Aim of this study was to evaluate whether the A736V TMPRSS6 polymorphism, a major genetic determinant of iron metabolism in healthy subjects, influences serum levels of hepcidin, the hormone regulating iron metabolism, and erythropoiesis in chronic hemodialysis (CHD).
To this end, we considered 199 CHD patients from Northern Italy (157 with hepcidin evaluation), and 188 healthy controls without iron deficiency, matched for age and gender. Genetic polymorphisms were evaluated by allele specific polymerase chain reaction assays, and hepcidin quantified by mass spectrometry.
Serum hepcidin levels were not different between the whole CHD population and controls (median 7.1, interquartile range (IQR) 0.55-17.1 vs. 7.4, 4.5-17.9 nM, respectively), but were higher in the CHD subgroup after exclusion of subjects with relative iron deficiency (p = 0.04). In CHD patients, the A736V TMPRSS6 polymorphism influenced serum hepcidin levels in individuals positive for mutations in the HFE gene of hereditary hemochromatosis (p < 0.0001). In particular, the TMPRSS6 736 V variant was associated with higher hepcidin levels (p = 0.017). At multivariate analysis, HFE and A736V TMPRSS6 genotypes predicted serum hepcidin independently of ferritin and C reactive protein (p = 0.048). In patients without acute inflammation and overt iron deficiency (C reactive protein <1 mg/dl and ferritin >30 ng/ml; n = 86), hepcidin was associated with lower mean corpuscular volume (p = 0.002), suggesting that it contributed to iron-restricted erythropoiesis. In line with previous results, in patients without acute inflammation and severe iron deficiency the “high hepcidin” 736 V TMPRSS6 variant was associated with higher erythropoietin maintenance dose (p = 0.016), independently of subclinical inflammation (p = 0.02).
The A736V TMPRSS6 genotype influences hepcidin levels, erythropoiesis, and anemia management in CHD patients. Evaluation of the effect of TMPRSS6 genotype on clinical outcomes in prospective studies in CHD may be useful to predict the outcomes of hepcidin manipulation, and to guide treatment personalization by optimizing anemia management.
Anemia; Chronic kidney disease; Erythropoietin; Genetics; Inflammation; Iron; Hemodialysis; Hepcidin; Hfe gene; Matriptase-2; Tmprss6
Despite fluctuations in dietary iron intake and intermittent losses through bleeding, the plasma iron concentrations in humans remain stable at 10–30 μM. While most of the iron entering blood plasma comes from recycling, appropriate amount of iron is absorbed from the diet to compensate for losses and maintain nontoxic amounts in stores. Plasma iron concentration and iron distribution are similarly regulated in laboratory rodents. The hepatic peptide hepcidin was identified as the systemic iron-regulatory hormone. In the efferent arc, hepcidin regulates intestinal iron absorption, plasma iron concentrations, and tissue iron distribution by inducing degradation of its receptor, the cellular iron exporter ferroportin. Ferroportin exports iron into plasma from absorptive enterocytes, from macrophages that recycle the iron of senescent erythrocytes, and from hepatocytes that store iron. In the more complex and less well understood afferent arc, hepatic hepcidin synthesis is transcriptionally regulated by extracellular and intracellular iron concentrations through a molecular complex of bone morphogenetic protein receptors and their iron-specific ligands, modulators and iron sensors. Through as yet undefined pathways, hepcidin is also homeostatically regulated by the iron requirements of erythroid precursors for hemoglobin synthesis. In accordance with the role of hepcidin-mediated iron redistribution in host defense, hepcidin production is regulated by inflammation as well. Increased hepcidin concentrations in plasma are pathogenic in iron-restrictive anemias including anemias associated with inflammation, chronic kidney disease and some cancers. Hepcidin deficiency causes iron overload in hereditary hemochromatosis and ineffective erythropoiesis. Hepcidin, ferroportin and their regulators represent potential targets for the diagnosis and treatment of iron disorders and anemias.
Iron overload; iron deficiency; anemia
The changes in iron status occurring during the course of heart failure (HF) and the underlying pathomechanisms are largely unknown. Hepcidin, the major regulatory protein for iron metabolism, may play a causative role. We investigated iron status in a broad spectrum of patients with systolic HF in order to determine the changes in iron status in parallel with disease progression, and to associate iron status with long-term prognosis.
Methods and results
Serum concentrations of ferritin, transferrin saturation (Tsat), soluble transferrin receptor (sTfR), and hepcidin were assessed as the biomarkers of iron status in 321 patients with chronic systolic HF [age: 61 ± 11 years, men: 84%, left ventricular ejection fraction: 31 ± 9%, New York Heart Association (NYHA) class: 72/144/87/18] at a tertiary cardiology centre and 66 age- and gender-matched healthy subjects. Compared with healthy subjects, asymptomatic HF patients had similar haematological status, but increased iron stores (evidenced by higher serum ferritin without distinct inflammation, P < 0.01) with markedly elevated serum hepcidin (P < 0.001). With increasing HF severity, patients in advanced NYHA classes had iron deficiency (ID) (reduced serum ferritin, low Tsat, high sTfR), iron-restricted erythropoiesis (reduced haemoglobin, high red cell distribution width), and inflammation (high serum high-sensitivity-C-reactive protein and interleukin 6), which was accompanied by decreased circulating hepcidin (all P < 0.001). In multivariable Cox models, low hepcidin was independently associated with increased 3-year mortality among HF patients (P < 0.001).
Increased level of circulating hepcidin characterizes an early stage of HF, and is not accompanied by either anaemia or inflammation. The progression of HF is associated with the decline in circulating hepcidin and the development of ID. Low hepcidin independently relates to unfavourable outcome.
Heart failure; Iron deficiency; Ferritin; Hepcidin; Prognosis
Hemojuvelin (HJV) is highly expressed in the liver, skeletal muscles, and heart, seems to play a role in iron absorption and release from cells, and has anti-inflammatory properties. Moreover, HJV plays an essential role in the regulation of hepcidin expression, specifically in the iron-sensing pathway. Hepcidin has emerged as a key regulator of iron homeostasis. In this study we tested for the first time the hypothesis that HJV is related to iron metabolism in hemodialysis (HD) patients.
Iron status, complete blood count, and serum creatinine, albumin, and lipids were assessed, using standard laboratory methods. Serum levels of soluble transferrin receptor (sTFR), high-sensitivity CRP, IL-6, hepcidin, and HJV were measured using commercially available kits.
Serum HJV, hepcidin, ferritin, IL-6, hsCRP, and serum creatinine were significantly higher (all P < 0.001), whereas serum iron, sTFR, transferrin, hemoglobin, and erythrocyte count were significantly lower in HD patients, compared to healthy volunteers (all P < 0.001). In univariate analysis, HJV was strongly correlated (P < 0.001) with ferritin, transferrin saturation, and TIBC, as well as with hsCRP, hepcidin, Kt/V (P < 0.01) and residual renal function, the presence of diabetes, APKD, and coronary heart disease. Predictors of HJV level in multiple regression analysis were ferritin (beta value was 0.50, P = 0.00004) and transferrin saturation (beta value was 0.47, P = 0.0002), explaining 81% of the HJV variations.
Serum HJV is elevated in HD patients and related predominantly to kidney function and iron metabolism. However, HJV is probably not correlated to inflammation. HJV appears to be a new player in iron metabolism in these patients.
Iron metabolism; Hemodialysis; Inflammation; Hepcidin; Hemojuvelin
Hepcidin is a central regulator of iron metabolism. Serum hepcidin levels are increased in patients with renal insufficiency, which may contribute to anemia. Urine hepcidin was found to be increased in some patients after cardiac surgery, and these patients were less likely to develop acute kidney injury. It has been suggested that urine hepcidin may protect by attenuating heme-mediated injury, but processes involved in urine hepcidin excretion are unknown.
To assess the role of tubular reabsorption we compared fractional excretion (FE) of hepcidin-25 with FE of β2-microglobulin (β2m) in 30 patients with various degrees of tubular impairment due to chronic renal disease. To prove that hepcidin is reabsorbed by the tubules in a megalin-dependent manner, we measured urine hepcidin-1 in wild-type and kidney specific megalin-deficient mice. Lastly, we evaluated FE of hepcidin-25 and β2m in 19 patients who underwent cardiopulmonary bypass surgery. Hepcidin was measured by a mass spectrometry assay (MS), whereas β2m was measured by ELISA.
In patients with chronic renal disease, FE of hepcidin-25 was strongly correlated with FE of β2m (r = 0.93, P <0.01). In megalin-deficient mice, urine hepcidin-1 was 7-fold increased compared to wild-type mice (p < 0.01) indicating that proximal tubular reabsorption occurs in a megalin- dependent manner. Following cardiac surgery, FE of hepcidin-25 increased despite a decline in FE of β2m, potentially indicating local production at 12–24 hours.
Hepcidin-25 is reabsorbed by the renal tubules and increased urine hepcidin-25 levels may reflect a reduction in tubular uptake. Uncoupling of FE of hepcidin-25 and β2m in cardiac surgery patients suggests local production.
AKI; β2-microglobulin; Hepcidin; Megalin; Kidney tubules
The metabolism of hepcidin is profoundly modified in chronic kidney disease (CKD). We investigated its relation to iron disorders, inflammation and hemoglobin (Hb) level in 199 non-dialyzed, non-transplanted patients with CKD stages 1–5. All had their glomerular filtration rate measured by 51Cr-EDTA renal clearance (mGFR), as well as measurements of iron markers including hepcidin and of erythropoietin (EPO). Hepcidin varied from 0.2 to 193 ng/mL. The median increased from 23.3 ng/mL [8.8–28.7] to 36.1 ng/mL [14.1–92.3] when mGFR decreased from ≥60 to <15 mL/min/1.73 m2 (p = 0.02). Patients with absolute iron deficiency (transferrin saturation (TSAT) <20% and ferritin <40 ng/mL) had the lowest hepcidin levels (5.0 ng/mL [0.7–11.7]), and those with a normal iron profile (TSAT ≥20% and ferritin ≥40), the highest (34.5 ng/mL [23.7–51.6]). In multivariate analysis, absolute iron deficiency was associated with lower hepcidin values, and inflammation combined with a normal or functional iron profile with higher values, independent of other determinants of hepcidin concentration, including EPO, mGFR, and albuminemia. The hepcidin level, although it rose overall when mGFR declined, collapsed in patients with absolute iron deficiency. There was a significant interaction with iron status in the association between Hb and hepcidin. Except in absolute iron deficiency, hepcidin’s negative association with Hb level indicates that it is not down-regulated in CKD anemia.
Iron overload is the hallmark of hereditary hemochromatosis and a complication of iron-loading anemias such as β-thalassemia. Treatment can be burdensome and have significant side effects, and new therapeutic options are needed. Iron overload in hereditary hemochromatosis and β-thalassemia intermedia is caused by hepcidin deficiency. Although transgenic hepcidin replacement in mouse models of these diseases prevents iron overload or decreases its potential toxicity, natural hepcidin is prohibitively expensive for human application and has unfavorable pharmacologic properties. Here, we report the rational design of hepcidin agonists based on the mutagenesis of hepcidin and the hepcidin-binding region of ferroportin and computer modeling of their docking. We identified specific hydrophobic/aromatic residues required for hepcidin-ferroportin binding and obtained evidence in vitro that a thiol-disulfide interaction between ferroportin C326 and the hepcidin disulfide cage may stabilize binding. Guided by this model, we showed that 7–9 N-terminal amino acids of hepcidin, including a single thiol cysteine, comprised the minimal structure that retained hepcidin activity, as shown by the induction of ferroportin degradation in reporter cells. Further modifications to increase resistance to proteolysis and oral bioavailability yielded minihepcidins that, after parenteral or oral administration to mice, lowered serum iron levels comparably to those after parenteral native hepcidin. Moreover, liver iron concentrations were lower in mice chronically treated with minihepcidins than those in mice treated with solvent alone. Minihepcidins may be useful for the treatment of iron overload disorders.
Excessive iron absorption is one of the main features of β-thalassemia and can lead to severe morbidity and mortality. Serial analyses of β-thalassemic mice indicate that while hemoglobin levels decrease over time, the concentration of iron in the liver, spleen, and kidneys markedly increases. Iron overload is associated with low levels of hepcidin, a peptide that regulates iron metabolism by triggering degradation of ferroportin, an iron-transport protein localized on absorptive enterocytes as well as hepatocytes and macrophages. Patients with β-thalassemia also have low hepcidin levels. These observations led us to hypothesize that more iron is absorbed in β-thalassemia than is required for erythropoiesis and that increasing the concentration of hepcidin in the body of such patients might be therapeutic, limiting iron overload. Here we demonstrate that a moderate increase in expression of hepcidin in β-thalassemic mice limits iron overload, decreases formation of insoluble membrane-bound globins and reactive oxygen species, and improves anemia. Mice with increased hepcidin expression also demonstrated an increase in the lifespan of their red cells, reversal of ineffective erythropoiesis and splenomegaly, and an increase in total hemoglobin levels. These data led us to suggest that therapeutics that could increase hepcidin levels or act as hepcidin agonists might help treat the abnormal iron absorption in individuals with β-thalassemia and related disorders.
Hepcidin regulation is linked to both iron and inflammatory signals and may influence iron loading in nonalcoholic steatohepatitis (NASH). The aim of this study was to examine the relationships among HFE genotype, serum hepcidin level, hepatic iron deposition and histology in nonalcoholic fatty liver disease (NAFLD). SNP genotyping for C282Y (rs1800562) and H63D (rs1799945) HFE mutations was performed in 786 adult subjects in the NASH Clinical Research Network (CRN). Clinical, histologic, and laboratory data were compared using nonparametric statistics and multivariate logistic regression. NAFLD patients with C282Y, but not H63D mutations, had lower median serum hepcidin levels (57 vs 65 ng/ml, p=0.01) and higher mean hepatocellular (HC) iron grades (0.59 vs 0.28, p<0.001), compared to wild type (WT) subjects. Subjects with hepatic iron deposition had higher serum hepcidin levels than subjects without iron for all HFE genotypes (p<0.0001). Hepcidin levels were highest among patients with mixed HC/reticuloendothelial system cell (RES) iron deposition. H63D mutations were associated with higher steatosis grades and NAFLD activity scores (OR≥1.4, CI >1.0≤2.5, p≤0.041), compared to WT, but not with either HC or RES iron. NAFLD patients with C282Y mutations had less ballooning or NASH (OR ≤0.62, 95% CI >0.39<0.94, p≤0.024) compared to WT subjects.
Presence of C282Y mutations in patients with NAFLD is associated with greater HC iron deposition and decreased serum hepcidin levels and there is a positive relationship between hepatic iron stores and serum hepcidin level across all HFE genotypes. These data suggest that body iron stores are the major determinant of hepcidin regulation in NAFLD regardless of HFE genotype. A potential role for H63D mutations in NAFLD pathogenesis is possible through iron-independent mechanisms.
NAFLD; Steatohepatitis; HFE; hepcidin; iron
The discovery of hepcidin clarified the basic mechanism of the control of systemic iron homeostasis. Hepcidin is mainly produced by the liver as a propeptide and processed by furin into the mature active peptide. Hepcidin binds ferroportin, the only cellular iron exporter, causing the internalization and degradation of both. Thus hepcidin blocks iron export from the key cells for dietary iron absorption (enterocytes), recycling of hemoglobin iron (the macrophages) and the release of storage iron from hepatocytes, resulting in the reduction of systemic iron availability. The BMP/HJV/SMAD pathway is the major regulator of hepcidin expression that responds to iron status. Also inflammation stimulates hepcidin via the IL6/STAT3 pathway with a support of an active BMP/HJV/SMAD pathway. In some pathological conditions hepcidin level is inadequately elevated and reduces iron availability in the body, resulting in anemia. These conditions occur in the genetic iron refractory iron deficiency anemia and the common anemia of chronic disease (ACD) or anemia of inflammation. Currently, there is no definite treatment for ACD. Erythropoiesis-stimulating agents and intravenous iron have been proposed in some cases but they are scarcely effective and may have adverse effects. Alternative approaches aimed to a pharmacological control of hepcidin expression have been attempted, targeting different regulatory steps. They include hepcidin sequestering agents (antibodies, anticalins, and aptamers), inhibitors of BMP/SMAD or of IL6/STAT3 pathway or of hepcidin transduction (siRNA/shRNA) or ferroportin stabilizers. In this review we summarized the biochemical interactions of the proteins involved in the BMP/HJV/SMAD pathway and its natural inhibitors, the murine and rat models with high hepcidin levels currently available and finally the progresses in the development of hepcidin antagonists, with particular attention to the role of heparins and heparin sulfate proteoglycans in hepcidin expression and modulation of the BMP6/SMAD pathway.
hepcidin; heparin; anemia of chronic diseases; inflammation; iron metabolism
Anemia of inflammation (AI) is a common complication of rheumatoid arthritis (RA) and has a negative impact on RA symptoms and quality of life. Upregulation of hepcidin by inflammatory cytokines has been implicated in AI. In this study, we evaluated and compared the effects of IL-6 and TNF-α blocking therapies on anemia, disease activity, and iron-related parameters including serum hepcidin in RA patients.
Patients (n = 93) were treated with an anti-IL-6 receptor antibody (tocilizumab) or TNF-α inhibitors for 16 weeks. Major disease activity indicators and iron-related parameters including serum hepcidin-25 were monitored before and 2, 4, 8, and 16 weeks after the initiation of treatment. Effects of tocilizumab and infliximab (anti-TNF-α antibody) on cytokine-induced hepcidin expression in hepatoma cells were analyzed by quantitative real-time PCR.
Anemia at base line was present in 66% of patients. Baseline serum hepcidin-25 levels were correlated positively with serum ferritin, C-reactive protein (CRP), vascular endothelial growth factor (VEGF) levels and Disease Activity Score 28 (DAS28). Significant improvements in anemia and disease activity, and reductions in serum hepcidin-25 levels were observed within 2 weeks in both groups, and these effects were more pronounced in the tocilizumab group than in the TNF-α inhibitors group. Serum hepcidin-25 reduction by the TNF-α inhibitor therapy was accompanied by a decrease in serum IL-6, suggesting that the effect of TNF-α on the induction of hepcidin-25 was indirect. In in vitro experiments, stimulation with the cytokine combination of IL-6+TNF-α induced weaker hepcidin expression than did with IL-6 alone, and this induction was completely suppressed by tocilizumab but not by infliximab.
Hepcidin-mediated iron metabolism may contribute to the pathogenesis of RA-related anemia. In our cohort, tocilizumab was more effective than TNF-α inhibitors for improving anemia and normalizing iron metabolism in RA patients by inhibiting hepcidin production.
Patients with chronic hepatitis C frequently have serum and hepatic iron overload, but the mechanism is unknown. Recently identified hepcidin, exclusively synthesized in the liver, is thought to be a key regulator for iron homeostasis and is induced by infection and inflammation. This study was conducted to determine the hepatic hepcidin expression levels in patients with various liver diseases. We investigated hepcidin mRNA levels of liver samples by real-time detection-polymerase chain reaction; 56 were hepatitis C virus (HCV) positive, 34 were hepatitis B virus (HBV) positive, and 42 were negative for HCV and HBV (3 cases of auto-immune hepatitis, 7 alcoholic liver disease, 13 primary biliary cirrhosis, 9 nonalcoholic fatty liver disease, and 10 normal liver). We analyzed the relation of hepcidin to clinical, hematological, histological, and etiological findings. Hepcidin expression levels were strongly correlated with serum ferritin (P < 0.0001) and the degree of iron deposit in liver tissues (P < 0.0001). Hepcidin was also correlated with hematological parameters (vs. hemoglobin, P = 0.0073; vs. serum iron, P = 0.0012; vs. transferrin saturation, P < 0.0001) and transaminase levels (P = 0.0013). The hepcidin-to-ferritin ratio was significantly lower in HCV+ patients than in HBV+ patients (P = 0.0129) or control subjects (P = 0.0080). In conclusion, hepcidin expression levels in chronic liver diseases were strongly correlated with either the serum ferritin concentration or degree of iron deposits in the liver. When adjusted by either serum ferritin values or hepatic iron scores, hepcidin indices were significantly lower in HCV+ patients than in HBV+ patients, suggesting that hepcidin may play a pivotal role in the pathogenesis of iron overload in patients with chronic hepatitis C.
Background and aims: The hepatic peptide hormone hepcidin, which has recently been isolated from human plasma and urine, is thought to be a central regulator of iron homeostasis. We investigated the presence and cellular localisation of hepcidin in the liver and developed a non-invasive assay to analyse its regulation in patients with hereditary haemochromatosis (HH), chronic renal insufficiency (CRI), and renal anaemia (RA).
Methods: Expression and localisation of hepcidin was shown by reverse transcription-polymerase chain reaction, western blot, immunocytochemistry, and immunofluorescence in human and guinea pig liver. Serum concentrations were determined in various groups of patients using a sensitive enzyme linked immunosorbent assay (ELISA).
Results: Western blot analysis with region specific antibodies identified a ~10 kDa peptide corresponding to the apparent molecular mass of pro-hepcidin. Localisation studies revealed that pro-hepcidin is expressed at the basolateral membrane domain of hepatocytes and is also present in blood. We developed a stable sensitive ELISA for detection and determination of pro-hepcidin in human serum. Mean pro-hepcidin level in human serum of healthy volunteers was 106.2 ng/ml. Enhanced levels of pro-hepcidin (148.1 ng/ml) were found in patients with CRI but normal haemoglobin values, indicating that the kidneys may metabolise and/or eliminate the circulating hormone. In contrast, concentrations of pro-hepcidin were significantly decreased in patients with HH (70.2 ng/ml) and also in patients with RA (115.0 ng/ml) compared with the CRI group.
Conclusions: From the detection of pro-hepcidin in human serum, we conclude that the prohormone may be involved in the regulation of iron metabolism in HH. Decreased pro-hepcidin levels could play an important role in the pathogenesis of HH.
hepcidin; chronic renal insufficiency; iron absorption; hereditary haemochromatosis; liver
Distortion of iron homeostasis may contribute to the pathogenesis of human immunodeficiency virus (HIV) infection and tuberculosis (TB). We studied the association of the central iron-regulatory hormone hepcidin with the severity of HIV and the association between hepcidin and other markers of iron homeostasis with development of TB.
Three groups of patients were selected from a prospective cohort of HIV-infected subjects in Bandung, Indonesia. The first group consisted of HIV-infected patients who started TB treatment more than 30 days after cohort enrollment (cases). The second group consisted of HIV-infected patients who were matched for age, gender and CD4 cell count to the cases group (matched controls). The third group consisted of HIV-infected patients with CD4 cell counts above 200 cells/mm3 (unmatched controls). Iron parameters including hepcidin were compared using samples collected at cohort enrollment, and compared with recently published reference values for serum hepcidin.
A total of 127 HIV-infected patients were included, 42 cases together with 42 matched controls and 43 unmatched controls. Patients with advanced HIV infection had elevated serum hepcidin and ferritin levels. Hepcidin levels correlated inversely with CD4 cells and hemoglobin. Cases had significantly higher hepcidin and ferritin concentrations at cohort enrollment compared to matched controls, but these differences were fully accounted for by the cases who started TB treatment between day 31 and 60 after enrollment. Hepcidin levels were not different in those with or without hepatitis C infection.
Iron metabolism is distorted in advanced HIV infection with CD4 cell counts correlating inversely with serum hepcidin levels. High serum hepcidin levels and hyperferritinemia were found in patients starting TB treatment shortly after cohort enrollment, suggesting that these parameters have a predictive value for development of manifest TB in HIV-infected patients.
Mass spectrometry (MS)-based assays for the quantification of the iron regulatory hormone hepcidin are pivotal to discriminate between the bioactive 25-amino acid form that can effectively block the sole iron transporter ferroportin and other naturally occurring smaller isoforms without a known role in iron metabolism. Here we describe the design, validation and use of a novel stable hepcidin-25+40 isotope as internal standard for quantification. Importantly, the relative large mass shift of 40 Da makes this isotope also suitable for easy-to-use medium resolution linear time-of-flight (TOF) platforms. As expected, implementation of hepcidin-25+40 as internal standard in our weak cation exchange (WCX) TOF MS method yielded very low inter/intra run coefficients of variation. Surprisingly, however, in samples from kidney disease patients, we detected a novel peak (m/z 2673.9) with low intensity that could be identified as hepcidin-24 and had previously remained unnoticed due to peak interference with the formerly used internal standard. Using a cell-based bioassay it was shown that synthetic hepcidin-24 was, like the -22 and -20 isoforms, a significantly less potent inducer of ferroportin degradation than hepcidin-25. During prolonged storage of plasma at room temperature, we observed that a decrease in plasma hepcidin-25 was paralleled by an increase in the levels of the hepcidin-24, -22 and -20 isoforms. This provides first evidence that all determinants for the conversion of hepcidin-25 to smaller inactive isoforms are present in the circulation, which may contribute to the functional suppression of hepcidin-25, that is significantly elevated in patients with renal impairment. The present update of our hepcidin TOF MS assay together with improved insights in the source and preparation of the internal standard, and sample stability will further improve our understanding of circulating hepcidin and pave the way towards further optimization and standardization of plasma hepcidin assays.
Patients with chronic hepatitis C (CHC) often have increased liver iron, a condition associated with reduced sustained response to antiviral therapy, more rapid progression to cirrhosis, and development of hepatocellular carcinoma. The hepatic hormone hepcidin is the major regulator of iron metabolism and inhibits iron absorption and recycling from erythrophagocytosis. Hepcidin decrease is a possible pathophysiological mechanism of iron overload in CHC, but studies in humans have been hampered so far by the lack of reliable quantitative assays for the 25-amino acid bioactive peptide in serum (s-hepcidin).
Using a recently validated immunoassay, we measured s-hepcidin levels in 81 untreated CHC patients and 57 controls with rigorous definition of normal iron status. All CHC patients underwent liver biopsy with histological iron score.
S-hepcidin was significantly lower in CHC patients than in controls (geometric means with 95% confidence intervals: 33.7, 21.5–52.9 vs. 90.9, 76.1–108.4 ng/mL, respectively; p < 0.001). In CHC patients, s-hepcidin significantly correlated with serum ferritin and histological total iron score, but not with s-interleukin-6. After stratification for ferritin quartiles, s-hepcidin increased significantly across quartiles in both controls and CHC patients (chi for trend, p < 0.001). However, in CHC patients, s-hepcidin was significantly lower than in controls for each corresponding quartile (analysis of variance, p < 0.001).
These results, together with very recent studies in animal and cellular models, indicate that although hepcidin regulation by iron stores is maintained in CHC, the suppression of this hormone by hepatitis C virus is likely an important factor in liver iron accumulation in this condition.
Chronic hepatitis C; Hemochromatosis; Hepcidin; Iron overload; Ferritin
Hepcidin, a key regulator of iron homeostasis, is increased in response to inflammation and some infections, but the in vivo role of hepcidin, particularly in children with iron deficiency anemia (IDA) is unclear. We investigated the relationships between hepcidin, cytokines and iron status in a pediatric population with a high prevalence of both anemia and co-morbid infections.
African refugee children <16 years were consecutively recruited at the initial post-resettlement health check with 181 children meeting inclusion criteria. Data on hematological parameters, cytokine levels and co-morbid infections (Helicobacter pylori, helminth and malaria) were obtained and urinary hepcidin assays performed. The primary outcome measure was urinary hepcidin levels in children with and without iron deficiency (ID) and/or ID anaemia (IDA). The secondary outcome measures included were the relationship between co-morbid infections and (i) ID and IDA, (ii) urinary hepcidin levels and (iii) cytokine levels. IDA was present in 25/181 (13.8%). Children with IDA had significantly lower hepcidin levels (IDA median hepcidin 0.14 nmol/mmol Cr (interquartile range 0.05–0.061) versus non-IDA 2.96 nmol/mmol Cr, (IQR 0.95–6.72), p<0.001). Hemoglobin, log-ferritin, iron, mean cell volume (MCV) and transferrin saturation were positively associated with log-hepcidin levels (log-ferritin beta coefficient (β): 1.30, 95% CI 1.02 to 1.57) and transferrin was inversely associated (β: −0.12, 95% CI −0.15 to −0.08). Cytokine levels (including IL-6) and co-morbid infections were not associated with IDA or hepcidin levels.
This is the largest pediatric study of the in vivo associations between hepcidin, iron status and cytokines. Gastro-intestinal infections (H. pylori and helminths) did not elevate urinary hepcidin or IL-6 levels in refugee children, nor were they associated with IDA. Longitudinal and mechanistic studies of IDA will further elucidate the role of hepcidin in paediatric iron regulation.
Anemia is a common complication of chronic kidney disease (CKD) that negatively impacts the quality of life and is associated with numerous adverse outcomes. Excess levels of the iron regulatory hormone hepcidin are thought to contribute to anemia in CKD patients by decreasing iron availability from the diet and from body stores. Adenine treatment in rats has been proposed as an animal model of anemia of CKD with high hepcidin levels that mirrors the condition in human patients.
We developed a modified adenine-induced kidney disease model with a higher survival rate than previously reported models, while maintaining persistent kidney disease and anemia. We then tested whether the small molecule bone morphogenetic protein (BMP) inhibitor LDN-193189, which was previously shown to lower hepcidin levels in rodents, mobilized iron into the plasma and improved iron-restricted erythropoiesis in this model.
Adenine-treated rats exhibited increased hepatic hepcidin mRNA, decreased serum iron, increased spleen iron content, low hemoglobin (Hb) and inappropriately low erythropoietin (EPO) levels relative to the degree of anemia. LDN-193189 administration to adenine-treated rats lowered hepatic hepcidin mRNA, mobilized stored iron into plasma and increased Hb content of reticulocytes.
Our data suggest that hepcidin lowering agents may provide a new therapeutic strategy to improve iron availability for erythropoiesis in CKD.
anemia; chronic kidney disease; hepcidin; iron; mouse model
Hepcidin is upregulated by inflammation and iron. Inherited (HFE genotype) and treatment-related factors (blood units (BU), Iron overload) affecting hepcidin (measured by C-ELISA) were studied in 42 consecutive patients with AML prior to and after allogeneic hematopoietic cell transplantation (HCT). Results. Elevated serum ferritin pre- and post-HCT was present in all patients. Median hepcidin pre- and post-HCT of 358 and 398 ng/mL, respectively, were elevated compared to controls (median 52 ng/mL) (P < .0001). Liver and renal function, prior chemotherapies, and conditioning had no impact on hepcidin. Despite higher total BU after HCT compared to pretransplantation (P < .0005), pre- and posttransplant ferritin and hepcidin were similar. BU influenced ferritin (P = .001) and hepcidin (P = .001). No correlation of pre- or posttransplant hepcidin with pretransplant ferritin was found. HFE genotype did not influence hepcidin. Conclusions. Hepcidin is elevated in AML patients pre- and post-HCT due to transfusional iron-loading suggesting that hepcidin synthesis remains intact despite chemotherapy and HCT.
Hepcidin has an important role in iron metabolism. We investigated whether hepcidin was involved in renal cell carcinoma (RCC).
We measured serum hepcidin-25 levels in 32 patients by liquid chromatograpy (LC)-mass spectrometry (MS)/MS, and assessed hepcidin mRNA expression in paired tumor and non-tumor tissue samples from the surgical specimens of 53 consecutive patients with RCC by real-time reverse transcription polymerase chain reaction.
The serum hepcidin-25 level was higher in patients with metastatic RCC than nonmetastatic RCC (P < 0.0001), and was positively correlated with the serum interleukin-6 and C-reactive protein levels (P < 0.001). Expression of hepcidin mRNA was lower in tumor tissues than in non-tumor tissues (P < 0.0001). The serum hepcidin-25 level was not correlated with the expression of hepcidin mRNA in the corresponding tumor tissue specimens from 32 patients. Hepcidin mRNA expression in tumor tissue was correlated with metastatic potential, but not with histological differentiation or tumor stage. Kaplan-Meier analysis showed that over expression of hepcidin mRNA was related to shorter overall survival in RCC patients. Univariate analysis (Cox proportional hazards model) showed that the hepcidin mRNA level was an independent prognostic factor for overall survival.
Our findings suggest that a high serum hepcidin-25 level may indicate the progression of RCC, and that upregulation of hepcidin mRNA expression in tumor tissue may be related to increased metastatic potential.
The results of recent randomized, controlled trials in patients with chronic kidney disease and anemia have suggested that hyporesponsiveness to erythropoiesis stimulating agents (ESA) is a significant predictor of poor patient outcomes. Functional iron deficiency (FID) is the most common cause of suboptimal ESA response, and intravenous iron administration (IVFe) efficiently raises hemoglobin (Hb) concentrations even under the condition of FID. Consequently, renal anemia correction has conceptually shifted from ‘higher Hb values with high ESA doses’ to ‘prevention of ESA hyporesponsiveness with IVFe’. The discovery of hepcidin has profoundly changed our understanding of the place of FID in renal anemia therapy. Hepcidin reduces the abundance of iron transport proteins which facilitate iron absorption from the gut and iron mobilization from macrophages. Serum hepcidin is mainly modulated by iron stores, as is serum ferritin. High hepcidin or ferritin levels block intestinal iron absorption and iron recycling in macrophages and decrease iron availability for erythropoiesis, leading to FID. Iron administration, especially IVFe, increases hepcidin levels and concomitantly inhibits iron supply to erythroid cells. This in turn could lead to a vicious circle, exacerbating FID and increasing iron demand. Therefore, physicians should be cautious with unrestricted IVFe to chronic kidney disease patients with FID.
Hepcidin; Iron; Renal anemia; Erythropoiesis stimulating agents; Ferritin
Hepcidin is a hormone central to the regulation of iron homeostasis in the body. It is believed to be produced exclusively by the liver. Ferroportin, an iron exporter, is the receptor for hepcidin. This transporter/receptor is expressed in Müller cells, photoreceptor cells, and retinal pigment epithelium (RPE) within the retina. Since the retina is protected by the retinal-blood barriers, we asked whether ferroportin in the retina is regulated by hepcidin in the circulation or whether the retina produces hepcidin for regulation of its own iron homeostasis. Here we show that hepcidin is expressed robustly in Müller cells, photoreceptor cells, and RPE, closely resembling the expression pattern of ferroportin. We also show that bacterial lipopolysaccharide (LPS) is a regulator of hepcidin expression in Müller cells and RPE, both in vitro and in vivo, and that the regulation occurs at the transcriptional level. The action of LPS on hepcidin expression is mediated by the Toll-like receptor-4. The upregulation of hepcidin by LPS occurs independent of Hfe (Human leukocyte antigen-like protein involved in Fe homeostasis). The increase in hepcidin levels in retinal cells in response to LPS treatment is associated with a decrease in ferroportin levels. The LPS-induced upregulation of hepcidin and consequent downregulation of ferroportin is associated with increased oxidative stress and apoptosis within the retina in vivo. We conclude that retinal iron homeostasis may be regulated in an autonomous manner by hepcidin generated within the retina and that chronic bacterial infection/inflammation of the retina may disrupt iron homeostasis and retinal function.
hepcidin; retina; hemochromatosis; lipopolysaccharide; ferroportin; retinal pigment epithelium
This study was designed to evaluate the levels of hepcidin in the serum of patients with chronic obstructive pulmonary disease (COPD).
In the study, 74 male patients (ages 45-75) in a stable period for COPD were grouped as Group I: Mild COPD (n:25), Group II: Moderate COPD (n:24), and Group III: Severe COPD (n:25). Healthy non-smoker males were included in Group IV (n:35) as a control group. The differences of hepcidin level among all the groups were examined. Also, in the patient groups with COPD, hepcidin level was compared with age, body mass index, cigarette (package/year), blood parameters (iron, total iron binding capacity, ferritin, hemoglobin, hematocrit [hct]), respiratory function tests, and arterial blood gas results.
Although there was no difference between the healthy control group and the mild COPD patient group (P=0.781) in terms of hepcidin level, there was a difference between the moderate (P=0.004) and the severe COPD patient groups (P=0.002). The hepcidin level of the control group was found to be higher than the moderate and severe COPD patient groups. In the severe COPD patients, hepcidin level increased with the increase in serum iron (P=0.000), hct (P=0.009), ferritin levels (P=0.012), and arterial oxygen saturation (SaO2, P=0.000).
The serum hepcidin level that is decreased in severe COPD brings into mind that it may play a role in the mechanism to prevent hypoxemia. The results suggest that serum hepcidin level may be a useful marker in COPD. Larger prospective studies are needed to confirm our findings between hepcidin and COPD.
Chronic obstructive pulmonary disease; hepcidin; hypoxemia
Recently, hepcidin expression in adipose tissue has been described and shown to be increased in patients with severe obesity. We tried to assess the effect of obesity on hepcidin serum levels and treatment outcome of iron deficiency anemia in children.
This was a case control study included 70 children with iron deficiency anemia "IDA" (35 obese and 35 non-obese) and 30 healthy non-obese children with comparable age and sex(control group). Parameters of iron status (Serum iron, ferritin, transferrin, total iron binding capacity and transferrin saturation) and serum hepcidin levels were assessed initially and after 3 months of oral iron therapy for IDA.
Compared to the control group, serum hepcidin was significantly lower in non-obese children with IDA(p < 0.01) and significantly higher in obese children with IDA (p < 0.01). Hepcidin increased significantly in non-obese children with IDA after 3 months of iron therapy (P < 0.01). On the other hand, obese children showed non-significant change in hepcidin level after iron therapy (p > 0.05). Although hepcidin showed significant positive correlations with Hb, serum iron and transferrin saturation in non-obese children with IDA, it showed significant negative correlations with Hb, serum iron and transferrin saturation in obese children with IDA (P < 0.05).
Obesity increased hepcidin levels and was associated with diminished response to oral iron therapy in childhood iron deficiency anemia.
Obesity; Hepcidin; Iron deficiency; Children
The anemia of chronic kidney disease and hemodialysis is characterized by chronic inflammation and release of cytokines, resulting in the upregulation of the iron hormone hepcidin, also increased by iron therapy and reduced glomerular filtration, with consequent reduction in iron absorption, recycling, and availability to the erythron. This response proves advantageous in the short-term to restrain iron availability to pathogens, but ultimately leads to severe anemia, and impairs the response to erythropoietin (Epo) and iron. Homozygosity for the common C282Y and H63D HFE polymorphisms influence iron metabolism by hampering hepcidin release by hepatocytes in response to increased iron stores, thereby resulting in inadequate inhibition of the activity of Ferroportin-1, inappropriately high iron absorption and recycling, and iron overload. However, in hemodialysis patients, carriage of HFE mutations may confer an adaptive benefit by decreasing hepcidin release in response to iron infusion and inflammation, thereby improving iron availability to erythropoiesis, anemia control, the response to Epo, and possibly survival. Therefore, anti-hepcidin therapies may improve anemia management in hemodialysis. However, HFE mutations directly favor hemoglobinization independently of hepcidin, and reduce macrophages activation in response to inflammation, whereas hepcidin might also play a beneficial anti-inflammatory and anti-microbic action during sepsis, so that direct inhibition of HFE-mediated regulation of iron metabolism may represent a valuable alternative therapeutic target. Genetic studies may offer a valuable tool to test these hypotheses and guide the research of new therapies.
Chronic kidney disease; Hemodialysis; Iron; HFE protein; Iron overload