Mark Busbridge

Plasma hepcidin levels are elevated but responsive to erythropoietin therapy in renal disease

Hepcidin is a critical inhibitor of iron export from macrophages, enterocytes, and hepatocytes. Given that it is filtered and degraded by the kidney, its elevated levels in renal failure have been suggested to play a role in the disordered iron metabolism of uremia, including erythropoietin resistance. Here, we used a novel radioimmunoassay for hepcidin-25, the active form of the hormone, to measure its levels in renal disease. There was a significant diurnal variation of hepcidin and a strong correlation to ferritin levels in normal volunteers. In 44 patients with mild to moderate kidney disease, hepcidin levels were significantly elevated, positively correlated with ferritin but inversely correlated with the estimated glomerular filtration rate. In 94 stable hemodialysis patients, hepcidin levels were also significantly elevated, but this did not correlate with interleukin-6 levels, suggesting that increased hepcidin was not due to a general inflammatory state. Elevated hepcidin was associated with anemia, but, intriguingly, the erythropoietin dose was negatively correlated with hepcidin, suggesting that erythropoietin suppresses hepcidin levels. This was confirmed in 7 patients when hepcidin levels significantly decreased after initiation of erythropoietin treatment. Our results show that hepcidin is elevated in renal disease and suggest that higher hepcidin levels do not predict increased erythropoietin requirements.

Erythropoietin administration in humans causes a marked and prolonged reduction in circulating hepcidin

Expression of hepcidin, the key hormone governing iron transport, is reduced by anemia in a manner which appears dependent on increased bone marrow activity. The temporal associations between plasma hepcidin and other iron parameters were examined in healthy humans after erythropoietin administration and venesection. Profound hepcidin suppression appeared abruptly 24 hours after subcutaneous erythropoietin (P=0.003), and was near maximal at onset, with peak (mid-afternoon) levels reduced by 73.2%, gradually recovering over the following two weeks. Minor changes in circulating iron, soluble transferrin receptor and growth differentiation factor-15 were observed after the reduction in hepcidin. Similar but more gradual changes in these parameters were observed after reducing hematocrit by removal of 250 mL blood. These human studies confirm the importance of a rapidly responsive marrow–hepcidin axis in regulating iron supply in vivo, and suggest that this axis is regulated by factors other than circulating iron, soluble transferrin receptor or growth differentiation factor-15.

Iron deficiency and raised hepcidin in idiopathic pulmonary arterial hypertension: clinical prevalence, outcomes, and mechanistic insights.

OBJECTIVES This study sought to understand the prevalence and clinical relevance of iron deficiency in patients with idiopathic pulmonary arterial hypertension (IPAH). BACKGROUND Iron availability influences the pulmonary vascular response to hypoxia in humans and may be significant in the pathogenesis of IPAH. METHODS Iron deficiency, defined by raised levels of soluble transferrin receptor (sTfR), was investigated in 98 patients with IPAH. Hepcidin and erythropoietin (EPO) levels were also measured. The effect of bone morphogenetic protein (BMP) receptor knockdown on BMP-6-stimulated hepcidin production was assessed in human hepatoma HepG2 cells. Relationships between sTfR and exercise capacity, functional class, and all-cause mortality were analyzed. RESULTS Circulating sTfR levels were raised in 63% of IPAH patients, indicating significant iron deficiency. Consistent with this, iron, ferritin, and transferrin saturation levels were reduced and red cell distribution width increased, without overt anemia. Hepcidin correlated inversely with sTfR and positively with increasing ferritin. Hepcidin was inappropriately raised in IPAH independent of the inflammatory marker interleukin-6. EPO levels were also raised and correlated inversely with hepcidin. BMP receptor-type 2 (BMPR2) knockdown in HepG2 cells increased BMP-6-stimulated hepcidin expression. sTfR increased with World Health Organization functional class (p < 0.05), correlated negatively with exercise capacity (p = 0.027), and values >28.1 nmol/l independently predicted survival (p = 0.011). CONCLUSIONS Iron deficiency is common in IPAH patients and associated with disease severity and poor clinical outcome. Inappropriately raised hepcidin levels, which impair iron absorption from the gut, may be a factor.

Sustained appetite improvement in malnourished dialysis patients by daily ghrelin treatment

Malnutrition is a common complication in patients on dialysis and is strongly associated with poor prognosis. Effective therapy could substantially improve morbidity and mortality, but neither enteral nor parenteral supplementation provide long-term benefit because of the strong appetite suppression seen in such patients. We performed a double-blinded randomized crossover study of a week-long treatment with daily subcutaneous ghrelin, a gut hormone that regulates hunger through the hypothalamus, in a group of 12 malnourished dialysis patients. Ghrelin administration increased ghrelin levels in circulation, modestly reduced blood pressure for up to 2 h, and immediately and significantly increased appetite, with an increase in energy intake noted at the first study meal. Persistence of this effect throughout the week was confirmed with food diaries and final study meals. Energy expenditure, measured with free-living pulse and motion monitors, was unchanged by ghrelin. Our study shows that daily treatment with ghrelin achieves a sustained positive change in energy balance in malnourished dialysis patients. Direct manipulation of appetite with ghrelin or its analogs represents an attractive and promising therapeutic strategy for this difficult clinical problem.

Development of a novel immunoassay for the iron regulatory peptide hepcidin.

Abstract To date there have been few published immunoassays for the important iron regulator hepcidin. This study describes a novel competitive radioimmunoassay (RIA) for the bioactive hepcidin peptide. A rabbit anti-hepcidin polyclonal antibody was produced using synthetic hepcidin radiolabelled with 125I to produce a competitive RIA. Normal patient (n=47) samples were collected and assayed for hepcidin to determine a reference range. Other patient groups collected were ulcerative colitis (UC; n=40), iron deficiency anaemia (IDA; n=15), chronic kidney disease not requiring dialysis (CKD; n=45) and chronic kidney disease requiring dialysis (HCKD; n=94). Detection limit of the assay was determined as 0.6 ng/mL. Intra-assay precision was 5 ng/mL (7.2%) and 50 ng/mL (5.8%), inter-assay precision was 5 ng/mL (7.6%) and 50 ng/mL (6.7%). Analytical recovery was 98% (5 ng/mL), 94% (10 ng/mL) and 97% (50 ng/mL). The assay was linear up to 200 ng/mL. No demonstrable cross-reactivity with human insulin, glucagon I, angiotensinogen I, β-defensin 1–4, α-defensin-1 and plectasin was observed. There was significant correlation (r=0.96, P≤0.0001) between the hepcidin RIA and an established hepcidin SELDI-TOF-MS method. Analysis of the normal human samples gave a reference range of 1.1–55 ng/mL for hepcidin. Further statistical evaluation revealed a significant difference between male and female hepcidin levels. There was significant correlation between hepcidin and ferritin in the control group (r=0.6, P≤0.0001). There was also a significant difference between the normal and disease groups (P≤0.0001). Healthy volunteers (n=10) showed a diurnal increase in plasma hepcidin at 4.00 pm compared to 8.00 am. A robust and optimised immunoassay for bioactive hepcidin has been produced and the patient sample results obtained further validates the important role of hepcidin in iron regulation, and will allow further investigation of this important peptide and its role in iron homeostasis.

Unexplained iron deficiency in idiopathic and heritable pulmonary arterial hypertension

Background Anaemia is common in left heart failure and is associated with a poorer outcome. Many patients with pulmonary arterial hypertension (PAH) are anaemic or iron-deficient. This study was performed to investigate the prevalence of iron deficiency in PAH and to identify possible causes. Methods All patients with idiopathic or heritable PAH diagnosed in 1995–2008 were identified. Controls were selected from patients with chronic thromboembolic pulmonary hypertension (CTEPH). Full blood counts were examined and any abnormality was investigated. Patients were excluded if they had a cause for iron deficiency. The prevalence study was based on 85 patients with idiopathic PAH and 120 with CTEPH. A separate group of 20 patients with idiopathic PAH and 24 with CTEPH with matching haemodynamics were prospectively investigated for serum factors affecting iron metabolism. Results The prevalence study identified a point prevalence of unexplained iron deficiency of 50% in premenopausal women with idiopathic PAH compared with 8% in premenopausal women with CTEPH (p=0.002); 14% in postmenopausal women with idiopathic PAH compared with 6% in postmenopausal women with CTEPH (p=0.16); 28% in men with idiopathic PAH men compared with 2% in men with CTEPH (p=0.002); and 60% in patients with heritable PAH. The serum study showed that patients with idiopathic PAH had lower serum iron and transferrin saturations than those with CTEPH. Interleukin-6 levels correlated with iron levels(r=−0.6, p=0.006) and transferrin saturations (r=−0.68, p=0.001) in idiopathic PAH but not in CTEPH. Conclusions The prevalence of unexplained iron deficiency is significantly higher in idiopathic PAH than in CTEPH. This may be linked to interleukin-6.

Hepcidin and inflammatory bowel disease: dual role in host defence and iron homoeostasis.

Objective Hepcidin is an endogenous antimicrobial peptide with a key role in iron homoeostasis. Hepcidin is similar to defensin, the deficiency of which is associated with Crohns disease. To date there has been no validated method to reliably assay serum hepcidin. We studied iron indices in inflammatory bowel disease (IBD) including hepcidin. Design We assessed serum hepcidin concentrations (using a newly developed competitive radioimmunoassay) and ferritin in patients with IBD. Haematinics including serum soluble transferrin receptor, serum iron, serum vitamin B12 and red cell folate levels were also measured. The hepcidin results were compared with a control group of healthy volunteers from the local community. Setting This study was based in a hospital. Patients Sixty-one patients with IBD (51 patients with ulcerative colitis and 10 with Crohns disease). Their mean hepcidin results were compared with 25 healthy controls. Main outcome measure: hepcidin concentration in serum samples in IBD patients compared with normal volunteers. Results We found significantly low serum hepcidin levels in patients with IBD. The hepcidin levels were low in IBD patients without iron deficiency anaemia as evidenced by normal ferritin and serum iron levels (n=41, mean hepcidin 6.81 ng/ml, SEM 1.2) and in IBD patients with iron deficiency anaemia (n=18, mean hepcidin 4.14 ng/ml, SEM 0.72) compared with healthy controls (n=25, mean hepcidin 15.3 ng/ml, SEM 3.14) (P=0.0045 and P=0.0050 on unpaired t-tests, respectively). We also measured IL-6 (enzyme-linked immunosorbent assay method, Abcam plc) in 21 of the 61 patients with IBD and compared the results with samples from 10 healthy volunteers. The IL-6 level was significantly higher (P=0.0222 on unpaired t-tests) in this group of IBD patients (n=21, IL-6 mean 2.94 pg/ml, SEM 0.64) compared with controls (n=10, IL-6 mean 0.663 pg/ml SEM 0.14). A significant positive correlation (Pearsons correlation coefficient r=0.6331) was present between hepcidin and IL-6, but not between hepcidin and serum soluble transferrin receptor (r=−0.235). Conclusion The low hepcidin results in IBD patients may reflect a causal or perpetuator effect on intestinal inflammation.

Hepcidin levels in diabetes mellitus and polycystic ovary syndrome

Increased body iron is associated with insulin resistance. Hepcidin is the key hormone that negatively regulates iron homeostasis. We hypothesized that individuals with insulin resistance have inadequate hepcidin levels for their iron load.

Hemorrhage-Adjusted Iron Requirements, Hematinics and Hepcidin Define Hereditary Hemorrhagic Telangiectasia as a Model of Hemorrhagic Iron Deficiency

Background Iron deficiency anemia remains a major global health problem. Higher iron demands provide the potential for a targeted preventative approach before anemia develops. The primary study objective was to develop and validate a metric that stratifies recommended dietary iron intake to compensate for patient-specific non-menstrual hemorrhagic losses. The secondary objective was to examine whether iron deficiency can be attributed to under-replacement of epistaxis (nosebleed) hemorrhagic iron losses in hereditary hemorrhagic telangiectasia (HHT). Methodology/Principal Findings The hemorrhage adjusted iron requirement (HAIR) sums the recommended dietary allowance, and iron required to replace additional quantified hemorrhagic losses, based on the pre-menopausal increment to compensate for menstrual losses (formula provided). In a study population of 50 HHT patients completing concurrent dietary and nosebleed questionnaires, 43/50 (86%) met their recommended dietary allowance, but only 10/50 (20%) met their HAIR. Higher HAIR was a powerful predictor of lower hemoglobin (p = 0.009), lower mean corpuscular hemoglobin content (p<0.001), lower log-transformed serum iron (p = 0.009), and higher log-transformed red cell distribution width (p<0.001). There was no evidence of generalised abnormalities in iron handling Ferritin and ferritin2 explained 60% of the hepcidin variance (p<0.001), and the mean hepcidinferritin ratio was similar to reported controls. Iron supplement use increased the proportion of individuals meeting their HAIR, and blunted associations between HAIR and hematinic indices. Once adjusted for supplement use however, reciprocal relationships between HAIR and hemoglobin/serum iron persisted. Of 568 individuals using iron tablets, most reported problems completing the course. For patients with hereditary hemorrhagic telangiectasia, persistent anemia was reported three-times more frequently if iron tablets caused diarrhea or needed to be stopped. Conclusions/significance HAIR values, providing an indication of individuals’ iron requirements, may be a useful tool in prevention, assessment and management of iron deficiency. Iron deficiency in HHT can be explained by under-replacement of nosebleed hemorrhagic iron losses.

Iron Homeostasis and Pulmonary Hypertension: Iron Deficiency Leads to Pulmonary Vascular Remodeling in the Rat

RATIONALE Iron deficiency without anemia is prevalent in patients with idiopathic pulmonary arterial hypertension and associated with reduced exercise capacity and survival. OBJECTIVES We hypothesized that iron deficiency is involved in the pathogenesis of pulmonary hypertension and iron replacement is a possible therapeutic strategy. METHODS AND RESULTS Rats were fed an iron-deficient diet (IDD, 7 mg/kg) and investigated for 4 weeks. Iron deficiency was evident from depleted iron stores (decreased liver, serum iron, and ferritin), reduced erythropoiesis, and significantly decreased transferrin saturation and lung iron stores after 2 weeks IDD. IDD rats exhibited profound pulmonary vascular remodeling with prominent muscularization, medial hypertrophy, and perivascular inflammatory cell infiltration, associated with raised pulmonary artery pressure and right ventricular hypertrophy. IDD rat lungs demonstrated increased expression of hypoxia-induced factor-1α and hypoxia-induced factor-2α, nuclear factor of activated T cells and survivin, and signal transducers and activators of transcription-3 activation, which promote vascular cell proliferation and resistance to apoptosis. Biochemical examination showed reduced mitochondrial complex I activity and mitochondrial membrane hyperpolarization in mitochondria from IDD rat pulmonary arteries. Along with upregulation of the glucose transporter, glucose transporter 1, and glycolytic genes, hk1 and pdk1, lung fluorine-18-labeled 2-fluoro-2-deoxyglucose ligand uptake was significantly increased in IDD rats. The hemodynamic and pulmonary vascular remodeling were reversed by iron replacement (ferric carboxymaltose, 75 mg/kg) and attenuated in the presence of iron deficiency by dichloroacetate and imatinib, 2 putative treatments explored for pulmonary arterial hypertension that target aerobic glycolysis and proliferation, respectively. CONCLUSIONS These data suggest a major role for iron in pulmonary vascular homeostasis and support the clinical evaluation of iron replacement in patients with pulmonary hypertension.Rationale: Iron deficiency without anemia is prevalent in patients with idiopathic pulmonary arterial hypertension and associated with reduced exercise capacity and survival. Objectives: We hypothesized that iron deficiency is involved in the pathogenesis of pulmonary hypertension and iron replacement is a possible therapeutic strategy. Methods and Results: Rats were fed an iron-deficient diet (IDD, 7 mg/kg) and investigated for 4 weeks. Iron deficiency was evident from depleted iron stores (decreased liver, serum iron, and ferritin), reduced erythropoiesis, and significantly decreased transferrin saturation and lung iron stores after 2 weeks IDD. IDD rats exhibited profound pulmonary vascular remodeling with prominent muscularization, medial hypertrophy, and perivascular inflammatory cell infiltration, associated with raised pulmonary artery pressure and right ventricular hypertrophy. IDD rat lungs demonstrated increased expression of hypoxia-induced factor-1α and hypoxia-induced factor-2α, nuclear factor of activated T cells and survivin, and signal transducers and activators of transcription-3 activation, which promote vascular cell proliferation and resistance to apoptosis. Biochemical examination showed reduced mitochondrial complex I activity and mitochondrial membrane hyperpolarization in mitochondria from IDD rat pulmonary arteries. Along with upregulation of the glucose transporter, glucose transporter 1, and glycolytic genes, hk1 and pdk1 , lung fluorine-18–labeled 2-fluoro-2-deoxyglucose ligand uptake was significantly increased in IDD rats. The hemodynamic and pulmonary vascular remodeling were reversed by iron replacement (ferric carboxymaltose, 75 mg/kg) and attenuated in the presence of iron deficiency by dichloroacetate and imatinib, 2 putative treatments explored for pulmonary arterial hypertension that target aerobic glycolysis and proliferation, respectively. Conclusions: These data suggest a major role for iron in pulmonary vascular homeostasis and support the clinical evaluation of iron replacement in patients with pulmonary hypertension. # Novelty and Significance {#article-title-52}