Impact of hepcidin, interleukin 6, and other inflammatory markers with respect to erythropoietin on anemia in chronic hemodialysis patients

Background/objective Hepcidin is a peptide hormone produced by the liver and appears to be the master regulator of iron homeostasis. This peptide is upregulated in inflammatory conditions, including uremia. Hepcidin functions to regulate (inhibit) iron transport across the gut mucosa, thereby preventing excess iron absorption and maintaining normal iron levels within the body. In this study, we aimed to investigate hepcidin levels and their relationship with the parameters of iron status, inflammation, anemia therapy, and parameters of dialysis efficiency in hemodialysis patients. Patients and methods Plasma hepcidin-25, inflammatory markers (high-sensitivity C-reactive protein and interleukin 6), and peripheral iron indices (serum iron, total iron-binding capacity, transferrin saturation and serum ferritin) were measured before hemodialysis in 40 end-stage renal disease (ESRD) patients treated with regular hemodialysis in a single dialysis unit as well as in 20 healthy individuals matched for age and sex serving as the control group. Results Plasma levels of hepcidin-25 were significantly higher in hemodialysis patients compared with controls. In a simple correlation analysis, plasma hepcidin levels were positively correlated with ferritin, transferrin saturation, CRP, and interleukin 6; however, it was negatively correlated with hemoglobin, dose of epoitin-α, and dose of iron. Conclusion Serum hepcidin levels were associated with iron status and inflammation in maintenance hemodialysis patients, and the high hepcidin serum levels, found in hemodialysis (HD) patients, are dependent on the magnitude of the inflammatory process and on recombinant human erythropoietin doses. Hepcidin and its regulatory pathways are potential therapeutic targets, which could lead to effective treatment of anemia in chronic hemodialysis.


Introduction
Anemia is a common complication in maintenance hemodialysis patients and contributes to reduced quality of life [1]. Despite the great success of recombinant human erythropoietin (EPO) in clinical practice for the treatment of anemia in dialysis patients [chronic kidney disease (CKD) patients], resistance to this therapy is ∼10-20% [2,3].
Hepcidin is a small defensin-like peptide produced largely by the liver but also by other cells, such as the macrophage and adipocyte [4]. In addition to its antimicrobial properties [5], it is the master regulator of iron metabolism, controlling the amount of dietary iron absorbed from the duodenum and also the release of iron from cells in the reticuloendothelial system (Kupffer cells, splenic macrophages, etc.) [6][7][8]. At a molecular level, hepcidin binds to the main iron exporter protein ferroportin, which controls iron efflux from duodenal enterocytes, hepatocytes, and macrophages suppressing iron absorption from the intestine and stimulating iron retention in macrophages and hepatocytes [9]. The key role of hepcidin in iron homeostasis and its disorders suggests that its assay in blood or urine could prove useful for the diagnosis and monitoring of iron disorders, and therefore potentially be used as a clinical marker to optimize treatment approaches [10,11].
Hepcidin levels are regulated in response to iron, erythropoietic demand, hypoxia, and inflammatory signals [7]. Iron administration upregulates hepcidin, thereby providing a feedback mechanism to limit further iron absorption, whereas anemia, iron deficiency, and hypoxia inhibit hepcidin, increasing iron availability for erythropoiesis. Hepcidin is also induced by inflammation, which is believed to be part of the host defense mechanism to fight infection and cancer by limiting iron availability to the bone marrow [6,8].

Impact of hepcidin, interleukin 6, and other inflammatory markers with respect to erythropoietin on anemia in chronic hemodialysis patients
The purpose of the present cross-sectional study was to investigate hepcidin levels in maintenance hemodialysis patients and their relationship with the parameters of iron status, inflammation, anemia therapy, and parameters of dialysis efficiency.

Participants and research design Eligibility
All patients were already on maintenance hemodialysis. The inclusion criteria for the studied patients were: (i) Chronic renal failure from any cause, (ii) Age more than 18 years and less than 70 years, (iii) Both sexes were involved, (iv) Dialysis duration more than 6 months, and (v) All patients treated by standard bicarbonate dialysis for 4 h, three times a week. Hemoglobin was measured monthly, and transferrin saturation (TSAT) index and ferritin were measured every 3 months. The target was to reach a hemoglobin value of at least 10.5 g/dl in more than 85% of patients. None of the patients received parenteral iron 10 days before enrollment. The last three epoitin and iron doses were recorded, and mean values were used in the analysis.

Study protocol and assessments
Protocol determinations were all carried out on the same day and included height, body weight, BMI (weight in kg/squared height in m 2 ), and systolic and diastolic blood pressure. All clinical examinations and evaluations were conducted under fasting conditions.
The quality of dialysis was assessed during the study period by calculating Kt/V for all the patients. Kt/V is defined as the dialyzer clearance of urea and was calculated according to the Daugirdas second generation formula [12].
Blood sampling was taken just before hemodialysis. Fasting blood samples (12 h) were obtained from controls in the morning as well as from hemodialysis patients before the first dialysis session of the midweek (mean Kt/V, a marker of dialysis adequacy, was 1.47 ± 0.3).

Biochemical assessments Sample collection and handling
Fasting blood samples (12 h) for laboratory analysis were collected on the same day as the examination of the patients. Blood samples were collected in EDTA tubes and centrifuged at 2000g at 2-8°C for 10 min within 30 min of sampling. The plasma was stored in aliquots at -70°C. Repeated freeze-thaw cycles were avoided.
To clarify the relationship of plasma hepcidin to the demographic, clinical, and biochemical characteristics among the hemodialysis patients, we subdivided the hemodialysis patients according to the median of hepcidin levels (calculated as 301 ng/ml) into two subgroups: subgroup 1, with hepcidin less than 301 ng/ml (lower hepcidin level) and subgroup 2, with hepcidin more than 301 ng/ml (higher hepcidin level). Table 4 shows the main clinical characteristics of the two subgroups where the duration of dialysis, TSAT, ferritin, hepcidin, hsCRP, IL-6, and spKt/V were significantly higher, whereas hemoglobin, TIBC, UF volume, weekly epoitin-a dose, and weekly iron dose were significantly lower in subgroup 2 than in subgroup 1 (Table 4 and Figs 2 and 3).
No correlations were observed between serum hepcidin and the various other clinical and laboratory parameters measured (Table 5 and Figs 4-8).

Discussion
Anemia in maintenance hemodialysis patients is a multifactorial condition and its clinical management remains challenging. The interactions between iron metabolism, EPO deficiency, and chronic inflammation are difficult to dissect and new markers are urgently needed to optimize treatment approaches [3].
Hepcidin was originally described as an antimicrobial peptide produced by the liver [5], but its main biological role is in the regulation of body iron homeostasis through interactions with ferroportin [6,7]. Hepcidin might, therefore, potentially be used as a clinical marker to optimize treatment approaches in several systemic iron disorders. It suppresses iron absorption from the intestine and stimulates iron retention in cells expressing ferroportin, notably macrophages and hepatocytes [11].
In agreement with previous data [3,20,21], the present study demonstrated that serum hepcidin levels were higher in maintenance hemodialysis patients than in healthy control individuals. The observation that there was a significant and independent correlation between hepcidin and ferritin levels suggests that it plays a major role in regulating iron homeostasis in this patient group. Our results also agreed with the results of Babitt et al. [8] who reported that hepcidin levels are likely to be higher in CKD patients due to limited hepcidin excretion, tissue iron overload, and inflammation.
This likely reflects the known regulation of hepcidin by iron stores, which was previously demonstrated

Figure 1
Comparison between plasma levels of hepcidin in hemodialysis patients and healthy controls.
in populations without CKD [22]. Previous studies that used mass spectrometry to measure hepcidin also demonstrated a correlation between ferritin and hepcidin in HD patients [23,24]. In contrast, Ashby et al. [25], using a radioimmunoassay, did not observe this correlation in HD patients. However, a significant relationship between serum ferritin and hepcidin levels Data are expressed as mean ± SD; Ca, calcium; Hb, hemoglobin; HDx, hemodialysis; hsCRP, high-sensitivity C-reactive protein; IL-6, interleukin 6; iPTH, intact parathormone; spKt/V, single pool urea clearance index (used to represent weekly dialysis dose, where K is the clearance of urea by the dialyser, t is the dialysis time, and V is the volume of distribution of urea); TIBC; total iron-binding capacity; TSAT, transferrin saturation; UF; ultrafiltration; *P-value is significant if <0.05.

Figure 4
Correlation between plasma hepcidin and high-sensitivity C-reactive protein (hsCRP) in hemodialysis patients.
is a consistent finding. Serum ferritin is a marker of iron stores in the liver and reticulo-endothelial system (RES) as well as being an acute phase protein. Fujita et al. [26] demonstrated that the serum ferritin level had a strong positive correlation with the hepatic level of hepcidin mRNA expression. However, it cannot be ruled out that hepcidin primarily regulates the liver iron content, which would in turn regulate serum ferritin levels by hepatic iron content, because hepatocytes and Kupffer cells also express ferroportin [27]. Further clarification of the relationship between hepcidin regulation and iron storage is needed.
In in-vitro studies, it has been observed that the transferrin concentration regulates hepcidin mRNA expression through a hemojuvelin/bone morphogenetic protein (BMP)-2/4-dependent pathway [28], which is thought to be the main signaling pathway for iron storederived and erythropoiesis-derived regulation [29]. In the present study, serum iron and TSAT were not significant predictors of hepcidin levels in multivariate analysis.
Patients undergoing continuous dialysis are in a chronic inflammatory state. In our study, all inflammation markers including hsCRP and IL-6 were found to be higher in hemodialysis patient compared with healthy controls. The causes of highly prevalent state of inflammation in HD patients are multiple, including decreased renal function, volume overload, comorbidity, and intercurrent clinical events [30]. The effects of inflammation on the synthesis of hepcidin are well understood [9,22] and are mediated, at least in part, by IL-6 through induction and binding of signal transducer and activator of transcription 3 (STAT3) to the hepcidin gene promoter [31]. There was a significant positive correlation between serum IL-6 and serum hepcidin levels in the present study, which would seem to support this proposed mechanism. In addition, there was a significant positive correlation between serum hsCRP and serum hepcidin levels. Our findings are supported by Costa et al. [32] who found that the high levels of hepcidin found in HD patients could be related to an underlying chronic inflammation. Moreover, correlations between hepcidin and inflammatory markers (CRP and IL-6) in the HD group are similar to observational data derived from the nonuremic population [9,22]. It was Zaritsky et al. [33] who found that the correlation between hsCRP and hepcidin provides further evidence in support of the relationship between both variables through all stages of CKD. Van Der Weerd et al. [34] showed a strong association between hepcidin-25 and hsCRP but not with IL-6 in chronic HD patients.
Our findings are in disagreement with the findings of Ashby et al. [25] who found no significant correlation between serum hepcidin levels and inflammatory markers (hsCRP and IL-6) in adults with CKD and hemodialysis patients. In addition, Kato et al. [23] found no association between hsCRP and hepcidin in hemodialysis patients, but this may be because of the different assay method.
It is now recognized that uremia, as a chronic inflammatory state, upregulates hepcidin, and in particular, dialysis patients have much higher serum Ca, calcium; Hb, hemoglobin; hsCRP, high-sensitivity C-reactive protein; iPTH, intact parathormone; IL-6, interleukin 6; spKt/V, single pool urea clearance index (used to represent weekly dialysis dose, where K is the clearance of urea by the dialyser, t is the dialysis time, and V is the volume of distribution of urea); TIBC, total iron-binding capacity; TSAT, transferrin saturation. hsCRP, high-sensitivity C-reactive protein; IL-6, interleukin 6; spKt/V, single pool urea clearance index (used to represent weekly dialysis dose, where K is the clearance of urea by the dialyser, t is the dialysis time, and V is the volume of distribution of urea); TIBC, total iron-binding capacity; *P-value is significant if <0.05

Figure 7
Correlation between plasma hepcidin and ferritin in hemodialysis patients and controls.

Figure 6
Correlation between plasma hepcidin and transferrin saturation (TSAT) in hemodialysis patients.

Figure 8
Correlation between plasma hepcidin and hemoglobin (Hb) in hemodialysis patients.
hepcidin levels than healthy individuals [35]. It is currently believed that this has a role in the pathogenesis of anemia in CKD by limiting iron availability to the bone marrow. At a molecular level, hepcidin binds to the main iron exporter protein ferroportin, which controls iron efflux from duodenal enterocytes, hepatocytes, and macrophages [9]. The regulation of hepcidin is complex, but one of the major stimuli to its production is IL-6, produced as part of the inflammatory response.
Other molecules, such as hemojuvelin and BMP-6, also have a role [36].
As with other inflammatory anemias, it has been hypothesized that antagonizing hepcidin may ameliorate the anemic state, and there is laboratory evidence to support this assumption. A group of scientists generated a monoclonal antibody against hepcidin and have shown that this improves anemia in an inflammatory mouse model [37]. An RNAbased antagonist of hepcidin also has been created.
It consists of a 44-nucleotide l-RNA oligonucleotide produced using so-called Spiegelmers technology (RNA molecules in which the ribose component is levorotatory, or the mirror image of the natural righthanded sugar moiety). The Spiegelmer is linked to a 40-kDa pegylation chain (NOX-H94), which has been shown to ameliorate anemia of inflammation in cynomolgus monkeys [38]. Rather than antagonizing the hepcidin molecule per se, another strategy could be to inhibit the production of hepcidin. This could be achieved by using antisense oligonucleotides or silencing messenger RNA transcribed from the hepcidin gene hepcidin antimicrobial peptide (HAMP). None of the strategies to suppress hepcidin production or antagonize this peptide have been subjected to clinical trials. A theoretical concern could be that inhibition of hepcidin might exacerbate the risk for infections, given its endogenous antimicrobial properties. However, there are counterarguments to this suggestion, and it may be possible to suppress hepcidin to 'safe' levels without obliterating hepcidin activity completely [39].
In the present study, we found that there was a significant negative correlation between serum hepcidin levels and weekly iron and EPO doses. The finding that EPO treatment decreases serum hepcidin levels in patients with chronic renal failure (CRF) is in accordance with the fact that increased erythropoiesis leads to suppression of hepcidin [29]. Costa et al. [32] reported that EPO downregulates liver hepcidin expression, acting, therefore, as a hepcidin-inhibitory hormone. The pathways through which EPO treatment modulates hepcidin levels are largely unknown [32].
Pinto et al. [40] suggested that it concerns a direct effect, mediated by a decrease in CEBPA binding to the hepcidin promoter after EPO supplementation. Alternatively, EPO administration may indirectly lead to suppression of hepcidin by increased levels of growth differentiation factor-15, which is secreted by erythroblasts [41], or by the molecule named twisted gastrulation (TWSG1). TWSG1 is expressed during erythropoiesis and acts by inhibiting BMP-induced expression of hepcidin [42]. Although we studied a relatively small and heterogeneous patient population, our results with respect to the hepcidin decrease in response to EPO are unambiguous. It seems that the signaling pathway that connects EPO with hepcidin is not markedly influenced by systemic disturbances seen in patients with kidney failure.
In our study, we found a positive correlation between spKt/V and serum hepcidin level. This unexpected finding was in contradiction with the findings of Zaritsky et al. [43] who reported that hepcidin could be cleared efficiently by hemodialysis as it is a very small molecule. This finding was consistent with other studies [20,25] that showed no reduction following a standard dialysis session. The cause of this variability remains unclear but might be attributable to differences in the membrane of the dialyser, residual renal function, or induction of hepcidin by the hemodialysis procedure [44]. Future studies will need to address the kinetics of hepcidin clearance during dialysis. Formal kinetic modeling of hepcidin clearance will allow for estimation of the generation rate and volume of distribution of hepcidin and would be valuable for predicting hepcidin removal through various dialysis regimens.

Conclusion
Our analyses suggest that serum hepcidin levels were associated with iron status, microinflammation, anemia therapy, and parameters of dialysis efficiency in maintenance hemodialysis patients. Availability of reliable, harmonized, and preferably automated assays (e.g. competitive ELISA) should become available for routine measurement of hepcidin before hepcidin can be used in clinical practice. The key role of hepcidin in iron homeostasis and its disorders suggests that its assay could prove useful for the diagnosis, monitoring, and prognosis of iron disorders, and therefore potentially be used as a clinical marker to optimize treatment approaches. However, large and well-designed studies exploiting harmonized assays are required to firmly establish the position of Hepcidin in diagnostic medicine. Finally, hepcidin-targeted therapies may improve treatment options for patients suffering from iron disorders.