Hepcidin Levels Research Articles

The hepatokine FGL1 regulates hepcidin and iron metabolism during anemia in mice by antagonizing BMP signaling

AbstractAs a functional component of erythrocyte hemoglobin, iron is essential for oxygen delivery to all tissues in the body. The liver-derived peptide hepcidin is the master regulator of iron homeostasis. During anemia, the erythroid hormone erythroferrone regulates hepcidin synthesis to ensure the adequate supply of iron to the bone marrow for red blood cell production. However, mounting evidence suggested that another factor may exert a similar function. We identified the hepatokine fibrinogen-like 1 (FGL1) as a previously undescribed suppressor of hepcidin that is induced in the liver in response to hypoxia during the recovery from anemia, and in thalassemic mice. We demonstrated that FGL1 is a potent suppressor of hepcidin in vitro and in vivo. Deletion of Fgl1 in mice results in higher hepcidin levels at baseline and after bleeding. FGL1 exerts its activity by directly binding to bone morphogenetic protein 6 (BMP6), thereby inhibiting the canonical BMP-SMAD signaling cascade that controls hepcidin transcription.

The use of hepcidin as a marker for diagnosing the type of anemia in patients with high activity of rheumatoid arthritis

Anemia is still one of the most common comorbidities that affects the prognosis of the underlying disease and the quality of life of patients.Objective: to evaluate the value of serum hepcidin level determination for the differential diagnosis of anemia of chronic disease/inflammation (ACD) in patients with active rheumatoid arthritis (RA).Material and methods. The study included 47 patients with RA with anemia consecutively admitted to V.A. Nasonova Research Institute of Rheumatology for inpatient treatment. According to WHO recommendations, the criterion for anemia was a decrease in hemoglobin level ˂ 120 g/l in women and ˂ 130 g/l in men. The control group consisted of 29 patients without anemia. In all patients, the DAS28 index was determined, and clinical and biochemical blood parameters were examined: serum iron, total iron-binding capacity of serum, hepcidin, cytokines, including interleukin (IL) 6 and tumor necrosis factor α (TNFα).Results and discussion. Of 47 patients with active RA and anemia, only 13 (28%) were diagnosed with isolated ACD. Iron deficiency anemia (IDA) was found in 17 (36%), the remaining 17 patients had a mixed genesis of anemia (ACD + IDA). Patients with isolated ACD had a statistically significant higher level of hepcidin(120.3±56.1 pg/ml) compared to the control group (90.3±37.9 pg/ml) and to patients with RA + IDA. In isolated ACD, the levels of IL6, TNFα, rheumatoid factor and antibodies to cyclic citrullinated peptide were 2 times higher (p<0.05) than in RA with iron deficiency (both in IDA and in mixed genesis of anemia). Only in isolated ACD did the hepcidin level correlate with the IL6 concentrations (r=0.8); no such correlation was found in patients with IDA and anemia of mixed origin or in patients without anemia. No correlation with TNFα levels was found in any subgroup. Conclusion. Hepcidin levels are an informative indicator for the differential diagnosis of the type of anemia during active inflammation. In RA patients with ACD, the maximum hepcidin concentration in blood serum was determined, and in IDA it was found to be lower than the reference values. The importance of the hepcidin – IL6 axis and the lack of influence of the proinflammatory cytokine TNFα on iron metabolism were demonstrated. Keywords: hepcidin; anemia; difficult-to-treat patient; rheumatoid arthritis> ˂ 0.05) than in RA with iron deficiency (both in IDA and in mixed genesis of anemia). Only in isolated ACD did the hepcidin level correlate with the IL6 concentrations (r=0.8); no such correlation was found in patients with IDA and anemia of mixed origin or in patients without anemia. No correlation with TNFα levels was found in any subgroup.Conclusion. Hepcidin levels are an informative indicator for the differential diagnosis of the type of anemia during active inflammation. In RA patients with ACD, the maximum hepcidin concentration in blood serum was determined, and in IDA it was found to be lower than the reference values. The importance of the hepcidin – IL6 axis and the lack of influence of the proinflammatory cytokine TNFα on iron metabolism were demonstrated.

Circulating microRNAs and hepcidin as predictors of iron homeostasis and anemia among school children: a biochemical and cross-sectional survey analysis

BackgroundMicroRNAs (miRNAs) can control several biological processes. Thus, the existence of these molecules plays a significant role in regulating human iron metabolism or homeostasis.PurposeThe study aimed to determine the role of circulating microRNAs and hepcidin in controlling iron homeostasis and evaluating possible anemia among school children.MethodsThe study was based on a biochemical and cross-sectional survey study that included three hundred fifty school children aged 12–18 years old. RT–PCR and immunoassay analysis were accomplished to estimate iron concentration, Hgb, serum ferritin (SF), soluble transferrin receptor (sTfR), total body iron stores (TIBs), total oxidative stress (TOS), total antioxidant capacity (TAC), α-1-acid glycoprotein (AGP), high sensitive C-reactive protein (hs-CRP), and miRNAs; miR-146a, miR-129b, and miR-122 in 350 school adolescents.ResultsIron disorders were cross-sectionally predicted in 28.54% of the study population; they were classified into 14.26% with ID, 5.7% with IDA, and 8.6% with iron overload. The overall proportion of iron depletion was significantly higher in girls (20.0%) than in boys (8.6%). MicroRNAs; miR-146a, miR-125b, and miR-122 were significantly upregulated with lower hepcidin expression in adolescence with ID and IDA compared to iron-overloaded subjects, whereas downregulation of these miRNAs was linked with higher hepcidin. Also, a significant correlation was recorded between miRNAs, hepcidin levels, AGP, hs-CRP, TAC, and other iron-related indicators.ConclusionMolecular microRNAs such as miR-146a, miR-125b, and miR-122 were shown to provide an additional means of controlling or regulating cellular iron uptake or metabolism either via the oxidative stress pathway or regulation of hepcidin expression via activating genes encoding Hfe and Hjv activators, which promote iron regulation. Thus, circulating miRNAs as molecular markers and serum hepcidin could provide an additional means of controlling or regulating cellular iron and be associated as valuable markers in diagnosing and treating cases with different iron deficiencies.

Nutritional Modulation of Hepcidin in the Treatment of Various Anemic States.

Twenty years after its discovery, hepcidin is still considered the main regulator of iron homeostasis in humans. The increase in hepcidin expression drastically blocks the flow of iron, which can come from one's diet, from iron stores, and from erythrophagocytosis. Many anemic conditions are caused by non-physiologic increases in hepcidin. The sequestration of iron in the intestine and in other tissues poses worrying premises in view of discoveries about the mechanisms of ferroptosis. The nutritional treatment of these anemic states cannot ignore the nutritional modulation of hepcidin, in addition to the bioavailability of iron. This work aims to describe and summarize the few findings about the role of hepcidin in anemic diseases and ferroptosis, as well as the modulation of hepcidin levels by diet and nutrients.

Loss of progesterone receptor membrane component 1 decreases hepatic hepcidin levels in animal model

Loss of progesterone receptor membrane component 1 decreases hepatic hepcidin levels in animal model

Efficacy and safety of hypoxia-inducible factor-prolyl hydroxylase inhibitor treatment for anemia in chronic kidney disease: an umbrella review of meta-analyses

The objective was to provide a comprehensive summary of existing evidence on the efficacy and safety of hypoxia-inducible factor-prolyl hydroxylase inhibitors (HIF-PHIs) for the treatment of anemia in chronic kidney disease (CKD). A systematic search was conducted in the Medline, Embase, and Cochrane databases. Only meta-analyses that evaluated the efficacy and safety of HIF-PHI treatment for anemia in CKD were included. The efficacy outcomes included hemoglobin levels and iron metabolism indices, while the safety outcomes were assessed by examining adverse events. The qualities of methodologies and evidence were assessed using the AMSTAR 2 system and the NutriGrade tool, respectively. Fourteen meta-analyses, comprising 105 distinct comparisons, were included. The comparisons were backed by evidence of high, moderate, and low levels, distributed in approximately equal proportions. None of the studies were deemed to possess a high level of confidence. In both the overall and individual treatment groups of HIF-PHI, there was an increase in the levels of hemoglobin, transferrin, and transferrin saturation, while the levels of hepcidin and total iron binding capacity decreased. Serum ferritin exhibited a reduction to some extent, while serum iron did not show significant alterations following HIF-PHI treatments. There were no notable disparities in safety outcomes between the HIF-PHI and erythropoietin stimulating agents or placebo groups. This umbrella review suggests that HIF-PHI treatment can effectively increase hemoglobin levels in CKD patients and enhance iron metabolism by decreasing hepcidin levels and improving iron transport. The safety profiles of HIF-PHIs were generally comparable to those of ESA therapies or placebos.

Hepcidin Reduction during Testosterone Therapy in Men with Type 2 Diabetes: A Randomized, Double-Blinded, Placebo-Controlled Study.

A randomized, placebo-controlled, double-blinded study at an academic tertiary-care medical center. Interventions were testosterone gel (TRT, n = 20) or placebo gel (n = 19) for 24 weeks. We included 39 men (50-70 years) with type 2 diabetes (T2D) on metformin monotherapy with bioavailable testosterone levels <7.3 nmol/L. Body composition was assessed with DXA- and MRI-scans; the main study outcomes were serum hepcidin-iron, FGF23, phosphate, klotho, and calprotectin. Hepcidin levels decreased during TRT (β = -9.5 ng/mL, p < 0.001), lean body mass (β = 1.9 kg, p = 0.001) increased, and total fat mass (β = -1.3 kg, p = 0.009) decreased compared to placebo. Delta hepcidin was not associated with changes in lean body mass or fat mass. Iron and the pathways of FGF23-phosphate-klotho and calprotectin were unchanged during TRT. During TRT, the reduction in hepcidin was not associated with circulating iron levels, lean body mass, or fat mass; these findings suggested a direct anti-inflammatory effect of TRT and no indirect effect mediated through these factors.

The Heparinoid Sevuparin Improves Anemia and Kidney Status in a Mouse Model of Chronic Kidney Disease

The control of systemic iron availability is essential to sustain the different needs of the body, without causing iron overload disease or deficiency (anemia) meaning that iron is highly regulated and the master regulator is hepcidin. High levels of hepcidin induce ferroportin degradation, inhibiting iron release from enterocytes and macrophages resulting in an internal iron deficiency, which underlies anemia of chronic diseases (ACD). Patients with chronic kidney disease (CKD) often have ACD due to a combination of inflammation, erythropoietin (EPO) insufficiency/unresponsiveness, elevated hepcidin levels, reduced kidney function and decreased iron availability. Treatments involve erythropoiesis-stimulating agents, that in a longer perspective often fail to ameliorate the anemia, highlighting the need for new therapeutic tools. Interestingly, it has been reported that hepcidin suppression improves anemia in rodent models of CKD, suggesting that normalising hepcidin levels may be a means to treat anemia in CKD and in general in ACD. It is known that heparins are potent hepcidin suppressors; sevuparin, a novel, clinical stage, low-molecular weight heparinoid compound with substantially reduced anticoagulant activity is a promising candidate drug to control hepcidin expression in patients with CKD. The authors previously found that sevuparin strongly suppressed basal, BMP6- and IL6-dependent hepcidin expression in vitro with a maximum effect after 6h (90% reduction) persisting for at least 16h. The hepcidin lowering effect of sevuparin was confirmed also in healthy mice. Furthermore, in healthy volunteers, a single dose of subcutaneous sevuparin caused a significant reduction of about 50% (3 mg/kg) and 80% (9 mg/kg) of serum hepcidin after 12 and 24h, respectively, providing a robust translational context to the sevuparin effects on hepcidin levels. The aim of the present work was to investigate the effect of sevuparin on hepcidin, hemoglobin and the kidney status in a CKD mouse model of high hepcidin anemia. Mice fed with “adenine rich diet” (ARD) (0.2% adenine; 0.9% phosphorus; 0.6% calcium; 20% casein) developed moderate or severe signs of kidney damage as manifested by weight loss, increased serum creatinine, high hepcidin levels, low serum iron and anemia (hemoglobin ≤ 12.5 g/dL) after 7-9 weeks. After the onset of CKD, the mice were chronically treated with PBS or sevuparin (10 mg/kg/daily) subcutaneously for 3 or 6 weeks alone or in combination with erythropoietin (EPO) administered intraperitoneally (50 U/mouse twice a week in the first 3 weeks followed by 3 weeks and then 25 U/mouse once a week to simulate EPO hypo-responsiveness). Sevuparin was provided by Modus Therapeutics AB, Sweden, whereas EPO was used in the form of the commercially available darbapoietin alfa (ARANESP ®). Sevuparin treatment alone increased hemoglobin, hematocrit and weight (Hb 11-12 g/dL; Ht 20%; 22 g vs baseline values of Hb 9-10 g/dL; Ht 18%; 14.5 g PBS) with adjoining modest decreases of serum hepcidin and creatinine. The reduction of creatinine was supported by a concomitant improvement of the kidney status visible as a reduction of collagen deposits indicative of reduced fibrosis compared to the mice treated with PBS only. The EPO/sevuparin combination showed a strong reduction from control of serum hepcidin levels (292 ng/mL vs 1382 ng/mL) after 3 weeks of treatment whereas after 6 weeks levels were increased up to 2000 ng/mL suggesting that the chronic treatment with EPO is not optimal, as observed in patients. Interestingly the concomitant benefit on anemia at 3w (about Hb 18 g/dL, Ht 54%) for the combination remained stable at 6w, further supported by the maintained high Ret-He (about 15.62 pg); suggesting a phased dependency on hepcidin in the development of CKD anemia. Furthermore, the improvement of the kidney status (histology and function) was also maintained throughout combination treatment. Our data in this mouse CKD model add new insights to the role of hepcidin in the development of CKD anemia over time. Furthermore, the results are promising and support further mechanistic and clinical studies to elucidate the therapeutic role for sevuparin as a treatment in anemia of inflammatory and/or chronic diseases in monotherapy or in combination with EPO and to explore its role in kidney protection.

Tmprss6 siRNA Treatment in Mice Stops Hepcidin Reduction across Pregnancy

Aim: Hepcidin levels are suppressed during human and murine pregnancy. It is unknown whether hepcidin levels are primarily the result of iron deficiency/utilisation or whether there is a pregnancy-specific mechanism for hepcidin suppression. We reasoned that Tmprss6 inhibition would cause constitutive BMP-SMAD signalling and help determine the mechanism of antenatal hepcidin suppression. Method: Female wildtype C57BL/6 mice were treated subcutaneously every 21 days with a GalNAc-siRNA conjugate targeting Tmprss6 (Silence Therapeutics GmbH, Berlin, Germany) or a non-targeting control (NTC) siRNA conjugate. Mice were mated and monitored for pregnancy. Samples were collected from pregnant mice humanely euthanised at E8.5 or E14.5, and unmated controls. Results: Treatment with Tmprss6 siRNA inhibited hepatic Tmprss6 mRNA expression (unmated fold change 0.077, p&amp;lt;0.0001). qPCR Hamp expression was significantly increased in Tmprss6 siRNA-treated mice, compared to NTC-treated mice, at all timepoints. Serum hepcidin, by ELISA, was higher in Tmprss6 siRNA-treated mice than in NTC-treated mice at E14.5 (1126ng/ml vs 178.5, p&amp;lt;0.0001). In unmated mice, serum hepcidin was not significantly elevated (640.9 vs 980.1ng/ml, p=0.224). Across NTC treated pregnancy, Hamp expression and serum hepcidin were reduced at E14.5 compared to unmated ( Hamp fold change E14.5 0.293, p=0.0367; serum hepcidin E14.5 178.5 vs 640.9, p=0.0247). In contrast, Tmprss6 siRNA treatment caused elevated hepcidin through pregnancy (E14.5 1126 vs unmated 980ng/ml, p=0.99); Fig 1a. Maternal liver iron was unchanged between groups. Tmprss6 siRNA reduced placental iron, fetal liver iron, and fetal weight (0.189 vs 0.226g, p&amp;lt;0.0001), Fig 1b. At E8.5/E14.5, Tmprss6 siRNA reduced Mean Cell Volume (E14.5: 41.42 vs 51.72fL, p&amp;lt;0.0001) and Mean Cell Haemoglobin (E14.5: 11.20 vs 15.68pg, p&amp;lt;0.0001); at E14.5 only, maternal anaemia was seen with Tmprss6 siRNA (haemoglobin 9.4 vs 13.85g/L, p=0.0004). Conclusion: Disruption of Tmprss6 increased hepcidin and ablated the hepcidin fall over pregnancy. This may suggest that any pregnancy-related factor which directly suppresses hepcidin cannot overcome Tmprss6 knockdown. Tmprss6 siRNA treatment resulted in iron-restricted erythropoiesis, reduced placental iron and impacted fetal development, despite unchanged liver iron stores.

Dysregulation of the Kynurenine Pathway, Cytokine Expression Pattern, and Proteomics Profile Link to Symptomology in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS).

Dysregulation of the kynurenine pathway (KP) is believed to play a significant role in neurodegenerative and cognitive disorders. While some evidence links the KP to myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS), further studies are needed to clarify the overall picture of how inflammation-driven KP disturbances may contribute to symptomology in ME/CFS. Here, we report that plasma levels of most bioactive KP metabolites differed significantly between ME/CFS patients and healthy controls in a manner consistent with their known contribution to symptomology in other neurological disorders. Importantly, we found that enhanced production of the first KP metabolite, kynurenine (KYN), correlated with symptom severity, highlighting the relationship between inflammation, KP dysregulation, and ME/CFS symptomology. Other significant changes in the KP included lower levels of the downstream KP metabolites 3-HK, 3-HAA, QUIN, and PIC that could negatively impact cellular energetics. We also rationalized KP dysregulation to changes in the expression of inflammatory cytokines and, for the first time, assessed levels of the iron (Fe)-regulating hormone hepcidin that is also inflammation-responsive. Levels of hepcidin in ME/CFS decreased nearly by half, which might reflect systemic low Fe levels or possibly ongoing hypoxia. We next performed a proteomics screen to survey for other significant differences in protein expression in ME/CFS. Interestingly, out of the seven most significantly modulated proteins in ME/CFS patient plasma, 5 proteins have roles in maintaining gut health, which considering the new appreciation of how gut microbiome and health modulates systemic KP could highlight a new explanation of symptomology in ME/CFS patients and potential new prognostic biomarker/s and/or treatment avenues.

Differential Effects of Monoferric Transferrin Forms on Erythropoiesis Are Mediated By TFR2

Transferrin (TF) is a bilobed 80kD glycoprotein with N- and C-lobe iron binding sites. TF circulates as four forms: unbound to iron (apo-TF), monoferric iron bound to the N-lobe or C-lobe (mono-TF) or to both lobes (holo-TF). All TF forms interact with TF receptor-1 (TFR1), which is ubiquitously expressed and serves as the main mechanism for cellular iron delivery. TF also interacts with TFR2 which is mainly expressed by hepatocytes and erythroid precursors to modulate cellular signaling events regulating hepcidin expression and erythropoiesis. We previously generated N-lobe blocked ( Tf N-bl) or C-lobe blocked ( Tf C-bl) TF mutant mice, and observed marked differences between them in iron parameters, RBC count and EPO sensitivity (Parrow et al., 2019). In this current work, we investigate the effects of the mutated TFs on ineffective erythropoiesis in murine β-thalassemia intermedia. We moreover examine the consequences of erythroid knockout of TFR2 in the TF N-blocked and C-blocked mice. Based on observations in the TF mutant mice, we hypothesized that Hbb th3/+ mice crossed with Tf C-bl would demonstrate improved erythropoietic and iron parameters compared with Hbb th3/+Tf N-bl. Hbb th3/+Tf C-bl mice demonstrated significantly increased RBC counts, elevated hemoglobin, improved erythrocyte morphology, decreased splenomegaly, fewer bone marrow erythroblasts, and improvement of IE. Additionally, serum erythroferrone (ERFE) was significantly reduced and hepcidin levels were increased in Hbb th3/+Tf C-bl relative to Hbb th3/+Tf +/+controls. By contrast, similar improvements in RBC counts and hemoglobin were not observed in Hbb th3/+Tf N-bl mice. Nonetheless, the Hbb th3/+Tf N-blmice displayed lower reticulocytes, increased platelets, decreased IE, decreased splenomegaly, and decreased serum ERFE compared to Hbb th3/+Tf +/+controls and thus displayed a phenotype intermediate between Hbb th3/+Tf C-bl and Hbb th3/+Tf +/+controls. Serum EPO was elevated in Hbb th3/+Tf N-blcompared to either Hbb th3/+Tf C-blor Hbb th3/+Tf +/+mice. Moreover, the increased hepcidin observed in Hbb th3/+Tf C-bl compared with Hbb th3/+Tf +/+was not observed in Hbb th3/+Tf N-bl mice. To examine the contribution of erythroid TFR2 to the different phenotypes of the N-blocked and C-blocked TF mice, we generated a mouse line expressing TFR2-3xFLAG that is flanked by loxp sites ( TFR2-3xFLAG fl/fl). These mice were used to generate mice with erythroid knockout of TFR2 in the TF mutant mice; i.e., EPOR Cre-tdtomTf N-blTFR2 cko and EPOR Cre-tdtomTf C-blTFR2 cko. N-blocked mice with conditional knockout of TFR2 demonstrated increases in RBC counts and Hb levels compared with the TF N-blocked mice ( EPOR Cre-tdtom Tf N-blTFR2 cko vs Tf N-bl/Tf N-blTFR2-3xFLAG fl/fl), and had values similar to the C-blocked mice ( Tf C-bl/Tf C-blTFR2-3xFLAG fl/fl or EPOR Cre-tdtomTf N-blTFR2 cko). Likewise, conditional KO of TFR2 in the N-blocked mice resulted in lower EPO levels, which were similar to those in the C-blocked mice. These data support the hypothesis that differences observed between the mono-TF forms at the erythroid level are mediated via TFR2. These data support a model by which lobe-specific iron occupancy of TF influences hematopoiesis via erythroid TFR2.

Iron Chelation Efficiency in Human Hepatocytes Is Enhanced By Exogenous Hepcidin

Iron chelation is clinically inefficient, with only a small percentage of the applied chelator being excreted in the iron-bound form. Chelation is also slow because only a small proportion of storage iron is in the chelatable labile iron pool (LIP) at any given moment. In principle, by increasing the magnitude of the LIP within hepatocyte, the major site of body storage iron in conditions of iron overload, the magnitude and hence the efficiency of chelation may be increased. Recent work has shown that SLN124, a RNAi-therapeutic inhibitor of Tmprss6 gene expression, increases iron chelation by deferiprone in a murine model of thalassemia (b-thalassemic Hbb th3/+ animals) (Vadolas et al Brit J Haematol 2021) and murine hereditary haemochromatosis (Altamura et al. HemaSphere 2019). We wished to determine whether the mechanism of increased chelation was consistent with the blocking of iron exit from hepatocyte through ferroportin channels by increased levels of hepcidin. Here we have examined whether application of exogenous hepcidin to a human hepatocyte cell line HepG2 can increase the chelation efficiency of deferiprone and whether this effect is also seen with other iron chelators such as deferasirox and deferoxamine. A model was developed from previous work (Vlachodimitropoulou et al. Blood 2017) where the net effect of iron uptake (from fetal bovine serum medium) versus intracellular iron removal by chelation, could be interrogated. The dose and time-dependent effect of exogenous hepcidin and its interaction with iron chelators on intracellular iron content was examined. Briefly, to load cells with iron, confluent HepG2 cells were incubated for 2h with 10% FBS followed by media change containing 10-30 uM iron binding equivalents (IBE) of therapeutic chelators, hepcidin, or control media. This loading method doubled the cellular iron; and subsequently increased cellular ferritin by at least 4-fold. Both effects could be completely inhibited with iron chelation. Following a pulse with hepcidin (100nM-10µM), intracellular iron content of HepG2 cells increased in a dose dependent manner, by 30-100% over the next 4h, followed by a decrease in cellular iron at 8 hours (Fig. 1A). This transient effect on iron retention could be prolonged with serial addition of hepcidin to the media (at 2 and 4h, data not shown). Co-treatment with the exclusively extracellular chelator CP40 demonstrated that while a substantial fraction of the hepcidin effect on iron retention was dependent on iron uptake from media (Fig 1B), the remaining increase in iron retention was independent of this effect and consistent with inhibition of iron egress from . Compared with control cells (without chelator but with or without hepcidin), a 50-200% enhancement of hepatocyte iron removal by 10uM IBE deferiprone was observed at 2 and 4 hours (Fig. 1C) with the addition of 300nM exogenous hepcidin. Correspondingly, cellular iron removal with deferoxamine (10-30 µM IBE) was enhanced by 250% at 4 h by exogenous hepcidin (Fig. 1D) whereas iron removal by deferasirox (30µM IBE) was enhanced by at least 50% at 2 and 4 h when hepcidin (300nM) was present (Fig. 1E), as compared to controls. Enhanced chelation with DFO was not observed until after 2h incubation, consistent with the known slower access of DFO to intracellular pools compared with deferiprone or deferasirox. Beyond 4h, the effect of hepcidin on enhanced chelation was not seen, suggesting that hepcidin is consumed or degraded in this culture system. We conclude that enhanced removal of intracellular iron by chelation is demonstrable with all three clinically available chelators following the application of exogenous hepcidin. This iron would be destined for increased excretion of chelate complexes in vivo and hence enhanced decrements in body iron. Two mechanisms for enhanced chelation are possible in this model; the first is by increased iron loading of hepatocytes incubated with hepcidin and secondly through the blocking by hepcidin of labile intracellular iron egress through ferroportin channels, thereby increasing the magnitude of chelatable intracellular iron pools. Future work in primary human hepatocytes will examine whether manipulation of intracellular hepcidin levels by SLN124 has similar chelation-enhancing effects.

Serum Hepcidin Is an Early Predictor of Iron Depletion in Non-Anemic Blood Donors

Introduction Iron deficiency is one of the major concerns in repeated whole blood donors. Although taking iron supplementation, frequent whole blood donors sometimes suffer from iron depletion which contributes to adverse impact on donor's health and may reduce the donor blood pool. Laboratory parameters identifying iron depleted status could help early recognize donors at risk of iron deficiency. Hepcidin is a key iron regulator, which blocks ferroportin to inhibit iron absorption and iron release from hepatocytes. In iron depletion, hepcidin down regulates to encourage iron absorption. Previous data reported that serum hepcidin at less than 10 ng/mL was significantly associated with iron deficiency. According to the biological function and physiological response of hepcidin, we hypothesize that hepcidin could be an early predictor of iron deficiency status in non-anemic individuals. Methods This cross-sectional study was conducted at the Blood Bank Center at Chiang Mai University Hospital, Chiang Mai, Thailand in May 2023. After the informed consent was obtained, ninety-seven healthy repeated whole blood donors who had donated blood at least twice in the past year were recruited. The medical records were reviewed. Basic characteristics and laboratories including complete blood count, reticulocyte indices, iron parameters, and serum hepcidin were evaluated. Donors who had serum ferritin level less than 30 and 30-100 ng/mL were categorized as iron deficiency, and low ferritin group, respectively. Those who had ferritin more than 100 ng/mL were the control group. Logistic regression analysis was done to determine the value of serum hepcidin in predicting iron deficiency status in each group. Results The total of 97 repeated whole blood donors were recruited, 48 (49.5%) were women. Mean age was 34.6 ± 10.7 years. There were 8 donors in the iron deficiency group, 38 in the low ferritin group, and 51 donors in the control group. Mean hemoglobin levels were 13.4 ± 1.1, 13.8 ± 1.2, and 14.2 ± 1.2 g/dL in the iron deficiency, low ferritin group and control, respectively, (p = 0.796). White blood cell and platelet count were not different in these groups. Similarly, red cell indices including mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), red cell distribution width (RDW), and reticulocyte indices showed no difference among groups. Serum hepcidin had a significant difference between the three groups. Median levels of hepcidin were 2.61 (1.37, 3.4) and 12.35 (10.12, 15.57) ng/mL in the iron deficiency and the low ferritin group, respectively, compared to 26.01 (21.36, 39.08) ng/mL in the control group, (p &amp;lt; 0.001). Low serum hepcidin (less than 10 ng/mL) were found in 14 (36.8 %) donors who had ferritin levels less than 100 ng/mL, compared to only 1 (2.4%) donor in the control group, (p &amp;lt; 0.001). In addition, serum hepcidin showed a strong correlation with ferritin levels (r = 0.779). Logistic regression model showed that only female sex (OR: 2.87; 95% CI: 1.26, 6.56) and hepcidin (OR: 0.76; 95% CI: 0.66, 0.86) were significant predictors of repeated donors who were at risk of iron depletion and had the ferritin levels less than 100 ng/mL. Conclusions Frequent blood donors are at risk of iron depletion. Serum hepcidin was a significant predictor of iron depleted status in non-anemic donors. Serum hepcidin also showed a significant correlation with ferritin levels. The downregulation of serum hepcidin was detected prior to the changes of red blood cell indices and reticulocyte responses. Early recognition and identification of donors at risk of iron depletion and prompt iron restoration are the key to ensuring the donor safety and preserving adequate blood donor pool.

Selective Inhibition of Repulsive Guidance Molecule C/Hemojuvelin (RGMc/HJV) with SRK-256 to Mobilize Iron in Functional Iron Deficiency

Functional iron deficiency is a serious condition marked by serum iron restriction in the presence of adequate iron stores that can lead to anemia, worsening quality of life, and leading to poor patient outcomes. Functional iron deficiency is characterized by abnormally high levels of hepcidin given serum iron status; it is frequently observed in diseases with chronic inflammation, such as anemia of chronic disease, chronic kidney disease, and myelofibrosis. Current treatments to address this patient population involve non-specifically targeting hepcidin and come with risks of off-target side effects. The secretion of hepcidin from the liver hepatocytes is linked to circulating serum iron levels and is controlled by the Bone Morphogenic Protein (BMP) 2/6 signaling pathway. Repulsive Guidance Molecule C or Hemojuvelin (RGMc/HJV) is a BMP 2/6 co-receptor in liver hepatocytes. Human genetic mutations in this gene result in juvenile hemochromatosis and genetic mouse studies have demonstrated that RGMc/HJV has a specific role in maintaining systemic iron homeostasis through modulation of hepcidin expression. We have generated a highly selective, fully human antibody (SRK-256) against RGMc/HJV which binds to the target with 39 pM affinity. SRK-256 has no detectable binding to RGMa or RGMb, thus avoiding potential side effects of targeting these gene family members with key physiological roles outside of iron metabolism. A high-resolution crystal structure (2.2 Å) elucidates not only the mechanism of action of SRK-256 directly competing for BMP binding to RGMc via contacts within the BMP binding domain, but also identifies binding to regions within this domain highly unique to RGMc over RGMa and RGMb, thus supporting the selectivity of the molecule. This mechanism of action is corroborated by in vitro assay data demonstrating that SRK-256 blocks signaling by BMP 2, 4, and 6. Pharmacodynamic and pharmacokinetic data supportive of a monthly subcutaneous dosing regimen in human patients has been generated in cynomolgus macaques, where SRK-256 demonstrates a dose-proportional duration of effect on suppression of hepcidin expression and elevation of transferrin saturation and achieves ~95% bioavailability via subcutaneous dosing. Furthermore, in the rat peptidoglycan-polysaccharide (PGPS) model of anemia of chronic disease, we have shown that in contrast to treatment with erythropoiesis stimulating agent alone (EPO, serum iron 47.7 ± 9.8 µg/dL), SRK-256 can restore serum iron to normal levels (Healthy control, serum iron 231.9 ± 12.0 µg/dL; SRK-256, serum iron 248.7 ± 60.9 µg/dL; and SRK-256+EPO, serum iron 277.6 ± 92.9 µg/dL) in the context of inflammation-driven, iron-restricted anemia (Figure 1). SRK-256 represents a potent and selective potential therapy for the treatment of patients with iron-restricted anemias across diseases of chronic inflammation.

Myelodysplastic Syndromes Benefit from Iron-Restricted Bone Marrow Transplant in Preclinical Mouse Model

Background: Hematopoietic stem cell transplantation (HSCT) remains the only fully curative therapy for diseases such as b-thalassemia, sickle cell disease, MDS and AML and represents a relevant procedure for gene therapy .Tissue iron overload and non-transferrin bound iron (NTBI) have emerged as adverse prognostic factors for post-transplant survival. Recently, we showed that - in line with observations in transplanted individuals - preparatory chemotherapy conditioning with a combination of busulfan and fludarabine in wild-type mice resulted in elevated serum iron and transferrin (Tf) saturation and elicited NTBI formation. Tf saturation and NTBI levels increased proportionally with conditioning intensity. Interestingly, we found that pre-conditioning TMPRSS6 silencing through a GalNAc-conjugated siRNA - by increasing hepcidin levels - reduced the peri-transplant elevation of Tf saturation and limited the appearance of NTBI. These results proved that NTBI formation is induced by conditioning chemotherapy in the peri-trasplant period independently of the presence of pre-existing iron overload. Aims &amp; Methods: Here we asked whether these observations hold true in conditions hallmarked by pre-existing iron overload and elevated NTBI and how iron restriction impacts on bone marrow transplant (BMT) outcome. To this aim, we applied the GalNAc TMPRSS6 siRNA as iron restriction strategy in a mouse model of myelodysplastic syndromes (MDS) and assessed its ability to reduce conditioning-elicited NTBI formation as well as its potential benefit on BMT. After TMPRSS6 siRNA administration, CD45.2 + MDS mice received conditioning chemotherapy and transplant of CD45.1 + wild-type bone marrow cells. Systemic iron parameters, engraftment and chimerism were then analyzed. Results: Importantly, conditioning exacerbated the elevated systemic iron levels in MDS mice, which was prevented by pre-conditioning TMPRSS6 silencing. Similar to our observation in wild-type mice, recipient CD45.2 + MDS mice showed a superior blood chimerism, resulting from increased bone marrow and spleen engraftment of CD45.1 + donor cells. Multilineage engraftment in bone marrow, spleen and peripheral blood was superior in iron-restricted recipient MDS mice compared to controls. Finally, donor chimerism of hematopoietic stem and progenitor cells (HSPCs) was higher in iron-restricted versus control MDS mice undergoing BMT. Conclusion: These findings demonstrate that pre-transplant conditioning profoundly alters iron homeostasis and restricting iron improves BMT outcome by increasing engraftment efficiency. Overall, these data suggest that conditioning-elicited NTBI is a critical mediator of peri-transplant toxicity and adversely impacts transplant outcome. Specifically, our observations prove that iron restriction improves donor HSPC engraftment in the recipient bone marrow, likely resulting in more efficient reconstitution of the HSPC pool and improved multilineage engraftment. Lastly, our results indicate that the peri-transplant application of novel iron restriction strategies aimed at reducing conditioning-induced NTBI - by positively affecting BMT outcome - is of potential benefit for patients undergoing BMT or gene therapy, presenting either with or without pre-transplant iron overload.

Rker-216 Reversed Microcytic Anemia in a Mouse Model of Iron Refractory Iron Deficiency Anemia

Background Iron refractory iron deficiency anemia (IRIDA) is an inherited disease caused by TMPRSS6 mutations that increase serum hepcidin and result in microcytic anemia from iron malabsorption. As hepcidin promotes ferroportin (Fpn) degradation to limit iron absorption, its production in anemia should be decreased to make up body iron deficits. In contrast to typical iron deficiency anemia that can be improved by oral iron supplementation or intravenous (IV) iron infusion, IRIDA patients tend to have no response to iron supplements and only transiently respond to iron infusion, resulting in lifelong anemia. Hepcidin is mainly regulated by the bone morphogenetic protein (BMP) pathway, in which BMP ligands activate transcription factors SMAD1/5/9 by binding to the BMP receptors, including ALK2. We previously showed that RKER-216 (m216), a research ALK2 neutralizing antibody, improved iron availability in mice with anemia of inflammation resulting from high hepcidin. Here, we explored m216 pharmacology in an IRIDA mouse model. Aim To examine 1) mechanism of action (MOA), 2) efficacy of IV iron infusion and m216-mediated iron absorption, and 3) dose response of m216 in resolving microcytic anemia in Tmprss6 knockout (KO) mice. Methods TMPRSS6 KO mice at 8-11 weeks (wk) were subcutaneously dosed with isotype control or m216 at 1 or 3 mg/kg in all studies. Mice were given a single dose for 6, 24 or 96 h in MOA and twice weekly for 9 or 21 d for efficacy studies. For IV iron, KO mice were given a single dose of iron dextran at 100 mg/kg and evaluated on day 9. Results TMPRSS6 KO mice at 8 wk had high serum hepcidin, low transferrin saturation (TSAT), and microcytic anemia as indicated by low mean corpuscular volume (MCV) and hemoglobin (Hb), key features in IRIDA. Liver SMAD1/5/9 phosphorylation, hepcidin ( Hamp) mRNA, and serum hepcidin levels were decreased similarly in KO mice dosed with 1 or 3 mg/kg of m216 for 6 h, compared to isotype controls. KO mice given both dose levels for 24 h decreased serum hepcidin by 88% and increased TSAT by 4-fold, compared to isotype mice, but only mice receiving 3 mg/kg had sustained effects by 96 h. These data suggest that m216 inhibited SMAD signaling to lower serum hepcidin, allowing more iron absorption to increase TSAT. To explore the relevance of m216 to current therapy, we compared the result of IV iron or 1 mg/kg of m216 in KO mice for 9 d. Compared to controls, liver iron, serum hepcidin, and TSAT were increased in mice given IV iron whereas mice receiving m216 exhibited reduced hepcidin with increased duodenal Fpn and TSAT, confirming target engagement. Although TSAT was higher in mice dosed with m216 compared to IV iron, anemia improved similarly (1-2 g/dl in Hb; 2 fl in MCV) in these two groups. These data suggest that despite high hepcidin, IV iron improved anemia via increased TSAT; however, the relationship between TSAT and anemia recovery is not always linear. We next examined KO mice dosed with m216 at 1 or 3 mg/kg for 3 wk. Hb and MCV improved from 9.2 g/dl and 26.2 fl in isotype controls to 11.5 g/dl and 33.7 fl in 1 mg/kg-m216 mice and further improved to 14.1 g/dl and 39.8 fl in the 3 mg/kg-m216 cohort, respectively, showing a dose proportional mitigation in improving microcytic anemia compared to isotype controls. In fact, Hb and MCV of the 3 mg/kg-m216 cohort were restored to the levels of WT mice, indicating that microcytic anemia was fully resolved in this group. Conclusion These data suggest that m216 inhibited the BMP pathway to increase iron absorption via hepcidin suppression and was able to provide similar effects as iron infusion in reversing microcytic anemia in a mouse model of IRIDA.

Combination Therapy with Luspatercept and the Ferroportin Inhibitor Vamifeport Is Superior to Either Drug Alone in Improving Anemia and Reducing Myeloid Skewing in MDS

Background: Patients with myelodysplastic syndromes (MDS) are prone to develop iron overload as a consequence of ineffective erythropoiesis and chronic transfusion therapy. Although iron overload is a common feature in MDS, it has remained unclear whether and how iron excess is detrimental for MDS pathophysiology. Recently, we have shown that iron restriction by the oral ferroportin (FPN) inhibitor vamifeport improves anemia and reduces myeloid skewing, being of benefit for MDS as single treatment. Luspatercept is a recombinant fusion protein acting as TGF-β superfamily ligands trap which has recently gained FDA approval for the treatment of anemia in transfusion-dependent low-risk MDS patients. Luspatercept promotes erythropoietin (EPO)-independent maturation of late-stage erythroid cells, resulting in increased hemoglobin and red blood cells (RBCs) count. However, this drug - due to a lack of improvement in myeloid skewing and AML evolution - is considered a non-disease-modifying therapy. Aims: In this study we investigated the effect of a combined therapy using luspatercept and the FPN inhibitor vamifeport in a preclinical MDS mouse model, with the hypothesis that both drugs could have additive effects with further benefit for the disease. Methods: To this end, we administered the FPN inhibitor vamifeport in NUP98-HOXD13 MDS mice for 3 months (0.5 mg/ml into drinking water), combined with luspatercept for the last 2 months (5 mg/kg twice a week, subcutaneous injection), starting at 3 months of age. Mice receiving the combined treatment were compared to those untreated or receiving single treatments. Results: At steady-state MDS mice develop anemia, neutropenia and lymphopenia, together with an iron overload phenotype, hallmarked by inappropriately low hepcidin levels, elevated serum iron and transferrin saturation, non-transferrin-bound iron (NTBI) formation and tissue iron deposition. Vamifeport administration in MDS mice reduced serum iron and NTBI formation and prevented tissue iron loading, either as a single or combined treatment with luspatercept. Luspatercept and vamifeport, as single-agent treatment, ameliorated anemia and the maturation of bone marrow and splenic RBCs in MDS mice. The combined therapy further improved anemia compared to the treatments with either drugs alone, as suggested by the higher hemoglobin levels, hematocrit and RBC count, and the decreased number of early erythroid precursors, sign of more effective erythropoiesis. Importantly, vamifeport, but not luspatercept, significantly altered myeloid skewing in MDS, revealing a major role of the iron status in myeloid expansion. Myeloid bias, monitored as percentage of CD11b + Gr1 + myeloid cells in the bone marrow was attenuated by iron restriction in vamifeport-treated MDS mice compared to control and luspatercept-treated MDS animals. The combined treatment merkedly reduced myeloid skewing in MDS mice, similarly to the single vamifeport treatment. Furthermore, the number of immature cKit + myeloid blasts in the peripheral blood of MDS animals were decreased by vamifeport alone and in combination with luspatercept, but not by luspatercept alone. Finally, splenomegaly was reduced in MDS mice treated with vamifeport alone or in combination with luspatercept, confirming that iron restriction improves ineffective erythropoiesis and limits myeloid skewing. Overall, the combination therapy with vamifeport and luspatercept showed superior effects in improving anemia and myeloid bias as compared to single treatments. Conclusion: In conclusion, our results show that iron restriction by vamifeport enhances the erythroid maturation action of luspatercept, likely by improving iron utilization in erythroid cells and ameliorating their survival. Furthermore, vamifeport improves the myeloid skewing in the MDS model, suggesting disease-modifying activity as single agent as well as combined therapy with luspatercept. Together, these data prove that combo therapies aimed at restricting iron and boosting erythroid maturation may offer additive beneficial effects in MDS and provide pre-clinical evidence for combining iron restriction and TFG-β superfamily ligand-trap approaches as more effective therapeutic strategies for the treatment of MDS.

Novel Anti-TMPRSS6 Monoclonal Antibody Provides a Potential Therapeutic Approach for the Treatment of Polycythemia Vera

Polycythemia vera (PV) is a myeloproliferative neoplasm (MPN) triggered by activating mutations in JAK2, an important kinase in the principal signaling pathway of the erythropoietin receptor. Most of the clinical manifestations of PV are caused by increased erythrocyte count, leading to increased risk of pulmonary hypertension and thrombosis. The mainstay of therapy for PV is therapeutic phlebotomy to reduce the hematocrit (HCT) level and minimize the risk of thrombosis, however, the effect of phlebotomy is transient until patients become iron deficient. Therefore, systemic iron restriction could be effective in treating PV patients. Transmembrane serine protease 6 (TMPRSS6) is a negative regulator hepcidin, the master regulator of iron homeostasis. Increasing genetic, preclinical and clinical evidence has demonstrated that inhibition of TMPRSS6 can potently elevated hepcidin levels, thereby achieving systemic iron restriction. Previously, we reported the generation of anti-TMPRSS6 antibody MWTx-003 and its efficacy in reducing iron overload and improving ineffective erythropoiesis in a mouse model of β-thalassemia. In this study, we explored therapeutic application of MWTx-003 in PV using a Jak2 V617F bone marrow transplantation mouse model. The mouse model exhibits typical PV phenotypes of erythrocytosis and elevated HCT levels. In the PV mice, treatment with MWTx-003 (2-10 mg/kg, i.p., every 4 days for 3 weeks) led to significantly reduced HCT levels, red blood cell (RBC) counts and hemoglobin (HGB) concentration that were comparable to that of wildtype control mice. These effects were evident in a dose-dependent manner and observed as early as two weeks into treatment. It is worth noting that in the 10 mg/kg dose group, a worsening splenomegaly and expansion of early erythroid progenitors were observed after 3 weeks of treatment, and these findings are typical of iron-restricted erythropoiesis in mice. Taken together, the anti-TMPRSS6 antibody offers a promising therapeutic approach in the management of PV, where erythrocytosis and high HCT levels are associated with poor outcomes. A Phase I clinical trial with MWTx-003 in healthy volunteers has been initiated in China.

Hepcidin and erythroferrone response to 3 weeks of exposure to normobaric hypoxia at rest in trained cyclists

Purpose: The effectiveness of altitude training on haematological adaptations is largely dependent on iron metabolism. Hepcidin and erythroferrone (ERFE) are key iron-regulating hormones, yet their response to altitude training is poorly understood. The aim of this study was to analyze changes in hepcidin and ERFE under the influence of 3 weeks of the Live High-Train Low (LH-TL) method.Methods: Twenty male trained cyclists completed a 3-week training program under normoxic conditions (NORM) or with passive exposure to normobaric hypoxia (LH-TL; FiO2 = 16.5%, ∼2000 m; 11–12 h/day). Hepcidin, ERFE, hypoxia inducible factor-2 (HIF-2), ferroportin (Fpn), erythropoietin (EPO), serum iron (Fe) and hematological variables were assessed at baseline (S1), then immediately after (S2) and 3 days after (S3) intervention.Results: In the LH-TL group, hepcidin decreased by 13.0% (p &amp;lt; 0.001) in S2 and remained at a reduced level in S3. ERFE decreased by 28.7% (p &amp;lt; 0.05) in S2 and returned to baseline in S3. HIF-2α decreased gradually, being lower by 25.3% (p &amp;lt; 0.05) in S3. Fpn decreased between S1 and S2 by 18.9% (p &amp;lt; 0.01) and remained lower during S3 (p &amp;lt; 0.01). In the NORM group, in turn, hepcidin levels increased gradually, being higher by 73.9% (p &amp;lt; 0.05) in S3 compared to S1. No statistically significant differences in EPO were observed in both groups.Conclusion: Three weeks of LH-TL suppresses resting hepcidin and ERFE levels in endurance athletes. We found no association between hepcidin and ERFE after LH-TL. Probably, ERFE is not the only factor that suppresses hepcidin expression in response to moderate hypoxia, especially in later stages of hepcidin downregulation. With the cessation of hypoxia, favorable conditions for increasing the availability of iron cease.

Anaemia in CKD-treatment standard.

Anaemia is one of the most common complications of chronic kidney disease (CKD), having a significant impact on quality of life, and is also associated with a number of adverse clinical outcomes. Its pathogenesis is multifactorial, caused largely by an inadequate production of erythropoietin from the diseased kidneys, with iron deficiency, inflammation, shortened red cell lifespan and enhanced blood loss also being contributory factors. The management of this condition was transformed in the late 1980s by the advent of recombinant human erythropoietin (epoetin), and treatment paradigms have developed over the last three decades, largely focusing on a combination of epoetin or its analogues (erythropoiesis-stimulating agents; ESAs) along with iron supplementation, often administered intravenously due to increased hepcidin levels limiting iron absorption from the gut. Indeed, in patients with early CKD and iron deficiency, iron per se may be sufficient to improve the anaemia, delaying the need for ESA therapy. Other causes of anaemia should be excluded and corrected (if possible) before resorting to treatment with ESAs and iron. More recently, the hypoxia-inducible factor-prolyl hydroxylase inhibitors have entered the therapeutic arena; these are orally active agents that upregulate endogenous erythropoietin production as well as a number of iron-regulatory genes which may also enhance erythropoiesis. The latter drugs are highly efficacious, and may have advantages in inflammatory conditions causing resistance to conventional ESA therapy, but concerns exist regarding their safety, particularly in the longer term. This article reviews the current standards of treatment, as well as recent novel developments in the management of anaemia in CKD.