Hepatic Hormone Hepcidin Research Articles

A single approach to targeting transferrin receptor 2 corrects iron and erythropoietic defects in murine models of anemia of inflammation and chronic kidney disease

Anemia is a common complication of systemic inflammation. Proinflammatory cytokines both decrease erythroblast sensitivity to erythropoietin (EPO) and increase the levels of the hepatic hormone hepcidin, sequestering iron in stores and causing functional iron deficiency. Anemia of chronic kidney disease (CKD) is a peculiar form of anemia of inflammation, characterized by impaired EPO production paralleling progressive kidney damage. Traditional therapy based on increased EPO (often in combination with iron) may have off-target effects due to EPO interaction with its non-erythroid receptors. Transferrin Receptor 2 (Tfr2) is a mediator of the iron-erythropoiesis crosstalk. Its deletion in the liver hampers hepcidin production, increasing iron absorption, whereas its deletion in the hematopoietic compartment increases erythroid EPO sensitivity and red blood cell production. Here, we show that selective hematopoietic Tfr2 deletion ameliorates anemia in mice with sterile inflammation in the presence of normal kidney function, promoting EPO responsiveness and erythropoiesis without increasing serum EPO levels. In mice with CKD, characterized by absolute rather than functional iron deficiency, Tfr2 hematopoietic deletion had a similar effect on erythropoiesis but anemia improvement was transient because of limited iron availability. Also, increasing iron levels by downregulating only hepatic Tfr2 had a minor effect on anemia. However, simultaneous deletion of hematopoietic and hepatic Tfr2, stimulating erythropoiesis and increased iron supply, was sufficient to ameliorate anemia for the entire protocol. Thus, our results suggest that combined targeting of hematopoietic and hepatic Tfr2 may be a therapeutic option to balance erythropoiesis stimulation and iron increase, without affecting EPO levels.

Functional role of endothelial transferrin receptor 1 in iron sensing and homeostasis.

Systemic iron homeostasis is regulated by the hepatic hormone hepcidin to balance meeting iron requirements while limiting toxicity from iron excess. Iron-mediated induction of bone morphogenetic protein (BMP) 6 is a central mechanism for regulating hepcidin production. Liver endothelial cells (LECs) are the main source of endogenous BMP6, but how they sense iron to modulate BMP6 transcription and thereby hepcidin is uncertain. Here, we investigate the role of endothelial cell transferrin receptor 1 (TFR1) in iron uptake, BMP6 regulation, and systemic iron homeostasis using primary LEC cultures and endothelial Tfrc (encoding TFR1) knockout mice. We show that intracellular iron regulates Bmp6 expression in a cell-autonomous manner, and TFR1 mediates iron uptake and Bmp6 expression by holo-transferrin in primary LEC cultures. In addition, endothelial Tfrc knockout mice exhibit altered iron homeostasis compared with littermate controls when fed a limited iron diet, as evidenced by increased liver iron and inappropriately low Bmp6 and hepcidin expression relative to liver iron. However, endothelial Tfrc knockout mice have a similar iron phenotype compared to littermate controls when fed an iron-rich standard diet. Finally, ferritin and non-transferrin bound iron (NTBI) are additional sources of iron that mediate Bmp6 induction in primary LEC cultures via TFR1-independent mechanisms. Together, our data demonstrate a minor functional role for endothelial cell TFR1 in iron uptake, BMP6 regulation, and hepatocyte hepcidin regulation under iron limiting conditions, and suggest that ferritin and/or NTBI uptake by other transporters have a dominant role when iron availability is high.

How Severe Anaemia Might Influence the Risk of Invasive Bacterial Infections in African Children.

Severe anaemia and invasive bacterial infections are common causes of childhood sickness and death in sub-Saharan Africa. Accumulating evidence suggests that severely anaemic African children may have a higher risk of invasive bacterial infections. However, the mechanisms underlying this association remain poorly described. Severe anaemia is characterized by increased haemolysis, erythropoietic drive, gut permeability, and disruption of immune regulatory systems. These pathways are associated with dysregulation of iron homeostasis, including the downregulation of the hepatic hormone hepcidin. Increased haemolysis and low hepcidin levels potentially increase plasma, tissue and intracellular iron levels. Pathogenic bacteria require iron and/or haem to proliferate and have evolved numerous strategies to acquire labile and protein-bound iron/haem. In this review, we discuss how severe anaemia may mediate the risk of invasive bacterial infections through dysregulation of hepcidin and/or iron homeostasis, and potential studies that could be conducted to test this hypothesis.

Serum markers of iron metabolism in chronic hepatitis C virus infections

Background Hepatitis C virus (HCV) infection is a common cause of chronic hepatitis, which leads to cirrhosis of the liver and hepatocellular carcinoma. Chronic hepatitis can cause iron buildup in the liver and result in liver injury. The major iron metabolism regulator, the hepatic hormone hepcidin, inhibits iron absorption and recycling, and as hepcidin is suppressed by the virus, it contributes to the pathogenesis of the liver. Aim To assess serum iron markers in patients with chronic hepatitis C (CHC) as opposed to people who are healthy and a summary of interactions of HCV and iron overload. Patients and methods This case–control study was performed on 30 hepatitis C-infected Egyptian patients (group I) and 15 apparently healthy control (group II). Routine laboratory investigations, as well as serum hepcidin and iron marker assessments were performed. Results Throughout this study, the serum hepcidin level in patients significantly decreased relative to the control group (P<0.001). The patients showed significantly higher serum iron, transferrin saturation, alanine aminotransferase, and aspartate aminotransferase compared with the control group (P<0.001). Serum albumin in patients’ group was considerably decreased in comparison with the control (P<0.05). There was a highly statistically significant lower platelet count value in patients compared with the control group (P<0.001). The interaction between hepcidin and iron, transferrin, and alanine aminotransferase is significantly negative. Conclusion Hepatic iron deposition is a joint feature in patients with CHC. Chronic HCV infection may reduce serum hepcidin, which may lead to iron overload in these patients. So hepcidin is a surrogate marker for evaluation of iron overload in patients with CHC.

S1629 ERFE‐A260S ACCOUNTS FOR HEPATIC IRON METABOLISM IMPAIRMENT BY ACTING ON THE BMP‐SMAD PATHWAY

Background:Iron overload is a major clinical challenge in the management of hereditary ineffective erythropoiesis ‐related anemia (iron‐loading anemia). To coordinate iron metabolism with hemoglobin (Hb) synthesis, erythropoietin (EPO)‐stimulated erythroid precursors release erythroferrone (ERFE), a soluble protein that suppresses the expression of hepcidin. Hepcidin is a hormone that controls iron entry into the circulation by blocking the iron exporter ferroportin.Congenital dyserythropoietic anemia type II (CDAII) is a bone marrow failure anemia characterized by mono‐lineage cytopenia and morphological abnormalities of erythroid precursor cells. The marked clinical variability observed in CDAII patients is related to its major complication, the iron overload associated with reduced expression of hepatic hormone hepcidin. We recently analyzed ERFE expression in both CDAII patients and in vitro model, demonstrating that in CDAII patients ERFE is overexpressed and related to abnormal erythropoiesis.Aims:To study possible modifier genes affecting the iron overload phenotype in CDAII.Methods:t‐NGS custom panel; genotyping; cells culture and transfection assays; qRT‐PCR; immunoblotting.Results:We herein identified a recurrent low‐frequency variant, A260S, in ERFE gene in 12.5% of CDAII patients showing severe phenotype by our 81‐genes target custom panel for modifier genes. We demonstrated that ERFE‐A260S variant leads to an increased expression of ERFE. We further characterized the effect of ERFE‐A260S in an in vitro human hepatic model, HuH7 cells, showing a marked impairment of iron regulation pathways by inhibition of phosphorylation of SMAD1/5/8 and consequent downregulation of the expression of BMP/SMADs pathway target genes: HAMP, ID1, ATHO8 and SMAD6.Summary/Conclusion:We herein described for the first time an ERFE polymorphism as modifier variant with a mild effect on disease expression under a multifactorial‐like model in a condition of iron‐loading anemia due to ineffective erythropoiesis. This is a novel paradigm that paves the ways to new investigations for evaluating the role of this ERFE variant as a modifier in many other related pathological conditions, as b‐thalassemia intermedia. Moreover, the discovery of the genetic basis of CDAII and its major complication, iron overload, will lead us to expand our understanding of the biology of ineffective erythropoiesis and to provide initiative for further investigation of new therapeutics. Indeed, ERFE is currently a candidate target for therapeutic inhibition, in iron loading anemia.

Regulators of hepcidin expression.

Iron, an essential nutrient, is required for many biological processes but is also toxic in excess. The lack of a mechanism to excrete excess iron makes it crucial for the body to regulate the amount of iron absorbed from the diet. This regulation is mediated by the hepatic hormone hepcidin. Hepcidin also controls iron release from macrophages that recycle iron and from hepatocytes that store iron. Hepcidin binds to the only known iron export protein, ferroportin, inducing its internalization and degradation and thus limiting the amount of iron released into the plasma. Important regulators of hepcidin, and therefore of systemic iron homeostasis, include plasma iron concentrations, body iron stores, infection and inflammation, hypoxia and erythropoiesis, and, to a lesser extent, testosterone. Dysregulation of hepcidin production contributes to the pathogenesis of many iron disorders: hepcidin deficiency causes iron overload in hereditary hemochromatosis and non-transfused β-thalassemia, whereas overproduction of hepcidin is associated with iron-restricted anemias seen in patients with chronic inflammatory diseases and inherited iron-refractory iron-deficiency anemia. The present review summarizes our current understanding of the molecular mechanisms and signaling pathways contributing to hepcidin regulation by these factors and highlights the issues that still need clarification.

Hepcidin as a potential predictor for preoperative anemia treatment with intravenous iron-A retrospective pilot study.

Preoperative anemia occurs in about one third of patients who undergo elective surgery and is associated with an impaired outcome. Therefore, screening of preoperative anemia was established in the context of a multidisciplinary Patient Blood Management (PBM) program at the University Hospital of Muenster, Germany. Anemic patients without contraindications were treated with intravenous (IV) iron (ferric carboxymaltose) to increase their hemoglobin (Hgb) levels and hence to treat anemia prior to surgery. Interestingly, we detected a large variability in the response of Hgb levels after IV iron administration. Systemic iron homeostasis is mainly regulated by the hepatic hormone hepcidin, which regulates the cell surface expression of the sole known iron exporter ferroportin. The objective of this retrospective pilot study was to analyze the potential of hepcidin to predict the response of anemic patients to preoperative IV iron treatment measured as increase in Hgb.Serum samples of non-anemic (n = 48), untreated anemic (n = 64) and anemic patients treated with IV iron (n = 79), in total 191 patients, were collected between October 2014 until June 2016. Serum hepcidin levels were determined and data were analyzed retrospectively.The analysis revealed at first a correlation between serum hepcidin levels and the parameters of the iron status. Second, patients treated with IV iron showed a noticeably higher increase in their delta Hgb level between PBM consultation and surgery (0.45g/dl [0.05, 1.05] compared to patients without IV iron (0.1g/dl [-0.48, 0.73], *p = 0.03). Patients were then grouped into ‘non-responders’, defined as delta Hgb <0.6g/dl and ‘responders’, with delta Hgb ≥0.6g/dl between the day of IV iron treatment and the day of surgery. Within normal ranges and clinically unapparent, a statistically noticeable difference between responders and non-responders was found for CRP and leukocytes. Serum hepcidin levels were higher in the group of non-responders (10.6ng/ml [3.93, 34.77]) compared to responders (2.1ng/ml [0.25, 7.97], *p = 0.04).To conclude, the data of this retrospective pilot study indicate that hepcidin might be a promising biomarker to predict a patient`s responsiveness to IV iron in preoperative anemia treatment. Prospective studies have to investigate serum hepcidin levels as a biomarker to guide physician`s decision on IV iron substitution.

Matriptase-2 suppresses hepcidin expression by cleaving multiple components of the hepcidin induction pathway

Systemic iron homeostasis is maintained by regulation of iron absorption in the duodenum, iron recycling from erythrocytes, and iron mobilization from the liver and is controlled by the hepatic hormone hepcidin. Hepcidin expression is induced via the bone morphogenetic protein (BMP) signaling pathway that preferentially uses two type I (ALK2 and ALK3) and two type II (ActRIIA and BMPR2) BMP receptors. Hemojuvelin (HJV), HFE, and transferrin receptor-2 (TfR2) facilitate this process presumably by forming a plasma membrane complex with BMP receptors. Matriptase-2 (MT2) is a protease and key suppressor of hepatic hepcidin expression and cleaves HJV. Previous studies have therefore suggested that MT2 exerts its inhibitory effect by inactivating HJV. Here, we report that MT2 suppresses hepcidin expression independently of HJV. In Hjv-/- mice, increased expression of exogenous MT2 in the liver significantly reduced hepcidin expression similarly as observed in wild-type mice. Exogenous MT2 could fully correct abnormally high hepcidin expression and iron deficiency in MT2-/- mice. In contrast to MT2, increased Hjv expression caused no significant changes in wild-type mice, suggesting that Hjv is not a limiting factor for hepcidin expression. Further studies revealed that MT2 cleaves ALK2, ALK3, ActRIIA, Bmpr2, Hfe, and, to a lesser extent, Hjv and Tfr2. MT2-mediated Tfr2 cleavage was also observed in HepG2 cells endogenously expressing MT2 and TfR2. Moreover, iron-loaded transferrin blocked MT2-mediated Tfr2 cleavage, providing further insights into the mechanism of Tfr2's regulation by transferrin. Together, these observations indicate that MT2 suppresses hepcidin expression by cleaving multiple components of the hepcidin induction pathway.

Transforming Growth Factor β1 (TGF-β1) Activates Hepcidin mRNA Expression in Hepatocytes

The hepatic hormone hepcidin is the master regulator of systemic iron homeostasis. Its expression level is adjusted to alterations in iron levels, inflammatory cues, and iron requirements for erythropoiesis. Bone morphogenetic protein 6 (BMP6) contributes to the iron-dependent control of hepcidin. In addition, TGF-β1 may stimulate hepcidin mRNA expression in murine hepatocytes and human leukocytes. However, receptors and downstream signaling proteins involved in TGF-β1-induced hepcidin expression are still unclear. Here we show that TGF-β1 treatment of mouse and human hepatocytes, as well as ectopic expression of TGF-β1 in mice, increases hepcidin mRNA levels. The hepcidin response to TGF-β1 depends on functional TGF-β1 type I receptor (ALK5) and TGF-β1 type II receptor (TβRII) and is mediated by a noncanonical mechanism that involves Smad1/5/8 phosphorylation. Interestingly, increasing availability of canonical Smad2/3 decreases TGF-β1-induced hepcidin regulation, whereas the BMP6-hepcidin signal was enhanced, indicating a signaling component stoichiometry-dependent cross-talk between the two pathways. Although ALK2/3-dependent hepcidin activation by BMP6 can be modulated by each of the three hemochromatosis-associated proteins: HJV (hemojuvelin), HFE (hemochromatosis protein), and TfR2 (transferrin receptor 2), these proteins do not control the ALK5-mediated hepcidin response to TGF-β1. TGF-β1 mRNA levels are increased in mouse models of iron overload, indicating that TGF-β1 may contribute to hepcidin synthesis under these conditions. In conclusion, these data demonstrate that a complex regulatory network involving TGF-β1 and BMP6 may control the sensing of systemic and/or hepatic iron levels.

Erfe-Encoding FAM132B in Congenital Dyserythropoietic Anemia Type II

Recessive mutations in SEC23B gene cause congenital dyserythropoietic anemia type II (CDAII), a rare hereditary disorder hallmarked by ineffective erythropoiesis, iron overload, and reduced expression of hepatic hormone hepcidin (Iolascon, 2013). The most recently described hepcidin regulator is the erythroblast-derived hormone erythroferrone (ERFE), a member of TNF-α superfamily that specifically inhibits hepcidin production in experimental models (Kautz, 2014). However, the function of ERFE in humans remains to be investigated.To determine whether dysregulation of ERFE expression is associated with ineffective erythropoiesis and iron-loading in CDAII, we studied the ERFE-encoding FAM132B gene expression in 48 SEC23B-related CDAII patients and 29 age and gender matched healthy controls (HCs). Twelve new cases and four novel SEC23B mutations were described. Samples were obtained after informed consent, according to the Declaration of Helsinki. Genomic DNA, mutational screening, RNA isolation, cDNA preparation, and qRT-PCR were performed as previously described (Russo, 2013).All patients were young adults (17.0±2.5 years at diagnosis), with increased serum ferritin (395.4±67.6 ng/mL) and transferrin saturation (71.9±5.4 %). We observed a statistically significant overexpression of FAM132B gene in peripheral blood mononuclear cells from CDAII patients (9.09±0.08) compared to HCs (8.32±0.12, p<0.0001). A similar trend was obtained when evaluating FAM132B expression in reticulocytes from a subset of patients and HCs. Of note, a statistically significant correlation between peripheral blood and reticulocyte FAM132B expression from the same patients was observed (Spearman ρ= 0.78, p=0.02). Although the role of ERFE in peripheral blood is still unknown, our observations suggested that the evaluation of FAM132B mRNA in peripheral blood is a reliable and easy-to-measure marker of ERFE levels.When we divided CDAII patients into two sub-groups accordingly to FAM132B gene expression, we observed a statistically significant reduction in hemoglobin (Hb) level in the high-FAM132B subset (8.6±0.4 g/dL) respect to low-FAM132B one (10.1±0.5 g/dL, p=0.02). Of note, the expression level of FAM132B did not correlate with the transfusion regimen. The higher amount of ERFE reflects the increased iron demand for Hb production as well as the expanding abnormal erythropoiesis, as attested by the increased RDW and sTfR (although not significant) in high-FAM132B patients. This in turn leads to reduced hepcidin in high-FAM132B group (4.2±1.8 nM) compared to low-FAM132B one (5.9±1.8 nM, p=0.05), resulting in augmented iron delivery to the erythron. Although the iron balance data do not differ significantly between the two groups, a tendency to decreased hepcidin/ferritin ratio and increased transferrin saturation was observed in high-FAM132B patients. Thus, FAM132B overexpression seems to contribute to the inappropriate suppression of hepcidin with subsequent hemosiderosis observed in CDAII.Consistent with our previous studies, we observed a reduced SEC23B expression in our patients compared to HC. Indeed, FAM132B and SEC23B gene expression exhibited an inverse correlation (Spearman ρ=-0.36, p=0.01). We confirmed the ex vivo data about inverse correlation between FAM132B and SEC23B expression observed in our patients by establishing K562 SEC23B-silenced cells. To knockdown SEC23B gene expression in K562 cells two different pGIPZ Lentiviral shRNAmir for SEC23B (shSEC23B-70/-74) were used. We observed a higher expression of FAM132B at 5 days of erythroid differentiation in K562 SEC23B-silenced cell compared to not-silenced ones. Conversely, SEC23B expression was lower in both shSEC23B compared to sh-CTR at 2 and 5 days of differentiation. Although the mechanisms of hemin-induced differentiation are quite different from EPO-induced ones, we can hypothesize that FAM132B over-expression is related to the maturative arrest and the subsequent increased number of erythroid precursors.This study provides the first analysis on ERFE regulation in humans. Our data suggest that ERFE over-expression in CDAII patients is the result of both physiological and pathological mechanisms leading to hepcidin suppression in condition of dyserythropoiesis. Nevertheless, it seems that ERFE cannot be the main erythroid regulator of hepcidin suppression, at least in CDAII patients. DisclosuresNo relevant conflicts of interest to declare.

Hepcidin and iron disorders: new biology and clinical approaches.

Hepatic hormone hepcidin is a principal regulator of iron homeostasis and a pathogenic factor in common iron disorders. Hepcidin deficiency causes iron overload in hereditary hemochromatosis and iron-loading anemias, whereas hepcidin excess causes or contributes to the development of iron-restricted anemia in inflammatory diseases, infections, some cancers, and chronic kidney disease. Because of this, hepcidin may become a useful tool for diagnosis and management of iron disorders. Furthermore, a number of strategies that target hepcidin, its receptor, and its regulators are under development as novel therapeutic approaches for diseases associated with iron dysregulation.

Multisystem, Induced Pluripotent Stem Cell (iPSC) Modeling Reveals a Role for Growth Differentiation Factors (GDFs) in the Etiology of β Thalassemia and Ineffective Erythropoiesis

β Thalassemia is one of the most common monogenic diseases in man encompassing a heterogeneous group of naturally occurring, inherited mutations characterized by abnormal globin gene expression. Iron overload is the principle cause of morbidity and mortality in β thalassemia. The hepatic hormone hepcidin regulates iron homeostasis modulating iron concentration in the plasma and its distribution in tissues throughout the body. Dysregulation of hepcidin production underlies many iron disorders with emerging evidence suggesting that deficiency of the hormone may result from the strong suppressive effect of high erythropoietic activity on hepcidin expression. Current treatment modalities for iron overload include phlebotomy and iron chelation. In β thalassemia, phlebotomy is not feasible and regular chelation is the principal treatment for iron overload. Iron chelators have side effects ranging from mild to very serious, and compliance is often suboptimal. Hepcidin diagnostics and the development of novel therapeutic options are clearly desirable and may help in the management of patients with β thalassemia.Hepcidin dysregulation, along with the ineffective erythropoiesis and anemia noted in β thalassemia highlight the need for a model capable of recapitulating the multisystem complexity of this clinically variable disease. Using induced pluripotent stem cell (iPSC) technology, cell lines can be established that are genetically identical to the individual from whom they are derived, allowing for disease modeling and the development of novel therapeutics in the exact genetic context of the patient. We have generated disease-specific iPSC lines from patients with β thalassemia major. Harnessing the pluripotency of iPSCs, we demonstrate the modeling of this multisystem disease through the directed differentiation of patient-specific iPSCs into hepatocytes that produce hepcidin as well as erythroblasts produced via a platform that allows for exponentially greater production of blood cells in comparison to existing methodologies (Smith et. al, Blood, 2013). We demonstrate that β thalassemia iPSC-derived erythroblasts secrete greater amounts of GDFs 11 and 15, and that exposure of the patient’s own iPSC-derived hepatocytes to disease-specific erythroblast supernatants results in a marked decrease in hepcidin expression recapitulating essential aspects of the disease in vitro. Furthermore, exposure of developing iPSC-derived erythroblasts to recombinant GDFs results in the production of immature cells that fail to reach maturity, providing a potential novel mechanism contributing to the development of ineffective erythropoiesis. Taken together, these results validate this iPSC-based, patient-specific in vitro system as a platform for testing new diagnostic approaches as well as novel therapeutic strategies targeting the correction of hepcidin dysregulation. DisclosuresNo relevant conflicts of interest to declare.

Unbiased RNAi screen for hepcidin regulators links hepcidin suppression to proliferative Ras/RAF and nutrient-dependent mTOR signaling

AbstractThe hepatic hormone hepcidin is a key regulator of systemic iron metabolism. Its expression is largely regulated by 2 signaling pathways: the “iron-regulated” bone morphogenetic protein (BMP) and the inflammatory JAK-STAT pathways. To obtain broader insights into cellular processes that modulate hepcidin transcription and to provide a resource to identify novel genetic modifiers of systemic iron homeostasis, we designed an RNA interference (RNAi) screen that monitors hepcidin promoter activity after the knockdown of 19 599 genes in hepatocarcinoma cells. Interestingly, many of the putative hepcidin activators play roles in signal transduction, inflammation, or transcription, and affect hepcidin transcription through BMP-responsive elements. Furthermore, our work sheds light on new components of the transcriptional machinery that maintain steady-state levels of hepcidin expression and its responses to the BMP- and interleukin-6–triggered signals. Notably, we discover hepcidin suppression mediated via components of Ras/RAF MAPK and mTOR signaling, linking hepcidin transcriptional control to the pathways that respond to mitogen stimulation and nutrient status. Thus using a combination of RNAi screening, reverse phase protein arrays, and small molecules testing, we identify links between the control of systemic iron homeostasis and critical liver processes such as regeneration, response to injury, carcinogenesis, and nutrient metabolism.

In Vivo Disruption Of The Hepcidin−Ferroportin Regulatory Circuitry Causes Fatal Systemic and Exocrine Pancreatic Iron Overload

Systemic iron levels are tightly controlled by the hepatic hormone hepcidin in response to iron availability, inflammation, hypoxia or the iron demand for erythropoiesis. Hepcidin binds to the iron export protein ferroportin (FPN1) to regulate iron release from exporting cells. A mutation of cysteine 326 (C326S) of FPN1 was reported in a patient with non−classical ferroportin disease (Sham et al, 2005) and shown to abrogate hepcidin binding in vitro (Fernandes et al, 2009).To study consequences of the disruption of the hepcidin−ferroportin interaction in vivo, we generated the first knock−in mouse model of C326S non−classical ferroportin disease.Mice with either heterozygous or homozygous C326S FPN alleles are viable and fertile. At 8−weeks of age both heterozygous and homozygous mice show profoundly increased transferrin saturation and serum ferritin levels as well as hepatic iron overload. Histological analysis by Perl's Prussian blue staining revealed that hepatic iron accumulation is restricted to hepatocytes and that Kupffer cells are spared of iron. In addition, splenic macrophages and duodenal enterocytes are iron−depleted.Macroscopically, C326S homozygous mice show progressive, brown discoloration of the pancreas that correlates with profound iron deposition. Histological analysis reveals that iron localizes exclusively to the exocrine pancreas sparing the islets of Langerhans. Consistently, C326S homozygous mice do not show any signs of diabetes. Pancreatic iron accumulation is closely associated with increased reactive oxygen species (ROS), degeneration of exocrine pancreatic cells, increased plasma lipase and exocrine pancreatic failure. Starting at the age of 33 weeks, pancreatic failure is accompanied by progressive wasting and death. We believe that C326S FPN mice represent the first example of fatal iron overload in an animal model, opening avenues to investigate the underlying molecular mechanisms.Sham R, Phatak PD, West C, et al. Autosomal dominant hereditary hemochromatosis associated with a novel ferroportin mutation and unique clinical features. Blood Cells Mol. Dis. 2005; 34:157−61.Fernandes A, Preza GC, Phung Y, et al. The molecular basis of hepcidin−resistant hereditary hemochromatosis. Blood. 2009;114:437−443. Disclosures:No relevant conflicts of interest to declare.

Hepcidin levels in chronic hemodialysis patients: a critical evaluation

Altered systemic iron metabolism is a key element of uremia, and functional iron deficiency mainly related to subclinical inflammation makes it difficult to maintain proper control of anemia in chronic hemodialysis patients (CHD). In the last decade, the hepatic hormone hepcidin has been progressively recognized as the master regulator of circulating iron levels through the modulation of cellular iron fluxes in response to iron stores, as well as to erythroid and inflammatory stimuli. Hepcidin is cleared by the kidney and progression of renal disease has been associated to increased serum hepcidin levels. This, in turn, reduces iron availability for erythropoiesis, suggesting anti-hepcidin strategies for improving anemia control. Moreover, hepcidin has been recently implicated in the pathogenesis of long-term complications of dialysis, like accelerated atherosclerosis. Initial studies almost invariably reported a sustained increase of serum hepcidin in chronic hemodialysis patients. Noteworthy, such studies included relatively few patients and controls that were poorly matched for major determinants of serum hepcidin at population level, i.e., age and gender. More recent data based on accurately matched larger series challenge the view that hepcidin is intrinsically increased in hemodialysis patients, showing a marked inter- and intra-individual variability of hormone levels. Here we take a critical look to the data published so far on hepcidin levels in CHD, analyze the reasons underlying the discrepancies in available studies and the hepcidin variability in CHD, and point out the need for further studies in large series of well-characterized CHD patients and controls.

The A736V TMPRSS6 Polymorphism Influences Hepatic Iron Overload in Nonalcoholic Fatty Liver Disease

Background & AimsHepatic iron accumulation due to altered trafficking is frequent in patients with nonalcoholic fatty liver disease (NAFLD), and is associated with more severe liver damage and hepatocellular carcinoma. The p.Ala736Val TMPRSS6 variant influences iron metabolism regulating the transcription of the hepatic hormone hepcidin, but its role in the pathogenesis of iron overload disorders is controversial. Aim of this study was to evaluate the whether the TMPRSS6 p.Ala736Val variant influences hepatic iron accumulation in a well-characterized series of Italian patients with histological NAFLD.Methods216 patients with histological NAFLD. TMPRSS6 and HFE variants were assessed by allele specific PCR, liver histology by the NAFLD activity score and Perls' staining for iron.ResultsHomozygosity for the p.736Val allele previously linked to higher hepcidin did not influence transferrin saturation (TS), but was associated with lower hepatic iron stores (p = 0.01), and ferritin levels (median 223 IQR 102–449 vs. 308 IQR 141–618 ng/ml; p = 0.01). Homozygosity for TMPRSS6 p.736Val was nearly associated with lower ballooning (p = 0.05), reflecting hepatocellular damage related to oxidative stress. The influence of TMPRSS6 on hepatic iron accumulation was more marked in patients negative for HFE genotypes predisposing to iron overload (p.Cys282Tyr + and p.His63Asp +/+; p = 0.01), and the p.736Val variant was negatively associated with hepatic iron accumulation independently of age, gender, HFE genotype, and beta-thalassemia trait (OR 0.59, 0.39–0.88).ConclusionsThe p.Ala736Val TMPRSS6 variant influences secondary hepatic iron accumulation in patients with NAFLD.

The Regulation of Hepcidin in β-Thalassemia

Abstract 901β-Thalassemia is a genetic disorder characterized by reduced or absent β-globin synthesis. Complications include anemia, ineffective erythropoiesis (IE), splenomegaly and iron overload. Homeostatic iron levels are maintained by the hepatic hormone hepcidin (HAMP), which degrades the iron exporter ferroportin. Iron-responsive transcriptional control of HAMP is normally mediated by the bone morphogenetic protein (BMP)/SMAD pathway. Despite the high organ iron load associated with β-thalassemia, low Hamp levels have been reported in patients with this disorder. HAMP levels are also low relative to organ iron load in mouse models of β-thalassemia. Although iron overload is a central component of the pathogenesis of this disease, the factors contributing to inadequate HAMP expression have not been fully described. We previously showed that moderate transgenic overexpression of HAMP increased hemoglobin levels, improved erythropoiesis, and decreased both splenomegaly and iron overload in a mouse model of β-thalassemia intermedia (th3/+). In order to define the pathways controlling HAMP transcription in β-thalassemia, initial studies examined HAMP expression in the context of BMP/SMAD pathway members and targets genes. qRT-PCR analyses confirm decreased HAMP expression in 2 month old th3/+ mice and indicate a significantly larger decrease in mice with β-thalassemia major (th3/th3) relative to wild-type (+/+) controls. A significant decrease in the common SMAD pathway member, SMAD4, is also evident in both strains. TMPRSS6, the transcript encoding the protease matriptase-2, is also decreased in th3/th3 mice. In contrast, at 5 months of age the transcriptional profile of these genes in th3/+ mice is indistinguishable from that of wild-type mice. By 12 months of age, there is a trend toward increased HAMP transcription corresponding to increases in the BMP/SMAD targets ID1 and ATOH8. Examination of the effect of a low-iron diet on the transcriptional levels of these genes over time in th3/+ mice indicates no significant differences from +/+ mice after the data were normalized to liver iron concentrations. In contrast, comparison of the transcriptional profile of these genes with +/+ mice fed a high-iron diet indicates that HAMP expression is blunted in thalassemic mice. Preliminary Western blot analyses indicate that phosphorylation of SMAD 1/5/8 in th3/+ and th3/th3 liver samples, although comparable to that in +/+ samples, is lower than the levels observed in mice fed a high-iron diet. These data indicate that iron sensing through the BMP/SMAD pathway is intact, but somewhat blunted in thalassemic mice. Thus an alternative mechanism, possibly mediated by the IE that is characteristic of this disease, is likely responsible for the suppression of HAMP. In order to gain support for this hypothesis, qRT-PCR analyses were performed on mice overexpressing transgenic erythropoietin (TgEpo) to examine the effect of erythropoiesis on HAMP expression. The data indicate suppression of HAMP transcription despite clear activation of the BMP/SMAD pathway, as evidenced by increases in BMP6 and ID1. Of note, SMAD7 transcription was not detected in these animals. Compared to +/+ mice, phlebotomized mice also show decreased transcription of HAMP and SMAD4 without corresponding decreases in the BMP/SMAD pathway targets ID1 and ATOH8. TMPRSS6 transcription was also decreased, but FURIN expression was increased. FURIN, a known hypoxia target gene, was also transcribed at a significantly higher rate in th3/th3 mice than in either th3/+ or +/+ mice. qRT-PCR data indicate that this is mediated by HIF-2α, as evidenced by decreased transcription of the HIF-1α target, pyruvate dehydrogenase kinase 1, and increased transcription of the HIF-2α target, erythropoietin (EPO), in th3/th3 liver samples. HIF2-α expression in th3/th3 liver lysates was confirmed by Western blot analysis. Collectively, these data indicate that HAMP expression in thalassemia is suppressed through a mechanism distinct from a BMP/SMAD feedback loop and provide support for the existence of an ‘erythroid factor’. Results also suggest the increased suppression of HAMP observed in th3/th3 mice is attributable to the activation of HIF2-α, which in turn would be expected to aggravate the production of an erythroid factor through the upregulation of EPO. Disclosures:No relevant conflicts of interest to declare.

Reduced serum hepcidin levels in patients with chronic hepatitis C

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 versus 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.

Hepcidin Suppression Relative to Iron Status in Patients with Chronic Hepatitis C.

Patients with chronic hepatitis C (CHC) often have increased liver iron deposits, 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 a major regulator of iron metabolism that 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 (Ganz T, Blood 2008, epub Aug 8), we measured s-hepcidin levels in 82 CHC naïve patients and 57 sex-matched healthy controls with rigorous definition of normal iron status. All CHC patients underwent liver biopsy with histological iron score (according to Deugnier YM, Gastroenterology 1992). S-hepcidin was much lower in CHC than in controls (geometric means with 95% CIs: 33.7, 21.5–52.9 versus 90.9, 76.1–108.4 ng/ml, respectively; P<0.001), while both serum ferritin and transferrin saturation were significantly higher in CHC than in controls, as expected. S-hepcidin strongly correlated with serum ferritin in both controls (r=0.741; P<0.001) and CHC patients (r = 0.718; P<0.001). In CHC patients, s-hepcidin also correlated with histological total iron score (r = 0.46; P<0.001), but not with serum interleukin-6 (r = −0.042; P = n.s.). After stratification for serum ferritin quartiles, s-hepcidin levels increased significantly across quartiles in both controls and CHC patients (chi for trend, P<0.001). However, in the latter group s-hepcidin levels were significantly lower than in controls for each corresponding quartile (ANOVA, P<0.001). In the lowest ferritin quartile, s-hepcidin was significantly inversely correlated with viral loading (e.g. serum HCV-RNA IU/ml; r = −0.526; P=0.036), while this association gradually disappeared with increasing ferritin quartiles. These results, together with very recent studies in animal and cellular models (Nishina S, Gastroenterology 2008; Miura K, Hepatology 2008), indicate that though hepcidin regulation by iron stores is maintained in CHC, the suppression of this hormone by HCV is likely an important factor in liver iron accumulation in this condition.