| Abstract|| |
Hepcidin may play a critical role in the response of patients with anemia to iron and erythropoiesis-stimulating agent therapy. To evaluate the factors affecting serum hepcidin levels and their relation to other indexes of anemia, iron metabolism and inflammation, as well as the dose of erythropoietin, we studied 80 maintenance hemodialysis (MHD) patients treated with recombinant human erythropoietin and their serum hepcidin levels were specifically measured by using a competitive enzyme-linked immunosorbent assay. In linear regression analysis, ferritin was found to be a significant predictor of hepcidin levels in all the study patients. In the absence of apparent inflammation, serum hepcidin levels correlated exclusively with ferritin levels in MHD patients, and it was also an independent marker of inflammation as highly sensitive C-reactive protein.
|How to cite this article:|
Sany D, Elsawy AE, Elshahawy Y. Hepcidin and regulation of iron homeostasis in maintenance hemodialysis patients. Saudi J Kidney Dis Transpl 2014;25:967-73
|How to cite this URL:|
Sany D, Elsawy AE, Elshahawy Y. Hepcidin and regulation of iron homeostasis in maintenance hemodialysis patients. Saudi J Kidney Dis Transpl [serial online] 2014 [cited 2021 Dec 8];25:967-73. Available from: https://www.sjkdt.org/text.asp?2014/25/5/967/139868
| Introduction|| |
Anemia occurs in the majority of patients with end-stage renal disease (ESRD) requiring dialysis therapy, and it can be corrected effectively using erythropoiesis-stimulating agents (ESA).  However, a considerable proportion of patients exhibit a suboptimal response to ESA, and iron deficiency has been identified as the major cause of this hyporesponsiveness. ,
Because of accelerated erythropoiesis driven by the ESA treatment (coupled with the ongoing uremia and dialysis-related iron losses), ESRD patients on ESA are at high risk of developing iron-restricted erythropoiesis because the rate at which iron is released from stores and delivered to the bone marrow fails to match the increased iron demand. This limited availability of iron to bone marrow can be corrected effectively by intravenous iron therapy, which improves hemoglobin (Hb) res-ponse.  On the other hand, the inflammation frequently seen in dialysis patients may also contribute to iron-restricted erythropoiesis by reducing the release of stored iron from the reticuloendothelial system to circulating trans-ferrin,  a condition that, unlike iron depletion, reduces the likelihood and extent of response to intravenous iron administration. ,
The observation that polymorphonuclear cells from patients on maintenance hemodialysis (MHD) had two- to three-times the iron content as cells of healthy subjects may reflect the defective regulation of iron transport proteins.  The accurate identification of patients who would benefit from iron therapy has relevant clinical and economic implications, as it enables a better response to ESA, while avoiding the risks associated with overzealous iron the-rapy. , Unfortunately, the laboratory tests used to evaluate iron status have revealed a sub-optimal accuracy in identifying cases that will respond to intravenous iron,  as their relationships with iron status tend to be confounded by other factors, such as inflammation as in the case of ferritin, transferrin saturation (TSAT) and the percentage of hypochromic red blood cells (%Hypo), ,, or erythropoietic activity as in the case of soluble transferrin receptors (sTfR). 
A recently discovered small peptide known as hepcidin, which is produced by hepatocytes and circulates in the plasma, plays a central role in regulating the iron status in the body.  Hepcidin binds to ferroportin, a cellular iron export channel protein, causing it to be internalized and degraded in lysosomes and preventing the efflux of iron from iron-exporting tissues into the plasma.  Excess of hepcidin leads to dysregulation of iron metabolism in chronic kidney disease (CKD) patients.  Production of hepcidin is induced by excess iron stores and by inflammation, and is suppressed by erythropoietic activity.  It has been hypothesized that measuring serum levels of hep-cidin may be useful as an additional tool for predicting and monitoring the need for iron supplementation. ,, Elevated serum levels of the bioactive 25-amino acid hepcidin isoform, hepcidin-25 (Hep-25), have been consistently reported in dialysis patients, ,,,,,,, probably due to the combination of an impaired renal excretion and an increased formation secondary to inflammation and iron overload.  Because Hep-25 blocks iron release from the macro-phages, its increase may contribute to the disordered iron homeostasis and ESA resistance in uremia by limiting iron availability for erythropoiesis. 
We aimed in the present study to measure serum hepcidin levels in MHD patients and to determine the correlation between hepcidin and parameters of iron metabolism and inflammation in order to assess the role of hepcidin as a biomarker for iron status and inflammation in MHD patients.
| Patients and Methods|| |
We studied 80 MHD patients who had been on standard bicarbonate HD for at least six months and were dialyzed for 3-4 h three times a week; 45 patients used low-flux synthetic membranes (polysulphone, Fresenius) and 35 patients used high-flux membranes.
All the study patients signed informed consent in accordance with the requirements of the Institutional Committee on Human Research, and the study protocol was approved by the Ain Shams University Hospital, Cairo committee.
Patients with malignant disease, chronic inflammatory disease, hemoglobinopathies and severe liver or lung disease, as well as patients using anti-inflammatory or immunosuppressive agents, were excluded from this study. None of the patients had experienced bleeding, hospitalization or blood transfusions in the previous three months.
All enrolled patients were on maintenance intravenous ESA (administered at the end of the dialysis session), aiming at maintain Hb within the range of 10.5-12.5 g/dL. Seventy patients were on epoetin α and 10 patients were on darbepoetin α. Before enrolment, the majority of patients were on maintenance low-dose intravenous iron aiming to maintain ferritin within 200-600 ng/mL and %Hypo <6%. However, none of the patients had received iron within the 12 weeks prior to the evaluation, and there were no changes in their ESA dosage during the 12 weeks prior to the study period.
Studied patients were sub-grouped for analysis according to the result of highly sensitive C-reactive protein (hs-CRP) (Group I: 30 patients with hs-CRP <3 mg/dL; Group II: 50 patients with hs-CRP ≥3 mg/dL).
Blood samples were drawn from the MHD patients upon starting the mid-week HD session. The levels of Hb, urea nitrogen, creatinine and iron were measured by standard laboratory methods using an auto-analyzer. Transferrin was measured by the Nitro-so-PSAP test and TSAT was calculated as serum iron/total iron-binding capacity. The serum levels of highly sensitive hs-CRP were measured by a latex photometric immunoassay. The levels of hepcidin were measured using enzyme-linked immuno-sorbent assay (ELISA) kits (Uscn Life Science Inc - E91979Hu 96 Tests- with a detection range: 62.5-4000 pg/ mL, sensitivity: 22.9 pg/mL), which have high sensitivity and excellent specificity for the detection of human hepcidin. No significant cross-reactivity or interference between human hepcidin and analogues was observed.
| Statistical Analysis|| |
All results are presented as the mean ± SD. Differences between groups were analyzed by the unpaired Student's "t" test. The Mann- Whitney Wilcoxon U test was used instead of unpaired t-test for non-parametric data (SD >50% of the mean). The correlation of clinical and laboratory variables and the serum hep-cidin levels were evaluated using Spearman's rho test. In multiple linear regression models used to investigate the association of biochemical parameters with hepcidin, variable selection was performed by the stepwise method. Statistical analyses were carried out with SPSS version 16.0 (SPSS Inc., Chicago, IL, USA). P-values <0.05 indicated statistical significance.
| Results|| |
The mean age in our study patients was 50.2 ± 15.8 years, and 48.8% of the patients were male. The median duration of HD for all patients was 3.5 years (interquartile range: 1-22). Etiology of CKD in the study patients is shown in [Figure 1].
The hs-CRP levels for group I and group II were medians of 1.9 mg/dL (interquartile range: 0.4-3.0) and 7.65 mg/dL (interquartile range: 3.2-46.0), respectively. Serum hepcidin levels were higher for group II than group I, with medians of 95.9 pmol/L (interquartile range: 16.5-473) and 65.7 pmol/L (interquartile range: 8.6 145.9), respectively (P = 0.008). Serum ferritin levels were higher for group II than group I at medians of 445.5 ug/L (interquartile range 33-2645) and 318.3 ug/L (interquartile range 27-730), respectively (P = 0.027).
The serum hepcidin levels in all MHD patients positively correlated with the levels of ferritin (R = 0.993, P <0.0001; [Figure 2], and they significantly correlated with ferritin levels in group I and group II patients (R = 0.983, P < 0.0001 and R = 0.994, P < 0.0001). In group I, the hepcidin levels significantly correlated with serum iron levels and T.SAT% (P <0.05).
|Figure 2: Scatter plot of correlation between hepcidin and ferritin in all patients (P <0.001).|
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The serum levels of hepcidin significantly correlated with hs-CRP in all the study patients (R = 0.321, P = 0.01). Although patients with infection or chronic inflammatory disease were excluded from this study, the hs-CRP levels of 50 MHD patients were >3 mg/dL, which suggested the presence of possible concealed inflammation; in these patients, the serum hs-CRP levels correlated with the hepcidin levels (R = 0.023, P <0.05).
The means of the Hb levels for group I and group II of the study patients were 10.7 ± 1.0 g/dL and 10.6 ± 1.3 g/dL, respectively [Table 1]; all these patients were treated with rHuEPO. The serum levels of hepcidin did not correlate with Hb (R = -0.03, P = 0.78) or the weekly dose of rHuEPO (R = -0.10, P = 0.38).
Linear regression analysis showed that hep-cidin levels highly significant correlated only with ferritin in all the study patients and subgroups (P <0.001).
| Discussion|| |
We demonstrated a strong correlation between markers of iron storage and serum hep-cidin levels in MHD patients. This likely reflects the known regulation of hepcidin of the iron stores, which was previously demonstrated in populations without CKD.  Previous studies that used mass spectrometry to measure hepcidin also demonstrated a correlation between ferritin and hepcidin in MHD pa-tients. , In contrast, Ashby et al,  using a radioimmunoassay, did not observe this correlation in adult MHD patients; however, target-driven intravenous iron therapy might have confounded those results. Univariate and linear regression analysis demonstrated ferritin as a significant predictor of the serum hepcidin levels.
In the present study, serum TSAT was not found to be a significant predictor of hepcidin levels in regression analysis. On the other hand, a significant correlation between serum ferritin and hepcidin levels was a consistent finding. Serum ferritin is a marker of iron stores in the liver and the reticuloendothelial system as well as being an acute phase protein. A similar observation has been reported in patients with various liver diseases. Fujita et al  demonstrated that the serum ferritin level had a strong positive correlation with the hepatic levels of hepcidin mRNA expression. In cultured cell models, a direct correlation between ferritin and hepcidin expression in hepatocytes has not been demonstrated so far. However, we cannot rule out the possibility that hepcidin primarily regulates the liver iron content, which would in turn regulate serum ferritin levels, because hepatocytes and Kupffer cells also express ferroportin. Further clarification of the correlation between hepcidin regulation and iron storage is needed.
Ashby et al  demonstrated that hepcidin levels (using a radioimmunoassay) were significantly elevated in MHD patients, but did not correlate with ferritin, in contrast with our results, and this could be explained by the fact that the ferritin levels of the patients in the former study were much higher than in ours.
In several clinical conditions, especially iron-deficiency anemia and thalassemia,  erythro-poietic regulation of hepcidin has been shown to be strong. Despite high iron storage levels in thalassemia patients, hepcidin shows a marked decrease in accordance with a higher serum level of sTfR.  However, the erythro-poietic activity of hepcidin does not correlate with its serum levels in dialysis patients, and the interaction between erythropoiesis and hepatic hepcidin synthesis might be abnormal in MHD patients, which is yet to be determined. A prospective longitudinal study is necessary to clarify the relation between serum hepcidin and doses of iron and ESA administration in MHD patients.
Previous studies of humans with chronic infections and severe inflammatory disease have shown markedly increased levels of hepcidin, strongly suggesting that elevated hepcidin levels play a key role in the anemia of inflammation and reticuloendothial blockade. , The correlation between hs-CRP and hepcidin in our study provides further evidence in support of the relationship between both variables through all stages of CKD.  Hepcidin may mediate the deleterious effects of inflammation on both iron homeostasis and erythropoiesis. It is important to note that the protocol of our study excluded patients with active illness or infection; therefore, a more robust correlation between inflammation and hepcidin may exist in MHD. In our study, in contrast to what is described previously, , serum hepci-din correlated with the inflammatory marker hs-CRP. Among several regulatory mechanisms, the effects of acute inflammation on the synthesis of hepcidin may be mediated at least partly by IL-6 through induction by the binding of a signal transducer and activator of transcription (STAT)-3 to the hepcidin promoter. 
We are aware that our study had some limitations, as it was a single-center study on a small sample of patients; therefore, it may be under-powered for evaluating the role of the different biomarkers in predicting iron status.
We conclude that serum hepcidin levels correlated with serum ferritin levels (an iron storage parameter) in MHD patients, which may have important diagnostic and potentially therapeutic implications. Hepcidin may, in the future, improve the targeting and timing of iron therapy by identifying patients during periods of reticuloendothelial blockage of iron transport, when they would likely not benefit from iron therapy.
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Dr. Dawlat Sany
University of Ain-Shams, Division of Renal Diseases, Cairo
[Figure 1], [Figure 2]