Yasushi Shimonaka

Determinants of Hepcidin in Patients on Maintenance Hemodialysis: Role of Inflammation

Background/Aim: Hepcidin could be one of the most important regulators for iron metabolism in patients on maintenance hemodialysis (MHD). The factors affecting serum hepcidin levels were evaluated among indexes of anemia, iron metabolism, or inflammation, as well as the dose of erythropoietin. Methods: 198 MHD patients treated with recombinant human erythropoietin were recruited and serum hepcidin-25 levels were specifically measured by liquid chromatography tandem mass spectrometry. Results: In multivariate analysis, only transferrin and ferritin were selected as significant predictors of hepcidin in all patients. In the selected patients with highly sensitive C-reactive protein of >0.3 mg/dl, however, ferritin as well as the IL-6 level were found to be significant predictors for serum hepcidin. The serum ferritin/hepcidin ratio was very similar among MHD and healthy volunteers, suggesting that uremic conditions do not affect the ratio. Serum hepcidin levels decreased by only 27% after a single hemodialysis session, but returned to basal levels 1 h after and remained so until the next hemodialysis session. Conclusions: In the absence of apparent inflammation, the serum hepcidin level could be exclusively associated with ferritin in MHD patients and was independent of inflammatory cytokines. Only in the presence of microinflammation, however, might IL-6 also affect hepcidin expression.

Erythropoietin stimulation decreases hepcidin expression through hematopoietic activity on bone marrow cells in mice

Erythropoiesis-stimulating agents (ESA) are now central to the treatment of renal anemia and are associated with improved clinical outcomes. It is well known that erythropoietin (EPO) is a key regulator of erythropoiesis through its promotion of red blood cell production. In order to investigate the role of ESA on iron metabolism, we analyzed the regulation of the iron regulatory hormone hepcidin by ESA treatment in a bone marrow transplant model in mouse. After treating C57BL/6 mice with continuous erythropoietin receptor activator (C.E.R.A.), recombinant human epoetin-β (rhEPO), or recombinant human carbamylated epoetin-β (rhCEPO), we investigated serum hepcidin concentrations and parameters of erythropoiesis. Serum hepcidin concentrations after rhEPO treatment were analyzed in mice subjected to total body irradiation followed by bone marrow transplantation. C.E.R.A. administration caused long-term downregulation of serum hepcidin levels. Serum hepcidin levels in rhEPO-treated mice decreased significantly, whereas there was no change in rhCEPO-treated mice. The reduction in circulating hepcidin levels after rhEPO administration was not observed in irradiated mice. Finally, bone marrow transplantation recovered the response to rhEPO administration that downregulates hepcidin concentration in irradiated mice. These results indicate that ESA treatment downregulates serum hepcidin concentrations, mainly by indirect mechanisms affecting hematopoietic activity in bone marrow cells.

Hepcidin as well as TNF-α are significant predictors of arterial stiffness in patients on maintenance hemodialysis

BACKGROUND Dysregulated iron metabolism has been suspected to be linked to anemia of chronic disease and to cardiovascular disease (CVD). For the purpose of clarifying the factors affecting arterial stiffness, we evaluated the relationship between iron metabolism, brachial-ankle (ba)-pulse wave velocity (PWV) and several risk factors for CVD in maintenance hemodialysis (MHD) patients. METHODS A total of 168 MHD patients were recruited, and the levels of iron parameters, hepcidin, CVD risk factors and ba-PWV were evaluated. The level of serum hepcidin-25 was specifically measured by liquid chromatography-tandem mass spectrometry. RESULTS Serum levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and hepcidin were higher in MHD patients, which was consistent with results from our previous study. ba-PWV significantly correlated with age (P < 0.01, R = 0.34), total cholesterol (T-CHO; P = 0.02, R = 0.21), TNF-α (P < 0.01, R = 0.24) and hepcidin (P < 0.01, R = 0.25) but not with other iron parameters and CVD risk factors. According to multiple regression analysis, age (β = 0.30), T-CHO (β = 0.24) TNF-α (β = 0.19) and hepcidin (β = 0.23) were selected as the significant predictors of ba-PWV in MHD patients. CONCLUSION Serum levels of both hepcidin and TNF-α are independently associated with arterial stiffness in MHD patients, suggesting that microinflammation and iron metabolism might affect the integrity of arterial walls.

Effects of recombinant human erythropoietin on haemolytic anaemia in mice

The effects of repeated administration of recombinant human erythropoietin (rHuEPO) were investigated in mice with haemolytic anaemia. Mice with haemolytic anaemia induced by phenylhydrazine (PHZ mice) were examined as an acute model and New Zealand black mice (NZB mice) at 13 months of age were examined as a chronic model. The plasma erythropoietin (EPO) level in PHZ mice was high and showed a strong inverse correlation with the Hb in the anaemia development period. However, it was relatively low in the recovery period from anaemia. On the other hand, the plasma EPO level in NZB mice showed a simple inverse correlation with the Hb. The rHuEPO was injected every day for a week into these mice. While a high plasma EPO level was maintained in PHZ mice, no significant effect was observed by injection with rHuEPO at dose of 600 IU/kg. However, in the recovery period from anaemia, RBC and haemoglobin in PHZ mice were increased by the rHuEPO treatment and recovered more quickly to their normal levels. In NZB mice, RBC and haemoglobin were also increased by treatment with rHuEPO at dose of 600 IU/kg. Anti‐RBC autoantibodies and anti‐EPO antibodies did not increase, while RBC and plasma EPO levels were increased by the rHuEPO treatment. These results suggest that some types of haemolytic anaemia are not always combined with high endogenous EPO levels and that exogenous rHuEPO may be effective for use in the treatment of haemolytic anaemia.

Branched-chain amino acids reduce hepatic iron accumulation and oxidative stress in hepatitis C virus polyprotein-expressing mice

Branched‐chain amino acids (BCAA) reduce the incidence of hepatocellular carcinoma (HCC) in patients with cirrhosis. However, the mechanisms that underlie these effects remain unknown. Previously, we reported that oxidative stress in male transgenic mice that expressed hepatitis C virus polyprotein (HCVTgM) caused hepatic iron accumulation by reducing hepcidin transcription, thereby leading to HCC development. This study investigated whether long‐term treatment with BCAA reduced hepatic iron accumulation and oxidative stress in iron‐overloaded HCVTgM and in patients with HCV‐related advanced fibrosis.

Heterogeneous expressions of hepcidin isoforms in hepatoma‐derived cells detected using simultaneous LC‐MS/MS

Hepcidin, a key regulator of iron homeostasis, is known to have three isoforms: hepcidin‐20, ‐22, and ‐25. Hepcidin‐25 is thought to be the major isoform and the only one known to be involved in iron metabolism; the physiological roles of other isoforms are poorly understood. Because of its involvement in the pathophysiology of hereditary hemochromatosis and the anemia of chronic disease, the regulatory mechanisms of hepcidin expression have been extensively investigated, but most studies have been performed only at the transcriptional level. Difficulty in detecting hepcidin has impeded in vitro research. In the present study, we developed a novel method for simultaneous quantification of hepcidin‐20, ‐22, and ‐25 in the media from hepatoma‐derived cell lines. Using this method, we determined the expression patterns of hepcidin isoforms and the patterns of responses to various stimuli in human hepatoma‐derived cultured cells. We found substantial differences among cell lines. In conclusion, a novel method for simultaneous quantification of hepcidin isoforms is presented. Heterogeneous expressions of hepcidin isoforms in human hepatoma‐derived cells were revealed by this method. We believe our method will facilitate quantitative investigation of the role hepcidin plays in iron homeostasis.

The three isoforms of hepcidin in human serum and their processing determined by liquid chromatography-tandem mass spectrometry (LC-tandem MS)

Hepcidin, the iron regulatory hormone, has three isoforms; -20, -22 and -25. While hepcidin-25 has been studied extensively, the physiological significance of other isoforms remains poorly understood. Using a quantitative method based on liquid chromatography-tandem mass spectrometry (LC-tandem MS) developed by our group, we quantified hepcidin isoforms in human serum to elucidate their characteristics, and investigated the role of hepatocytes in isoform processing. Hepcidin isoforms in serum obtained from 40 healthy volunteers were quantified. Synthetic hepcidin peptides were added to healthy serum, and to HepG2 culture media, and hepcidin isoform concentrations determined. All three hepcidin isoforms were detected in human serum; however, hepcidin-25 concentrations were highest. The three hepcidin isoforms showed a strong positive correlation with each other and with serum ferritin. Additionally, while hepcidin-20 was strongly correlated with serum creatinine, the other isoforms were not. Hepcidin-20 and -25 levels were also increased in chronic kidney disease (CKD) serum. Hepcidin-22 rapidly degraded into hepcidin-20, whereas hepcidin-25 remained relatively stable. Finally, hepcidin-22 degradation into hepcidin-20 was accelerated in the presence of HepG2. This method has enabled us to reveal fundamental characteristics of the three hepcidin isoforms in serum and may be a powerful tool for quantifying hepcidin isoform expression and processing.

Epoetin beta pegol (C.E.R.A.) promotes utilization of iron for erythropoiesis through intensive suppression of serum hepcidin levels in mice.

Epoetin beta pegol (C.E.R.A.) is a novel, long-acting, erythropoiesis-stimulating agent. We investigated iron utilization associated with erythropoiesis, and how it is affected by the long-term suppression of hepcidin following C.E.R.A. treatment in mice. C57BL/6N mice were administered a single intravenous injection of C.E.R.A. Hemoglobin (Hb) levels were significantly elevated following C.E.R.A. administration, while serum hepcidin levels were continuously suppressed. Expression levels of duodenal iron transporters, ferroportin (FPN) which is internalized by hepcidin binding and divalent metal transporter 1 (DMT1), increased after C.E.R.A. administration. To evaluate the contribution of dietary iron absorption to erythropoiesis following C.E.R.A. treatment, C.E.R.A.-treated mice were fed either an iron-deficient diet to restrict dietary iron absorption or a control diet. The restriction of dietary iron absorption resulted in impaired Hb elevation after C.E.R.A. administration. To investigate the utilization of stored iron for erythropoiesis following C.E.R.A. treatment, iron indices were analyzed in C.E.R.A.-treated mice given an intraperitoneal pre-treatment injection of iron-dextran. Serum hepcidin levels and splenic hemosiderin deposition decreased after C.E.R.A. administration. These results indicate that C.E.R.A. promotes utilization of iron for erythropoiesis both through enhancement of dietary iron absorption and mobilization of iron storage from reticuloendothelial cells.

Treatment with anti-IL-6 receptor antibody prevented increase in serum hepcidin levels and improved anemia in mice inoculated with IL-6–producing lung carcinoma cells

BackgroundHepcidin, a key regulator of iron metabolism, is produced mainly by interleukin-6 (IL-6) during inflammation. A mechanism linking cancer-related anemia and IL-6 through hepcidin production is suggested. To clarify the hypothesis that overproduction of IL-6 elevates hepcidin levels and contributes to the development of cancer-related anemia, we evaluated anti-IL-6 receptor antibody treatment of cancer-related anemia in an IL-6–producing human lung cancer xenograft model.MethodsNude mice were subcutaneously inoculated with cells of the IL-6–producing human lung cancer cell line LC-06-JCK and assessed as a model of cancer-related anemia. Mice bearing LC-06-JCK were administered rat anti-mouse IL-6 receptor antibody MR16-1 and their serum hepcidin levels and hematological parameters were determined.ResultsLC-06-JCK–bearing mice developed anemia according to the production of human IL-6 from xenografts, with decreased values of hemoglobin, hematocrit, and mean corpuscular volume (MCV) compared to non–tumor-bearing (NTB) mice. LC-06-JCK–bearing mice showed decreased body weight and serum albumin with increased serum amyloid A. MR16-1 treatment showed significant inhibition of decreased body weight and serum albumin levels, and suppressed serum amyloid A level. There was no difference in tumor volume between MR16-1-treated mice and immunoglobulin G (IgG)-treated control mice. Decreased hemoglobin, hematocrit, and MCV in LC-06-JCK–bearing mice was significantly relieved by MR16-1 treatment. LC-06-JCK–bearing mice showed high red blood cell counts and erythropoietin levels as compared to NTB mice, whereas MR16-1 treatment did not affect their levels. Serum hepcidin and ferritin levels were statistically elevated in mice bearing LC-06-JCK. LC-06-JCK–bearing mice showed lower values of MCV, mean corpuscular hemoglobin (MCH), and serum iron as compared to NTB mice. Administration of MR16-1 to mice bearing LC-06-JCK significantly suppressed levels of both serum hepcidin and ferritin, with increased values of MCV and MCH.ConclusionsOur results suggest that overproduction of hepcidin by IL-6 signaling might be a major factor that leads to functionally iron-deficient cancer-related anemia in the LC-06-JCK model. We demonstrated that inhibition of the IL-6 signaling pathway by MR16-1 treatment resulted in significant recovery of iron-deficiency anemia and alleviation of cancer-related symptoms. These results indicate that IL-6 signaling might be one possible target pathway to treat cancer-related anemia disorders.

Hepcidin/ferroportin expression levels involve efficacy of pegylated-interferon plus ribavirin in hepatitis C virus-infected liver

AIM To investigate the relationship between the iron-metabolism-related gene expression profiles and efficacy of antiviral therapy in chronic hepatitis C patients. METHODS The hepatic expression profile of iron-metabolism-related genes was analyzed and its association with virological response to pegylated-interferon plus ribavirin combination therapy was evaluated. A hundred patients with chronic hepatitis C (genotype1b, n = 50; genotype 2, n = 50) were enrolled and retrospectively analyzed. Liver biopsy samples were subjected to quantitative polymerase chain reaction for iron-metabolism-related genes and protein expression (Western blotting analysis) for ferroportin. As a control, normal liver tissue was obtained from 18 living donors of liver transplantation. Serum hepcidin level was measured by sensitive liquid chromatography/electrospray ionization tandem mass spectrometry. RESULTS Iron overload is associated with liver damage by increasing oxidative stress and hepatitis C virus (HCV) is reported to induce iron accumulation in hepatocytes in vivo. Conversely, iron administration suppresses HCV replication in vitro. Therefore, the association between HCV infection and iron metabolism remains unclear. Compared with controls, patients had significantly higher gene expression for transferrin, iron-regulatory proteins 1 and 2, divalent metal transporter 1, and ferroportin, but similar for transferrin receptors 1 and 2, and hepcidin. When the expression profiles were compared between sustained virological response (SVR) and non-SVR patients, the former showed significantly lower transcription and protein expression of hepcidin and ferroportin. Expression of hepcidin-regulating genes, BMPR1, BMPR2, and hemojuvelin, was significantly increased, whereas BMP2 was decreased in HCV-infected liver. BMPR2 and hemojuvelin expression was significantly lower in the SVR than non-SVR group. HCV infection affects the expression of iron-metabolism-related genes, leading to iron accumulation in hepatocytes. CONCLUSION Decreased expression of hepcidin and ferroportin in SVR patients indicates the importance of hepatocytic iron retention for viral response during pegylated-interferon plus ribavirin treatment.