HEPCIDIN-25 - A USEFUL CLINICAL GUIDE FOR IRON-RESTRICTED ERYTHROPOIESIS DETECTION IN HAEMODIALYSIS PATIENTS Lavinia-Oltița Brătescu 1, Liliana Bârsan 3, Liliana Gârneață 2,3, Ana Stanciu 3, Mariana Lipan 1,Simona Stancu 2,3 and Gabriel Mircescu 2,3 1“Sf Pantelimon“ International Healthcare Systems Nephrology and Dialysis Medical Center, Bucharest, Romania 2. Internal Medicine and Nephrology Dept. "Carol Davila" University of Medicine and Pharmacy, Bucharest, Romania; 3 - Nephrology Dept. "Dr Carol Davila" Teaching Hospital of Nephrology, Bucharest, Romania

Hepcidin 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 is produced by the liver as prohepcidin (84 aa) and thereafter converted to the active peptide hepcidin (25 aa) Hepcidin is produced by the liver as prohepcidin (84 aa) and thereafter converted to the active peptide hepcidin (25 aa) Hepcidin accelerates the degradation of ferroportin - the main cellular iron transporter in enterocyte, hepatocyte and macrophage - and induces hypoferrinemia by inhibiting intestinal iron absorbtion and iron recycling Hepcidin accelerates the degradation of ferroportin - the main cellular iron transporter in enterocyte, hepatocyte and macrophage - and induces hypoferrinemia by inhibiting intestinal iron absorbtion and iron recycling Yujie Cui, Qingyu Wu and Yiqing Zhou. Kidney Int. 23 Sept 2009; doi: /ki

Hepcidin actions are thought to Hepcidin actions are thought to prevent iron overload by inhibiting intestinal iron absorbtion prevent iron overload by inhibiting intestinal iron absorbtion protect against infections, by hypoferrinemia which limits the iron-dependent multiplication of invading bacteria. protect against infections, by hypoferrinemia which limits the iron-dependent multiplication of invading bacteria. Hepcidin regulation reflects this dual role Hepcidin regulation reflects this dual role low hepcidin levels were associated with anemia, depleted iron stores, increased iron needs and active erythropoiesis, while high hepcidin levels were found in case of increased iron stores low hepcidin levels were associated with anemia, depleted iron stores, increased iron needs and active erythropoiesis, while high hepcidin levels were found in case of increased iron stores hepcidin is an acute phase reactant, modulated by IL-6. hepcidin is an acute phase reactant, modulated by IL-6. Hepcidin excretion is unknown, but renal or dialysis depuration could be important in defining hepcidin levels. Hepcidin excretion is unknown, but renal or dialysis depuration could be important in defining hepcidin levels. Hepcidin integrates iron absorbtion, storage and mobilisation from storage with the requirements of erythropoiesis Hepcidin integrates iron absorbtion, storage and mobilisation from storage with the requirements of erythropoiesis

Hepcidin in CKD High hepcidin levels were reported in CKD patients and could result from Inflammation Inflammation Increased iron stores resulting either from reduced (low epoetin driven) or ineffective erythropoiesis as well as overzealous iron therapy Increased iron stores resulting either from reduced (low epoetin driven) or ineffective erythropoiesis as well as overzealous iron therapy Decreased hepcidin depuration by the failing kidney Decreased hepcidin depuration by the failing kidney could be involved in iron restricted erythropoiesis could be a marker of iron status, useful to guide therapy

Absolute iron deficiency Functional iron deficiency Blocked iron stores Iron restricted erythropoiesis Anaemia ESA responsiveness Anaemia Iron therapy

Hypothesis…controversis Iron metabolism abnormalities are common in renal anemia Iron metabolism abnormalities are common in renal anemia Iron restricted erythropoiesis is determined in almost half of cases by the association between iron deficiency and iron immobilization in macrophages; Iron restricted erythropoiesis is determined in almost half of cases by the association between iron deficiency and iron immobilization in macrophages; 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 deficiency Hepcidin could be a diagnostic tool and a therapeutic target in iron deficiency The therapeutic test (parenteral iron supplementation) allows iron therapy tailoring The therapeutic test (parenteral iron supplementation) allows iron therapy tailoring Iron deficiency is the most common manageable cause of low ESA responsiveness; Iron deficiency is the most common manageable cause of low ESA responsiveness; The correction of iron-restricted erythropoiesis allows to avoid (30-40%) or to reduce ESA doses by 20-40%. The correction of iron-restricted erythropoiesis allows to avoid (30-40%) or to reduce ESA doses by 20-40%. Iron metabolism abnormalities are common in renal anemia Iron metabolism abnormalities are common in renal anemia Iron restricted erythropoiesis is determined in almost half of cases by the association between iron deficiency and iron immobilization in macrophages; Iron restricted erythropoiesis is determined in almost half of cases by the association between iron deficiency and iron immobilization in macrophages; 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 deficiency Hepcidin could be a diagnostic tool and a therapeutic target in iron deficiency The therapeutic test (parenteral iron supplementation) allows iron therapy tailoring The therapeutic test (parenteral iron supplementation) allows iron therapy tailoring Iron deficiency is the most common manageable cause of low ESA responsiveness; Iron deficiency is the most common manageable cause of low ESA responsiveness; The correction of iron-restricted erythropoiesis allows to avoid (30-40%) or to reduce ESA doses by 20-40%. The correction of iron-restricted erythropoiesis allows to avoid (30-40%) or to reduce ESA doses by 20-40%.

FIRST STUDY: IS HEPCIDIN AN IMPORTANT PLAYER IN IRON METABOLISM IN HAEMODIALYSIS PATIENTS? The hypothesis The hypothesis Hep-25 assessement could be useful in identifying iron- restricted erythropoiesis hemodialysis patients Study objective Study objective To investigate in hemodialysis the relationships between hepcidin-25 levels and: Peripheral iron indices (TSAT, ferritin) Peripheral iron indices (TSAT, ferritin) Parameters of inflammation (CRP, IL-6) Parameters of inflammation (CRP, IL-6) Iron supplements and darbepoietin doses Iron supplements and darbepoietin doses Parameters of dialysis efficiency (spKt/V) Parameters of dialysis efficiency (spKt/V) FIRST STUDY: IS HEPCIDIN AN IMPORTANT PLAYER IN IRON METABOLISM IN HAEMODIALYSIS PATIENTS? The hypothesis The hypothesis Hep-25 assessement could be useful in identifying iron- restricted erythropoiesis hemodialysis patients Study objective Study objective To investigate in hemodialysis the relationships between hepcidin-25 levels and: Peripheral iron indices (TSAT, ferritin) Peripheral iron indices (TSAT, ferritin) Parameters of inflammation (CRP, IL-6) Parameters of inflammation (CRP, IL-6) Iron supplements and darbepoietin doses Iron supplements and darbepoietin doses Parameters of dialysis efficiency (spKt/V) Parameters of dialysis efficiency (spKt/V)

Study design A cross-sectional study on patients treated by HD in a single International Healthcare Systems dialysis unit A cross-sectional study on patients treated by HD in a single International Healthcare Systems dialysis unit Patients: 80 HD patients Patients: 80 HD patients Median age 54 (range 21-80) years Median age 54 (range 21-80) years Gender 59%M Gender 59%M Median HD vintage 3.16 (range ) years Median HD vintage 3.16 (range ) years Primary kidney disease Primary kidney disease GN – 45%; IN – 21%; ADPKD 8%; VN 9%; Diabetic nephropathy – 4%; Other 13% GN – 45%; IN – 21%; ADPKD 8%; VN 9%; Diabetic nephropathy – 4%; Other 13% Residual kidney function - 28% Residual kidney function - 28% Inclusion criteria: Inclusion criteria: Stable HD patients for at least 3 months, with age >18 years, regardless of degree of anaemia, inflammation, iron status or anaemia therapy (IV iron, dabepoietin-α) Stable HD patients for at least 3 months, with age >18 years, regardless of degree of anaemia, inflammation, iron status or anaemia therapy (IV iron, dabepoietin-α) A cross-sectional study on patients treated by HD in a single International Healthcare Systems dialysis unit A cross-sectional study on patients treated by HD in a single International Healthcare Systems dialysis unit Patients: 80 HD patients Patients: 80 HD patients Median age 54 (range 21-80) years Median age 54 (range 21-80) years Gender 59%M Gender 59%M Median HD vintage 3.16 (range ) years Median HD vintage 3.16 (range ) years Primary kidney disease Primary kidney disease GN – 45%; IN – 21%; ADPKD 8%; VN 9%; Diabetic nephropathy – 4%; Other 13% GN – 45%; IN – 21%; ADPKD 8%; VN 9%; Diabetic nephropathy – 4%; Other 13% Residual kidney function - 28% Residual kidney function - 28% Inclusion criteria: Inclusion criteria: Stable HD patients for at least 3 months, with age >18 years, regardless of degree of anaemia, inflammation, iron status or anaemia therapy (IV iron, dabepoietin-α) Stable HD patients for at least 3 months, with age >18 years, regardless of degree of anaemia, inflammation, iron status or anaemia therapy (IV iron, dabepoietin-α)

Parameters Hepcidin [competitive ELISA assay, with anti-Hepcidin-25 antibody, using a commercial kit produced by Peninsula Laboratories, LLC (Bachem, UK) – for the first time in a clinical study Hepcidin [competitive ELISA assay, with anti-Hepcidin-25 antibody, using a commercial kit produced by Peninsula Laboratories, LLC (Bachem, UK) – for the first time in a clinical study Anemia Anemia Hemoglobin Hemoglobin Darbepoetin alpha dose Darbepoetin alpha dose Iron sucrose dose Iron sucrose dose Peripheral iron indices Peripheral iron indices Transferrin saturation index (TSAT) Transferrin saturation index (TSAT) serum Ferritin serum Ferritin Acute phase reactants Acute 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 albumin Serum albumin HD therapy HD therapy spKt/V spKt/V Ultrafiltration volume per session Ultrafiltration volume per session Residual diuresis Residual diuresis

General data

Hepcidin levles in 80 stage 5CKD HD patients AuthorsPatientsAssayHepcidin Ashby et al 2009 Stage 5HD BCR 94 pts. RIA Anti Hep-25 Bachem 58,5 ng/ml [96,7% CI 27,6-158] Zaritsky et al 2009 Stage 5PD BCR 26 pts. ELISA Anti Hep-25 Bachem 652,4 ng/ml Bratescu et al 2009 Stage 5HD BCR 78 pts. ELISA Anti Hep-25 Bachem 112,95 ng/ml [96,5% CI 107,10-122,15] ng/mL Hepcidin (ng/ml) median [96.7% CI ]

Hepcidin, anemia and peripheral iron indices Only ferritin was significantly higher in q4 than in q1 of hepcidin

Hepcidin – iron stores relationship Hepcidin (ng/ml) 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

Hepcidin –iron available for erythropoiesis 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

Hepcidin-25 - inflammation, anaemia and HD therapy 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

Predictors of Hepcidin variation

First study conclusions 1)Iron-restricted erythropoiesis – low Hb and ferritin, high EPO doses and ERI – were associated with low Hep-25 levels Hepcidin25 could be a useful marker of iron restricted erythropoiesis 2)Inflammation (CRP and IL-6) appears to have less influence on Hep-25 levels. 3)Depuration (renal or extra-renal) could influence Hep-25 level, because higher level of Hep-25 were seen in patients without residual diuresis and in those with lower volumes of ultrafiltration. 1)Iron-restricted erythropoiesis – low Hb and ferritin, high EPO doses and ERI – were associated with low Hep-25 levels Hepcidin25 could be a useful marker of iron restricted erythropoiesis 2)Inflammation (CRP and IL-6) appears to have less influence on Hep-25 levels. 3)Depuration (renal or extra-renal) could influence Hep-25 level, because higher level of Hep-25 were seen in patients without residual diuresis and in those with lower volumes of ultrafiltration.

SECOND STUDY EFFECTS OF ADDITIONAL IRON DOSES ON HEPCIDIN-25 LEVEL IN HAEMODIALYSIS PATIENTS WITHOUT EVIDENT IRON DEFICIENCY Premises Iron-restricted response to ESAs is difficult to identify; the recommended test is the Hb response to intravenous (IV) iron supplementation. Iron-restricted response to ESAs is difficult to identify; the recommended test is the Hb response to intravenous (IV) iron supplementation. Hep-25 could be a useful marker. Giving more iron to properly selected patients would restore the erythropoietic activity which would decrease Hep-25 levels, as there are some indices that hepcidin is directly regulated by the intensity of erythropoietic activity, not only by iron stores or inflammation. Hep-25 could be a useful marker. Giving more iron to properly selected patients would restore the erythropoietic activity which would decrease Hep-25 levels, as there are some indices that hepcidin is directly regulated by the intensity of erythropoietic activity, not only by iron stores or inflammation. Study hypotesis Giving additional iron doses to anemic HD patients without evident iron deficiency would restore erythropoietic activity, what would reduce serum levels of Hep-25. SECOND STUDY EFFECTS OF ADDITIONAL IRON DOSES ON HEPCIDIN-25 LEVEL IN HAEMODIALYSIS PATIENTS WITHOUT EVIDENT IRON DEFICIENCY Premises Iron-restricted response to ESAs is difficult to identify; the recommended test is the Hb response to intravenous (IV) iron supplementation. Iron-restricted response to ESAs is difficult to identify; the recommended test is the Hb response to intravenous (IV) iron supplementation. Hep-25 could be a useful marker. Giving more iron to properly selected patients would restore the erythropoietic activity which would decrease Hep-25 levels, as there are some indices that hepcidin is directly regulated by the intensity of erythropoietic activity, not only by iron stores or inflammation. Hep-25 could be a useful marker. Giving more iron to properly selected patients would restore the erythropoietic activity which would decrease Hep-25 levels, as there are some indices that hepcidin is directly regulated by the intensity of erythropoietic activity, not only by iron stores or inflammation. Study hypotesis Giving additional iron doses to anemic HD patients without evident iron deficiency would restore erythropoietic activity, what would reduce serum levels of Hep-25.

Study design Design: prospective interventional, unicentric study, with 2 periods: observation (3 months) and intervention (3 months) Design: prospective interventional, unicentric study, with 2 periods: observation (3 months) and intervention (3 months) Subjects: 41 HD patients from a single hemodialysis unit, constantly treated with IV iron and ESAs >3 months and without evident iron deficiency at baseline (ferritin, TSAT), selected from 80 patients participating in the previously study Subjects: 41 HD patients from a single hemodialysis unit, constantly treated with IV iron and ESAs >3 months and without evident iron deficiency at baseline (ferritin, TSAT), selected from 80 patients participating in the previously study Investigated parameters: Investigated parameters: Hepcidin-25 (competitiv ELISA method), ferritin, transferin, TSAT, CRP, serum albumin, evaluated at (-) 3 months, baseline and (+) 3 months Hepcidin-25 (competitiv ELISA method), ferritin, transferin, TSAT, CRP, serum albumin, evaluated at (-) 3 months, baseline and (+) 3 months Hemoglobin, iron and darbepoetin doses – evaluated monthly Hemoglobin, iron and darbepoetin doses – evaluated monthly Design: prospective interventional, unicentric study, with 2 periods: observation (3 months) and intervention (3 months) Design: prospective interventional, unicentric study, with 2 periods: observation (3 months) and intervention (3 months) Subjects: 41 HD patients from a single hemodialysis unit, constantly treated with IV iron and ESAs >3 months and without evident iron deficiency at baseline (ferritin, TSAT), selected from 80 patients participating in the previously study Subjects: 41 HD patients from a single hemodialysis unit, constantly treated with IV iron and ESAs >3 months and without evident iron deficiency at baseline (ferritin, TSAT), selected from 80 patients participating in the previously study Investigated parameters: Investigated parameters: Hepcidin-25 (competitiv ELISA method), ferritin, transferin, TSAT, CRP, serum albumin, evaluated at (-) 3 months, baseline and (+) 3 months Hepcidin-25 (competitiv ELISA method), ferritin, transferin, TSAT, CRP, serum albumin, evaluated at (-) 3 months, baseline and (+) 3 months Hemoglobin, iron and darbepoetin doses – evaluated monthly Hemoglobin, iron and darbepoetin doses – evaluated monthly

Methods Observation period Iron and EPO administration was made according to the The Romanian Nephrology Society Best Practice Guidelines Iron and EPO administration was made according to the The Romanian Nephrology Society Best Practice Guidelines Intervention period Additional IV iron doses were administered, according to Hb and iron indices at baseline: Additional IV iron doses were administered, according to Hb and iron indices at baseline: Iron dose was increased by 25% for each 0.5g/dL drop in haemoglobin if baseline ferritin was ng/mL, regardless of TSAT Iron dose was increased by 25% for each 0.5g/dL drop in haemoglobin if baseline ferritin was ng/mL, regardless of 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/dL Darbepoetin doses were adjusted targeting a Hb of 11g/dL Increased by 25% if Hb decreased with >0.5 g/dL as compared to the rolling average of previous three Hb levels or when the rolling average was 0.5 g/dL as compared to the rolling average of previous three Hb levels or when the rolling average was <11g/dl Decreased by 25% if Hb increased more than 0.5g/dL or was >12g/dL Decreased by 25% if Hb increased more than 0.5g/dL or was >12g/dL Halved if Hb was over 13g/dL Halved if Hb was over 13g/dL Observation period Iron and EPO administration was made according to the The Romanian Nephrology Society Best Practice Guidelines Iron and EPO administration was made according to the The Romanian Nephrology Society Best Practice Guidelines Intervention period Additional IV iron doses were administered, according to Hb and iron indices at baseline: Additional IV iron doses were administered, according to Hb and iron indices at baseline: Iron dose was increased by 25% for each 0.5g/dL drop in haemoglobin if baseline ferritin was ng/mL, regardless of TSAT Iron dose was increased by 25% for each 0.5g/dL drop in haemoglobin if baseline ferritin was ng/mL, regardless of 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/dL Darbepoetin doses were adjusted targeting a Hb of 11g/dL Increased by 25% if Hb decreased with >0.5 g/dL as compared to the rolling average of previous three Hb levels or when the rolling average was 0.5 g/dL as compared to the rolling average of previous three Hb levels or when the rolling average was <11g/dl Decreased by 25% if Hb increased more than 0.5g/dL or was >12g/dL Decreased by 25% if Hb increased more than 0.5g/dL or was >12g/dL Halved if Hb was over 13g/dL Halved if Hb was over 13g/dL

Results  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 increased  TSAT decreased  Ferritin decreased  Hepcidin-25 decreased with 70%  EPO doses and darbepoietin resistance index decreased with 29% and 15% After iron doses increased with 75%:  Transferrin increased  TSAT decreased  Ferritin decreased  Hepcidin-25 decreased with 70%  EPO doses and darbepoietin resistance index decreased with 29% and 15% Erythropoietic activity restoration as a 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 variations Relationships between Hep-25, TSAT and ferritin variations

Predictors of hepcidin level at the 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 seems to be influenced by the iron administration, which activates erythropoiesis, resulting in a paradoxically decrease in iron stores (lower ferritin) and increase in iron available for erythropoiesis (higher TSAT).

Limits of hepcidin assay Ganz et alELISA, anti-Hepcidin-25 antibody (Bachem) 2009range of detection ng/ml Ashby et alRIA, anti-Hepcidin-25 antibody (Bachem) 2009lower limit of detection 0,6ng/ml liniar up to 200ng/ml Zaritsky et al C-ELISA, anti-Hepcidin-25 antibody (Bachem) 2009 Mamon et alHPLC–(MS/MS) 2009linearity from 0.1 to 80nmoL/L

Second study conclusions Administration of additional iron doses to HD EPO treated patients, but without evident iron deficiency was associated with significant reduction in serum Hep-25, increase in transferrin and decrease in ferritin, what suggests correction of “hidden” functional iron deficiency and activation of erythropoiesis. Administration of additional iron doses to HD EPO treated patients, but without evident iron deficiency was associated with significant reduction in serum Hep-25, increase in transferrin and decrease in ferritin, what suggests correction of “hidden” functional iron deficiency and activation of erythropoiesis. Reducing in Hep-25 levels after iron augmentation could be clinically useful to identify HD patients with iron- restricted erythropoiesis, EPO treated, who could benefit of additional IV iron, but it is necessary confirmation by further controlled studies. Reducing in Hep-25 levels after iron augmentation could be clinically useful to identify HD patients with iron- restricted erythropoiesis, EPO treated, who could benefit of additional IV iron, but it is necessary confirmation by further controlled studies. Administration of additional iron doses to HD EPO treated patients, but without evident iron deficiency was associated with significant reduction in serum Hep-25, increase in transferrin and decrease in ferritin, what suggests correction of “hidden” functional iron deficiency and activation of erythropoiesis. Administration of additional iron doses to HD EPO treated patients, but without evident iron deficiency was associated with significant reduction in serum Hep-25, increase in transferrin and decrease in ferritin, what suggests correction of “hidden” functional iron deficiency and activation of erythropoiesis. Reducing in Hep-25 levels after iron augmentation could be clinically useful to identify HD patients with iron- restricted erythropoiesis, EPO treated, who could benefit of additional IV iron, but it is necessary confirmation by further controlled studies. Reducing in Hep-25 levels after iron augmentation could be clinically useful to identify HD patients with iron- restricted erythropoiesis, EPO treated, who could benefit of additional IV iron, but it is necessary confirmation by further controlled studies.

Final conclusions Serum Hep-25 levels in HD patients are determined by iron stores, erythropoietic activity and inflammation. Serum Hep-25 levels in HD patients are determined by iron stores, erythropoietic activity and inflammation. Hep-25 seems to be a good marker of iron stores and iron available for erythropoiesis. Hep-25 seems to be a good marker of iron stores and iron available for erythropoiesis. Hep-25 could be a useful marker of “hidden” functional iron deficiency: Hep-25 could be a useful marker of “hidden” functional iron deficiency: Low levels of hepcidin in HD patients with high ERI would indicate iron-restricted erythropoiesis, even when inflammation is present; Low levels of hepcidin in HD patients with high ERI would indicate iron-restricted erythropoiesis, even when inflammation is present; A downward trend in Hep-25 levels when iron doses are increased, in HD patients without evident iron deficiency would suggest correction of iron-restricted erythropoiesis, so would guide iron therapy, in EPO treated patients, when iron indices are not indicative of iron deficiency. A downward trend in Hep-25 levels when iron doses are increased, in HD patients without evident iron deficiency would suggest correction of iron-restricted erythropoiesis, so would guide iron therapy, in EPO treated patients, when iron indices are not indicative of iron deficiency. Serum Hep-25 levels in HD patients are determined by iron stores, erythropoietic activity and inflammation. Serum Hep-25 levels in HD patients are determined by iron stores, erythropoietic activity and inflammation. Hep-25 seems to be a good marker of iron stores and iron available for erythropoiesis. Hep-25 seems to be a good marker of iron stores and iron available for erythropoiesis. Hep-25 could be a useful marker of “hidden” functional iron deficiency: Hep-25 could be a useful marker of “hidden” functional iron deficiency: Low levels of hepcidin in HD patients with high ERI would indicate iron-restricted erythropoiesis, even when inflammation is present; Low levels of hepcidin in HD patients with high ERI would indicate iron-restricted erythropoiesis, even when inflammation is present; A downward trend in Hep-25 levels when iron doses are increased, in HD patients without evident iron deficiency would suggest correction of iron-restricted erythropoiesis, so would guide iron therapy, in EPO treated patients, when iron indices are not indicative of iron deficiency. A downward trend in Hep-25 levels when iron doses are increased, in HD patients without evident iron deficiency would suggest correction of iron-restricted erythropoiesis, so would guide iron therapy, in EPO treated patients, when iron indices are not indicative of iron deficiency.