Hepcidin-25 in Chronic Hemodialysis Patients Is Related to Residual Kidney Function and Not to Treatment with Erythropoiesis Stimulating Agents

Hepcidin-25, the bioactive form of hepcidin, is a key regulator of iron homeostasis as it induces internalization and degradation of ferroportin, a cellular iron exporter on enterocytes, macrophages and hepatocytes. Hepcidin levels are increased in chronic hemodialysis (HD) patients, but as of yet, limited information on factors associated with hepcidin-25 in these patients is available. In the current cross-sectional study, potential patient-, laboratory- and treatment-related determinants of serum hepcidin-20 and -25, were assessed in a large cohort of stable, prevalent HD patients. Baseline data from 405 patients (62% male; age 63.7±13.9 [mean SD]) enrolled in the CONvective TRAnsport STudy (CONTRAST; NCT00205556) were studied. Predialysis hepcidin concentrations were measured centrally with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Patient-, laboratory- and treatment related characteristics were entered in a backward multivariable linear regression model. Hepcidin-25 levels were independently and positively associated with ferritin (p<0.001), hsCRP (p<0.001) and the presence of diabetes (p = 0.02) and inversely with the estimated glomerular filtration rate (p = 0.01), absolute reticulocyte count (p = 0.02) and soluble transferrin receptor (p<0.001). Men had lower hepcidin-25 levels as compared to women (p = 0.03). Hepcidin-25 was not associated with the maintenance dose of erythropoiesis stimulating agents (ESA) or iron therapy. In conclusion, in the currently studied cohort of chronic HD patients, hepcidin-25 was a marker for iron stores and erythropoiesis and was associated with inflammation. Furthermore, hepcidin-25 levels were influenced by residual kidney function. Hepcidin-25 did not reflect ESA or iron dose in chronic stable HD patients on maintenance therapy. These results suggest that hepcidin is involved in the pathophysiological pathway of renal anemia and iron availability in these patients, but challenges its function as a clinical parameter for ESA resistance

Serum hepcidin following autologous hematopoietic cell transplantation : an illustration of the interplay of iron status, erythropoiesis and inflammation

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Effect of Deferoxamine on Post-Transfusion Iron, Inflammation, and In Vitro Microbial Growth in a Canine Hemorrhagic Shock Model: A Randomized Controlled Blinded Pilot Study

Red blood cell (RBC) transfusion is associated with recipient inflammation and infection, which may be triggered by excessive circulating iron. Iron chelation following transfusion may reduce these risks. The aim of this study was to evaluate the effect of deferoxamine on circulating iron and inflammation biomarkers over time and in vitro growth of Escherichia coli (E. coli) following RBC transfusion in dogs with atraumatic hemorrhage. Anesthetized dogs were subject to atraumatic hemorrhage and transfusion of RBCs, then randomized to receive either deferoxamine or saline placebo of equivalent volume (n = 10 per group) in a blinded fashion. Blood was sampled before hemorrhage and then 2, 4, and 6 h later. Following hemorrhage and RBC transfusion, free iron increased in all dogs over time (both p \u3c 0.001). Inflammation biomarkers interleukin-6 (IL6), CXC motif chemokine-8 (CXCL8), interleukin-10 (IL10), and keratinocyte-derived chemokine (KC) increased in all dogs over time (all p \u3c 0.001). Logarithmic growth of E. coli clones within blood collected 6 h post-transfusion was not different between groups. Only total iron-binding capacity was different between groups over time, being significantly increased in the deferoxamine group at 2 and 4 h post-transfusion (both p \u3c 0.001). In summary, while free iron and inflammation biomarkers increased post-RBC transfusion, deferoxamine administration did not impact circulating free iron, inflammation biomarkers, or in vitro growth of E. coli when compared with placebo

Effect of Deferoxamine on Post-Transfusion Iron, Inflammation, and In Vitro Microbial Growth in a Canine Hemorrhagic Shock Model: A Randomized Controlled Blinded Pilot Study

Red blood cell (RBC) transfusion is associated with recipient inflammation and infection, which may be triggered by excessive circulating iron. Iron chelation following transfusion may reduce these risks. The aim of this study was to evaluate the effect of deferoxamine on circulating iron and inflammation biomarkers over time and in vitro growth of Escherichia coli (E. coli) following RBC transfusion in dogs with atraumatic hemorrhage. Anesthetized dogs were subject to atraumatic hemorrhage and transfusion of RBCs, then randomized to receive either deferoxamine or saline placebo of equivalent volume (n = 10 per group) in a blinded fashion. Blood was sampled before hemorrhage and then 2, 4, and 6 h later. Following hemorrhage and RBC transfusion, free iron increased in all dogs over time (both p &lt; 0.001). Inflammation biomarkers interleukin-6 (IL6), CXC motif chemokine-8 (CXCL8), interleukin-10 (IL10), and keratinocyte-derived chemokine (KC) increased in all dogs over time (all p &lt; 0.001). Logarithmic growth of E. coli clones within blood collected 6 h post-transfusion was not different between groups. Only total iron-binding capacity was different between groups over time, being significantly increased in the deferoxamine group at 2 and 4 h post-transfusion (both p &lt; 0.001). In summary, while free iron and inflammation biomarkers increased post-RBC transfusion, deferoxamine administration did not impact circulating free iron, inflammation biomarkers, or in vitro growth of E. coli when compared with placebo

Quantification of hepcidin isoforms using hepcidin-25⁺⁴⁰ as internal standard.

<p><b>A.</b> Linearity (range 0–40 nM) for hepcidin-25, hepcidin-24, hepcidin-22, and hepcidin-20 as determined by hepcidin-25⁺⁴⁰ as internal standard. Linearity curves are assessed in different runs. Blank serum (hep-24, hep-20 and hep-25) or heparin plasma (used for hep-22 as serum yields an interfering peak near the position of this isoform) was used as matrix for the addition of the synthetic hepcidin isoforms (PI) to end concentrations of 0, 0.5,1, 2, 3, 5, 7.5, 10, 15, 20 and 40 nM. Since there is a small interfering peak at 2191.8 Da in blank serum, the linearity curve of hepcidin-20 was corrected for the base line hepcidin-20 peak (data not shown). Description of the lines: hepcidin-25, Y = 0.964X+0.064 (R² = 0.9950); hepcidin-24, Y = 1.145X−0.767 (R² = 0.9975); hepcidin-22, Y = 1.100X−0.197 (R² = 0.9998); hepcidin-20, Y = 0.867X+0.055 (R² = 0.9998). <b>B.</b> WCX-TOF MS profile of blank plasma that was spiked with 10 nM of each of the synthetic hepcidin-20, -22, -24, -25, and -25⁺⁴⁰ peptides, which illustrates that all these hepcidin analogues have the same WCX-binding characteristics and flight behavior during TOF MS.</p

WCX-TOF MS profiles of sample pools of patients with presumed hepcidin isoforms.

<p>Panel <b>A</b>, peptide profile of a heparin plasma pool of IC patients; Panel <b>B/C</b>, peptide profile of heparin plasma pool of nephrology patients that were untreated (<b>B</b>) or pre-incubated with 1 molar excess of the anti-hepcidin molecule PRS-080 prior to WCX-TOF MS analysis (<b>C</b>); Panels <b>D/E</b>, control peptide profile of plasma from patients with juvenile hemochromatosis and iron deficiency anemia, respectively, that lack hepcidin-25. Positions in the spectrum: hepcidin-25⁺⁴⁰ (internal standard), m/z 2829.4; hepcidin-25, m/z 2789.4; hepcidin-24, m/z 2673.9; hepcidin-22, m/z 2436.1; and hepcidin-20, m/z 2191.8. It should be noted that: i) profiles from IC and nephrology patients both clearly contain the m/z 2673.9 peak at the presumed position of hepcidin-24; ii) this peak disappears completely from the profile after incubation with PRS-080, similar to hepcidin-25/-22; iii) hepcidin-25⁺⁴⁰ does not disappear from the profile as it was added after the PRS-080 incubation period, which limits complex formation; iv) the intensity of the presumed peak of hepcidin-20 at m/z 2191.8 after PRS-080 incubation decreases but does not disappear completely suggesting that another hepcidin-unrelated peptide is also present at this position; v) the peptide spectra of patient that lack hepcidin-25 also contain a peak at m/z 2191.8 (calculated between 1–2 nM), providing further evidence for the unlikeliness that this peak is solely derived from hepcidin-20.</p

Characteristics of commercial hepcidin peptides used.

*<p>catalog product of Peptide International but manufactured by Peptide Institute (Osaka, Japan);</p>∧<p>vialed at 100% peptide content;</p>†<p>due to an isotope content of 98% the actual mass is 1 Da less than the theoretical mass of this peptide.</p>#<p>delivered as 0.10 mg per vial; personal communication revealed that ≈ 0.11 mg was pipetted in each vial. PI, Peptide International (Louisville, KY, USA); B, Bachem LTD (St. Helens, UK); n.a., not applicable.</p

Hepcidin-mediated ferroportin internalization.

<p>Different concentrations of synthetic hepcidin-25, -24, -22 and -20 (indicated in nM on the horizontal axis) were added to the growth medium of a stable cell line that expresses green fluorescent protein-fused ferroportin (GFP-FPN). Hepcidin-mediated GFP-FPN internalization and degradation was quantified by measuring cellular fluorescence levels in arbitrary units.</p