In this cross-sectional study in a cohort of stable prevalent HD patients, hepcidin-25 levels were shown to be independently and positively associated with iron stores (as reflected by ferritin levels), inflammation (hsCRP) and the presence of diabetes, and inversely with erythropoiesis (sTfR and reticulocyte count), residual kidney function (eGFR) and male gender. Of note, no relations between hepcidin-25 and either ESA dose or iron supplementation were observed.
In our study, ferritin was the strongest determinant of hepcidin, which has been well established before in healthy controls 
, CKD patients 
and in patients with ESRD treated with HD and peritoneal dialysis 
. Notably, the studies in HD patients included mostly low patient numbers. As can be seen from , the relation between hepcidin-25 and ferritin was present irrespective of the level of inflammation. However, whether hepcidin is upregulated in response to increased ferritin levels cannot be concluded from our study.
sTfR has proven to be a valuable tool to assess bone marrow erythropoietic activity and iron stores in HD patients treated with ESA 
. However, it could not predict a response of intravenous iron administration on the hemoglobin level 
. In our study, an inverse association between either sTfR and reticulocyte count, and hepcidin levels was observed, after multivariable adjustments. Whether low hepcidin levels enhance erythropoiesis, or whether increased bone marrow erythropoietic activity suppresses expression of hepcidin, cannot be concluded from this cross-sectional study.
We showed a strong association between hepcidin-25 and hsCRP, but not with IL-6. Several studies have demonstrated a relation between CRP 
or IL-6 
in small groups of chronic HD patients, whereas others did not 
. The explanation for the association between hepcidin-25 and hsCRP, and not IL-6, is not readily apparent, especially as transcription of hepatic hepcidin is activated by binding of IL-6 to its receptor complex 
. However, in a murine and human model investigating various factors associated with hepcidin expression, the role of IL-6 was limited 
. Furthermore, the IL-6 assay used in our study showed a wide inter-assay variability, especially in the lower range. This could have resulted in less accurately measured values of IL-6 as compared to hsCRP, and hence less precision in the estimation.
We are the first to report an independent association between eGFR and both the active hepcidin-25 and the inactive isoform hepcidin-20 in HD patients. As we used a mass-spectrometry assay that specifically measures hepcidin-25, our results indicate that the observed association between eGFR and hepcidin-25 was not due to the concurrent measurement of inactive isoforms. To date, studies on the association between eGFR and hepcidin levels in CKD patients have been conflicting 
. Low hepcidin levels (measured with a radioimmunoassay) were reported in HD and PD patients with residual diuresis 
, but RKF was not quantified in that study. Whether the high hepcidin levels in chronic HD patients were exclusively caused by decreased renal clearance, or whether other mechanisms are involved, cannot be concluded from our data.
In our study population, hepcidin-25 levels were significantly lower in men as compared to women. This can be explained by the fact that most women in our study will be post-menopausal, in whom higher hepcidin levels have been demonstrated 
. Furthermore, we showed that diabetic patients had higher hepcidin levels. In one study, diabetic patients had higher levels of hepcidin than healthy age-matched controls, although this relation was not adjusted for possible confounders 
Interestingly, we did not observe an interaction between ESA dose and hemoglobin levels in relation to hepcidin-25 as has been demonstrated before by Ashby et al 
. Therefore, it appears that hepcidin, measured with a mass spectrometric assay in chronic HD patients on maintenance therapy with ESA, is not a marker of ESA resistance. Whether hepcidin-25 can predict an ESA response, as has been shown in patients with the cardio-renal syndrome 
, cannot be concluded from our cross-sectional data. Nevertheless, in a study in 24 HD patients, hepcidin levels of ESA responsive patients did not differ from those who were ESA resistant 
, which is in accordance with our results.
Concerning iron supplementation in HD patients, various effects of iron loading on hepcidin levels have been reported 
. We did not observe a relationship between hepcidin and iron supplementation, which can be explained by the fact that patients in our study received maintenance iron therapy instead of a (single) loading dose. Recently, it was shown that hepcidin-25 levels did not predict a response to the administration of intravenous iron supplementation in HD patients on ESA maintenance therapy 
. Hence, it appears that hepcidin is more a marker of iron stores than a predictor of the effect of iron therapy.
A number of studies showed that hepcidin levels could be lowered over a single HD session 
, although concentrations were back to baseline only one hour after the treatment 
. Lowering of hepcidin by HD can be partly explained by appearance of (low) levels of hepcidin in the ultrafiltrate 
. In addition, it has been shown that hepcidin can attach to the membrane of the dialyzer 
, which can be explained by the amphipathic and protein-bound structure of hepcidin 
. Prospective research is needed to draw any conclusions on the effect of different dialyzers on hepcidin-levels.
Limitations and Strengths
Our study is limited by its cross-sectional design, which impedes assessing causal relationships. Furthermore, a specific treatment protocol for ESA and iron administration and timing of blood sampling was not provided. We tried to compensate for this by adjusting the regression models for participating center in an additional analysis, as the intervals between ESA and/or iron administration and blood sample withdrawal are supposed to be similar within a single treatment center. Since this did not change our results, we conclude that the dosing schedule was not a major confounder in our study. Another potential limitation of our study is the patient selection, based on centers where blood sampling and storage was logistically feasible. This might have introduced a selection bias of which the magnitude and direction cannot be estimated. As selective participation or non-participation must have occurred based on a logistical aspect, which is most probable not related to factors associated with hepcidin or determinants of hepcidin, selection bias seems unlikely.
The strength of our study is the large sample size and the prospective data collection. As far as we know, our study comprises the largest cohort of HD patients in which hepcidin measurements were performed, currently published. The large sample size facilitates multivariable statistics, which is an important method when examining the complex regulation of hepcidin 
. Moreover, hepcidin measurements have been performed with a validated mass spectrometric technique, enabling specific quantification of the bioactive hepcidin-25.
In this cohort of chronic, stable HD patients, hepcidin-25 levels were independently associated with iron stores (as reflected by ferritin levels), erythropoiesis (reticulocyte count and sTfR), inflammation (hsCRP), eGFR, the presence of diabetes and gender. Hepcidin-25 was strongly correlated with its bio-inactive isoform hepcidin-20, and similar associations with hepcidin-20 were identified. Of note, hepcidin-25 was not associated with the maintenance dose of ESA or iron therapy.
Our findings confirm the role of hepcidin as a biomarker of iron stores and erythropoiesis in chronic HD patients and indicate that hepcidin is not a biomarker of ESA resistance in patients on ESA maintenance therapy. Furthermore, it underscores the potential important role of (limited) RKF in HD patients. However, whether low hepcidin levels in HD patients are associated with a favorable outcome in terms of morbidity and mortality is not clear yet. Furthermore, whether hepcidin measurements in HD patients provide additional information concerning anemia management compared to current available markers such as ferritin is questionable.