelslevels

Hepcidin25 could be Hepcidin25 could be a useful marker of iron restricted a useful marker of iron restricted erythropoiesiserythropoiesis

2)2) Inflammation Inflammation (CRP and IL-6) (CRP and IL-6) appears to appears to have less have less influenceinfluence oon n Hep-25Hep-25 levels levels. .

3)3) Depuration (renal or extraDepuration (renal or extra--renal) could influence Hep-25 renal) could influence Hep-25 level, because higher level of Hep-25 level, because higher level of Hep-25 were seen in were seen in patients without patients without residual diuresis and residual diuresis and in those with in those with lower lower volumes of volumes of ultrafiltrationultrafiltration..

SECOND STUDYSECOND STUDY

EFFECTS OF ADDITIONAL IRON DOSES ON HEPCIDIN-25 EFFECTS OF ADDITIONAL IRON DOSES ON HEPCIDIN-25 LEVEL IN HAEMODIALYSIS PATIENTS WITHOUT EVIDENT LEVEL IN HAEMODIALYSIS PATIENTS WITHOUT EVIDENT IRON DEFICIENCYIRON DEFICIENCY

PremisesPremises

Iron-restricted response to ESAs is difficult to identify; the Iron-restricted response to ESAs is difficult to identify; the recommended test is the Hb response to intravenous (IV) iron recommended test is the Hb response to intravenous (IV) iron supplementation. supplementation.

Hep-25 could be a useful marker. Hep-25 could be a useful marker. Giving more iron to properlGiving more iron to properlyy selected patients would restore the eselected patients would restore the eryryththrropoietic activitopoietic activity y which wowhich wouuld decrease Hep-25 levels, as there are some indices ld decrease Hep-25 levels, as there are some indices that hepcidin is directly regulated by the intensity of that hepcidin is directly regulated by the intensity of erythropoerythropoiietic activity, not only by iron stores or inflammation.etic activity, not only by iron stores or inflammation.

Study hypotesisStudy hypotesis

Giving additional iron doses to anemic HD patients without Giving additional iron doses to anemic HD patients without evident iron deficiency would restore erythropoietic activity, evident iron deficiency would restore erythropoietic activity, what would reduce serum levels of Hep-25.what would reduce serum levels of Hep-25.

SECOND STUDYSECOND STUDY

EFFECTS OF ADDITIONAL IRON DOSES ON HEPCIDIN-25 EFFECTS OF ADDITIONAL IRON DOSES ON HEPCIDIN-25 LEVEL IN HAEMODIALYSIS PATIENTS WITHOUT EVIDENT LEVEL IN HAEMODIALYSIS PATIENTS WITHOUT EVIDENT IRON DEFICIENCYIRON DEFICIENCY

PremisesPremises

Iron-restricted response to ESAs is difficult to identify; the Iron-restricted response to ESAs is difficult to identify; the recommended test is the Hb response to intravenous (IV) iron recommended test is the Hb response to intravenous (IV) iron supplementation. supplementation.

Hep-25 could be a useful marker. Hep-25 could be a useful marker. Giving more iron to properlGiving more iron to properlyy selected patients would restore the eselected patients would restore the eryryththrropoietic activitopoietic activity y which wowhich wouuld decrease Hep-25 levels, as there are some indices ld decrease Hep-25 levels, as there are some indices that hepcidin is directly regulated by the intensity of that hepcidin is directly regulated by the intensity of erythropoerythropoiietic activity, not only by iron stores or inflammation.etic activity, not only by iron stores or inflammation.

Study hypotesisStudy hypotesis

Giving additional iron doses to anemic HD patients without Giving additional iron doses to anemic HD patients without evident iron deficiency would restore erythropoietic activity, evident iron deficiency would restore erythropoietic activity, what would reduce serum levels of Hep-25.what would reduce serum levels of Hep-25.

Study designStudy design

DesignDesign: : prospective interventional, unicentric studyprospective interventional, unicentric study, , with 2 with 2 periodsperiods: observation (3 months) and intervention (3 months): observation (3 months) and intervention (3 months)

Subjects:Subjects: 41 HD patients from a single hemodialysis unit, 41 HD patients from a single hemodialysis unit, constantly treated with IV iron and ESAs >3 months and without constantly treated with IV iron and ESAs >3 months and without evident iron deficiency at baseline (ferritin, TSAT), selected from evident iron deficiency at baseline (ferritin, TSAT), selected from 80 patients participating in the previously study80 patients participating in the previously study

Investigated parameters:Investigated parameters: Hepcidin-25 (competitiv ELISHepcidin-25 (competitiv ELISA A method), ferritinmethod), ferritin,, transferin, TSAT, transferin, TSAT,

CRP, serum albumin, evaluated at CRP, serum albumin, evaluated at (-) 3 (-) 3 monthsmonths, , baseline and baseline and (+) 3 (+) 3 monthsmonths

HHemoglobin, iron and darbepoetin dosesemoglobin, iron and darbepoetin doses – – evaluated monthlyevaluated monthly

DesignDesign: : prospective interventional, unicentric studyprospective interventional, unicentric study, , with 2 with 2 periodsperiods: observation (3 months) and intervention (3 months): observation (3 months) and intervention (3 months)

Subjects:Subjects: 41 HD patients from a single hemodialysis unit, 41 HD patients from a single hemodialysis unit, constantly treated with IV iron and ESAs >3 months and without constantly treated with IV iron and ESAs >3 months and without evident iron deficiency at baseline (ferritin, TSAT), selected from evident iron deficiency at baseline (ferritin, TSAT), selected from 80 patients participating in the previously study80 patients participating in the previously study

Investigated parameters:Investigated parameters: Hepcidin-25 (competitiv ELISHepcidin-25 (competitiv ELISA A method), ferritinmethod), ferritin,, transferin, TSAT, transferin, TSAT,

CRP, serum albumin, evaluated at CRP, serum albumin, evaluated at (-) 3 (-) 3 monthsmonths, , baseline and baseline and (+) 3 (+) 3 monthsmonths

HHemoglobin, iron and darbepoetin dosesemoglobin, iron and darbepoetin doses – – evaluated monthlyevaluated monthly

MetMethodshodsObservation periodObservation period Iron and EPO aIron and EPO administradministration was made according to the The tion was made according to the The

Romanian Nephrology Society Best Practice GuidelinesRomanian Nephrology Society Best Practice Guidelines

Intervention periodIntervention period Additional IV iron doses Additional IV iron doses were administered, according to were administered, according to

Hb and iron indices at baseline:Hb and iron indices at baseline: Iron dose was increased by 25% for each 0.5g/dL drop in Iron dose was increased by 25% for each 0.5g/dL drop in

haemoglobin if baseline ferritin was 200-800ng/mLhaemoglobin if baseline ferritin was 200-800ng/mL, regardless , regardless of TSATof TSAT

Iron was discontinued when Hb was stable or increased >13g/dL. Iron was discontinued when Hb was stable or increased >13g/dL. Darbepoetin doses were adjusted targeting a Hb of 11g/dLDarbepoetin doses were adjusted targeting a Hb of 11g/dL

Increased by 25% if Hb decreased with >0.5 g/dL as Increased by 25% if Hb decreased with >0.5 g/dL as compared compared to the rolling average of previous three Hb levels or when the to the rolling average of previous three Hb levels or when the rolling average was <11g/dlrolling average was <11g/dl

Decreased by 25% if Hb increased more than 0.5g/dL or was Decreased by 25% if Hb increased more than 0.5g/dL or was >12g/dL>12g/dL

Halved if Hb was over 13g/dLHalved if Hb was over 13g/dL

Observation periodObservation period Iron and EPO aIron and EPO administradministration was made according to the The tion was made according to the The

Romanian Nephrology Society Best Practice GuidelinesRomanian Nephrology Society Best Practice Guidelines

Intervention periodIntervention period Additional IV iron doses Additional IV iron doses were administered, according to were administered, according to

Hb and iron indices at baseline:Hb and iron indices at baseline: Iron dose was increased by 25% for each 0.5g/dL drop in Iron dose was increased by 25% for each 0.5g/dL drop in

haemoglobin if baseline ferritin was 200-800ng/mLhaemoglobin if baseline ferritin was 200-800ng/mL, regardless , regardless of TSATof TSAT

Iron was discontinued when Hb was stable or increased >13g/dL. Iron was discontinued when Hb was stable or increased >13g/dL. Darbepoetin doses were adjusted targeting a Hb of 11g/dLDarbepoetin doses were adjusted targeting a Hb of 11g/dL

Increased by 25% if Hb decreased with >0.5 g/dL as Increased by 25% if Hb decreased with >0.5 g/dL as compared compared to the rolling average of previous three Hb levels or when the to the rolling average of previous three Hb levels or when the rolling average was <11g/dlrolling average was <11g/dl

Decreased by 25% if Hb increased more than 0.5g/dL or was Decreased by 25% if Hb increased more than 0.5g/dL or was >12g/dL>12g/dL

Halved if Hb was over 13g/dLHalved if Hb was over 13g/dL

ResultsResultsParameter Baseline Assessment p

Mean (Median)

95% CI Mean (Median)

95% CI

Dry weight* 72.0 64.8 80.2 71.5 65.8 80.2 0.03 Kt/V 1.44 1.34 1.53 1.49 1.42 1.56 0.10 Hemoglobin (g/dL) 11.41 11.08 11.75 11.77 11.46 12.07 0.08 Transferrin(mg/dL) 142 134 150 171 161 181 <0.0001 Transferrin saturation index (%)* 40 35 50 25 21 29 <0.0001 Ferritin (ng/mL) 769 702 836 717 660 774 0.05 Iron status‡ Optimal (%) 21% - - 44% - - 0.06 Absolute deficiency (%) 0% - - 0% - - >0.5 Functional iron deficiency (%) 0% - - 24% - - 0.001 Iron overload (%) 15% - - 0% - - 0.03 Total darbepoetin dose (mcg)* 800 800 900 400 300 700 <0.0001 Darbepoetin dose (mcg/kg per week)* 0.40 0.26 0.56 0.31 0.19 0.36 0.0003 Darbepoetin resistance index (mcg/g Hb)* 0.03 0.02 0.05 0.0261 0.02 0.03 <0.0001 Total iron dose (mg)* 400 300 500 700 600 1000 <0.0001 Iron mean dose (mg/month)* 133.3 100 166.7 233.3 200 333.3 <0.0001 Hepcidin (ng/mL)* 112.8 105 121.8 66.5 31.3 89.0 0.0001 C-reactive protein (mg/L)* 5.0 5.0 8.0 6.0 5.0 10.0 0.04 Serum albumin (g/dL) 4.2 4.1 4.3 4.0 3.9 4.1 0.0003

‡ optimal iron status: transferrin saturation index 20%-40%; ferritin 200-800 ng/mL; absolute iron deficiency: transferrin saturation index <20%; ferritin <200 ng/mL; functional iron deficiency: transferrin saturation index <20%; ferritin >200 ng/mL and <800 ng/mL; iron overload: transferrin saturation index >50%; ferritin >800 ng/mL * non-normal distribution † paired t- Student

Iron doses were increased with 75%. The proportion of patients with „funcţional” iron deficiency increased

from 0 to 24% and no patient has presented iron overload.

Iron doses were increased with 75%. The proportion of patients with „funcţional” iron deficiency increased

from 0 to 24% and no patient has presented iron overload.

After iron doses increased with 75%:Transferrin increasedTSAT decreasedFerritin decreased Hepcidin-25 decreased with 70%EPO doses and darbepoietin resistance index decreased with 29% and 15%

After iron doses increased with 75%:Transferrin increasedTSAT decreasedFerritin decreased Hepcidin-25 decreased with 70%EPO doses and darbepoietin resistance index decreased with 29% and 15%

Erythropoietic activity restoration as a Erythropoietic activity restoration as a result of iron deficiency correction!result of iron deficiency correction!

Erythropoietic activity restoration as a Erythropoietic activity restoration as a result of iron deficiency correction!result of iron deficiency correction!

After 75% iron doses augmentation, the increase in Hep-25 variation was proportional to the increase in TSAT and ferritin variationsAfter 75% iron doses augmentation, the increase in Hep-25 variation was proportional to the increase in TSAT and ferritin variations

Relationships between Hep-25, TSAT and ferritin variationsRelationships between Hep-25, TSAT and ferritin variations

Predictors of hepcidin level at Predictors of hepcidin level at the assessmentthe assessment

• The model (baseline versus assesment ratio) predicted 26% of Hep-25 level after iron doses augmentation.

• The independents significant predictors of Hep-25 levels were baseline vs. assessment variations in transferrin and ferritin levels, although iron dose was retained in the model.

• The model (baseline versus assesment ratio) predicted 26% of Hep-25 level after iron doses augmentation.

• The independents significant predictors of Hep-25 levels were baseline vs. assessment variations in transferrin and ferritin levels, although iron dose was retained in the model.The level of Hep-25 The level of Hep-25 seems to be seems to be influenced by the iron administrinfluenced by the iron administrationation, , which activates erythropoiesis, resulting in a paradoxically which activates erythropoiesis, resulting in a paradoxically decreasedecrease in in

iron stores iron stores (lower ferritin) (lower ferritin) and increase and increase in in iron available for erythropoiesisiron available for erythropoiesis (higher TSAT).(higher TSAT).

The level of Hep-25 The level of Hep-25 seems to be seems to be influenced by the iron administrinfluenced by the iron administrationation, , which activates erythropoiesis, resulting in a paradoxically which activates erythropoiesis, resulting in a paradoxically decreasedecrease in in

iron stores iron stores (lower ferritin) (lower ferritin) and increase and increase in in iron available for erythropoiesisiron available for erythropoiesis (higher TSAT).(higher TSAT).

* Assessment versus baseline ratio. Dependent variable: hepcidin at evaluation. Predictors (Constant): age, gender, HD vintage, Kt/V ratio, mean iron dose, C reactive protein ratio, albumin ratio, ferritin ratio, hemoglobin ratio, transferrin ratio, darbepoetin dose, hepcidin at baselin

Determinanți Beta 95%CI of beta Sig.

(Constant) 56.82 to 243.14 0.002

Mean iron dose -0.09 -0.22 to 0.04 0.151

Transferrin ratio* -106.05 -176.96 to -35.13 0.004

Ferritin ratio* 76.49 32.02 to 120.96 0.001

Limits of hepcidin assayLimits of hepcidin assay

Ganz et al ELISA, anti-Hepcidin-25 antibody (Bachem)

2009 range of detection 5-4000ng/ml Ashby et al RIA, anti-Hepcidin-25 antibody

(Bachem) 2009 lower limit of detection 0,6ng/ml

liniar up to 200ng/ml Zaritsky et al C-ELISA, anti-Hepcidin-25 antibody

(Bachem)2009

Mamon et al HPLC–(MS/MS)2009 linearity from 0.1 to 80nmoL/L

Second study conclusionsSecond study conclusions

AdministraAdministration of additional iron doses to HD EPO tion of additional iron doses to HD EPO treatedtreated patients, but without evident iron deficiency patients, but without evident iron deficiency was associated with significant was associated with significant reduction reduction in serum in serum Hep-25Hep-25, , increasincrease in e in transferrin and decreastransferrin and decrease in e in ferritinferritin, , what suggests correction of what suggests correction of ““hiddenhidden”” functional iron deficiency functional iron deficiency and activation of and activation of erythropoiesis.erythropoiesis.

ReducReducing in Hep-25 levels after iron augmentation ing in Hep-25 levels after iron augmentation could be could be clinically useful to identify HD patients with clinically useful to identify HD patients with iron-restricted erythropoiesis, EPO treated, who iron-restricted erythropoiesis, EPO treated, who could benefit of additional IV ironcould benefit of additional IV iron,, but it is necessary but it is necessary coconnfirmation by further controlled studies.firmation by further controlled studies.

AdministraAdministration of additional iron doses to HD EPO tion of additional iron doses to HD EPO treatedtreated patients, but without evident iron deficiency patients, but without evident iron deficiency was associated with significant was associated with significant reduction reduction in serum in serum Hep-25Hep-25, , increasincrease in e in transferrin and decreastransferrin and decrease in e in ferritinferritin, , what suggests correction of what suggests correction of ““hiddenhidden”” functional iron deficiency functional iron deficiency and activation of and activation of erythropoiesis.erythropoiesis.

ReducReducing in Hep-25 levels after iron augmentation ing in Hep-25 levels after iron augmentation could be could be clinically useful to identify HD patients with clinically useful to identify HD patients with iron-restricted erythropoiesis, EPO treated, who iron-restricted erythropoiesis, EPO treated, who could benefit of additional IV ironcould benefit of additional IV iron,, but it is necessary but it is necessary coconnfirmation by further controlled studies.firmation by further controlled studies.

Final conclusionsFinal conclusions

Serum Hep-25 levels in HD patients are determined by iron Serum Hep-25 levels in HD patients are determined by iron stores, erythropoietic activity and inflammation. stores, erythropoietic activity and inflammation.

Hep-25 Hep-25 seems to be a good marker of iron stores and iron seems to be a good marker of iron stores and iron available for erythropoiesis.available for erythropoiesis.

Hep-25 Hep-25 could be a useful marker of “hidden” functional could be a useful marker of “hidden” functional iron iron deficdeficiencyiency: :

Low levels of hepcidin in HD patients with high Low levels of hepcidin in HD patients with high ERI ERI would would indicate iron-restricted erythropoiesis, even when indicate iron-restricted erythropoiesis, even when inflammation is presentinflammation is present;;

A downward trend in Hep-25 levels when iron doses are A downward trend in Hep-25 levels when iron doses are increased, in HD patients witincreased, in HD patients withhout evident iron deficiency out evident iron deficiency would suggest would suggest correction of iron-restricted erythropoiesiscorrection of iron-restricted erythropoiesis, , so would guide iron therapy, in EPO treated patients, so would guide iron therapy, in EPO treated patients, when iron indices are not indicative of iron deficiency.when iron indices are not indicative of iron deficiency.

Serum Hep-25 levels in HD patients are determined by iron Serum Hep-25 levels in HD patients are determined by iron stores, erythropoietic activity and inflammation. stores, erythropoietic activity and inflammation.

Hep-25 Hep-25 seems to be a good marker of iron stores and iron seems to be a good marker of iron stores and iron available for erythropoiesis.available for erythropoiesis.

Hep-25 Hep-25 could be a useful marker of “hidden” functional could be a useful marker of “hidden” functional iron iron deficdeficiencyiency: :

Low levels of hepcidin in HD patients with high Low levels of hepcidin in HD patients with high ERI ERI would would indicate iron-restricted erythropoiesis, even when indicate iron-restricted erythropoiesis, even when inflammation is presentinflammation is present;;

A downward trend in Hep-25 levels when iron doses are A downward trend in Hep-25 levels when iron doses are increased, in HD patients witincreased, in HD patients withhout evident iron deficiency out evident iron deficiency would suggest would suggest correction of iron-restricted erythropoiesiscorrection of iron-restricted erythropoiesis, , so would guide iron therapy, in EPO treated patients, so would guide iron therapy, in EPO treated patients, when iron indices are not indicative of iron deficiency.when iron indices are not indicative of iron deficiency.