Predictors of Serum Hepcidin Levels in Patients on Maintenance Hemodialysis Reference: Kuragano T, Shimonaka Y, Kida A, et al. Determinants of hepcidin in patients on maintenance hemodialysis: Role of infl ammation. Am J Nephrol. 2010;31:534–40.

Introduction Iron is an essential element and plays a vital role in oxygen transport by hemoglobin (Hb) and myoglobin; and by contributing to several enzymatic activities involved in mitochondrial respiratory chain and DNA synthesis in mammalian species. In cells which damage systems, vital for oxidation, free iron plays an important role as a pro-oxidant cofactor associated with hydroxyl radical production. Moreover, free-iron becomes important for bacterial growth. This may impact virulence to the host and may be due to the physiology of iron sequestration in the face of infection. Hence, iron regulation under physiological conditions becomes essential. Dysregulation of iron metabolism often reported in patients with chronic kidney disease (CKD) may lead to anemia of chronic disease (ACD) (see Fig. 1).

Hepcidin Hepcidin is a recently discovered small peptide that circulates in the plasma. It is produced by hepatocytes and plays a central role in iron regulation. It prevents the efflux of iron from iron-exporting tissues into the plasma by binding to ferroportin and causing it to internalize and degrade in lysosome. However, hepcidin has emerged as a key pathogenic feature in ACD in CKD patients because it is believed that iron metabolism dysregulation in CKD patients is as a result of excess of hepcidin.

Hepcidin Regulation While excess iron stores and infl ammation induces the production of hepcidin, erythropoietic activity suppresses it. These regulatory processes are crucial to the issue of ACD in CKD and dialysis patients. Another potential mechanism for regulating levels of serum hepcidin in CKD patients is its excretion in urine and removal by hemodialysis (HD). Accumulation of hepcidin is evident in patients with renal failure, suggesting the metabolic importance of renal clearance of hepcidin. Moreover, hepcidin should be effi ciently cleared by HD and isolated from plasma ultrafi ltrate owing to its very small molecular size.

Maintenance Hemodialysis and Serum Hepcidin Levels The study enrolled 198 maintenance hemodialysis (MHD) patients, on at least 1 year standard bicarbonate HD, dialyzed for 3–4 h three times a week; and 33 healthy human volunteers (HHV) from the medical staff with no evidence of any chronic disease. On initiating the fi rst HD session for the week, blood samples were drawn from the MHD patients and several laboratory investigations were performed (see Table 1). Response to treatment was calculated with ESA resistance index. Liquid chromatography tandem mass spectrometry (LC-MS/MS) was used to determine the serum hepcidin level (see Fig. 2), which was measured at the beginning and end of HD in 10 MHD patients to evaluate the changes in hepcidin level during single dialysis session. Hepcidin levels were also measured at 60 and 180 minutes after the HD session and at the start of session. Moreover, for each individual patient, the ultrafi ltrate volume was determined.

The baseline characteristics of the subjects enrolled in the study are detailed in Table 2. The Hb level ranged from 7.9 to 13.4 g/dL in MHD patients. While the mean serum hepcidin levels were signifi cantly high in MHD patients when compared with HHVs, no signifi cant difference in serum levels of transferrin, unsaturated iron-binding capacity, TSAT, and ferritin was observed. Linear regression analysis for identifying the potential association between hepcidin and iron parameters or infl ammatory parameters showed that serum hepcidin level was positively correlated with iron levels and negatively correlated with unsaturated iron binding capacity and transferrin. A signifi cant correlation of hepcidin and ferritin levels was demonstrated in HHV and MHD patients. However, no difference in the ferritin/hepcidin ratio was appreciated between the two groups.

While serum hepcidin levels were correlated with iron parameters and sTfR, no correlation was cited with age, uremia-related factors, Hb, dose of erythropoietin, or the indexes of infl ammation. On investigating predictors of serum hepicidin levels, ferritin and transferring level showed to be signifi cant determinants in contrast with IL-6, TNF-α, sTfR and hCRP which were not signifi cant. Despite no evident correlation between hepcidin levels and IL-6, TNF-α or hCRP; the levels of IL-6 and TNF-α were signifi cantly higher in patients with MHD. Further, the high levels of hCRP in MHD patients were suggestive of microinfl ammation.

Moreover, signifi cant correlation between IL-6 and hepcidin level was demonstrated in patients with hCRP levels ≥0.3 mg/dL (see Fig. 3). All patients were treated with rHuEPO and sTfR levels were signifi cantly higher in MHD patients. Serum hepcidin levels were negatively correlated with sTfR, but not with Hb or the ESA resistance index. The serum hepcidin levels decreased signifi cantly after HD. However, the basal levels were achieved 1 h after HD and remained constant until the next HD session.

Hepcidine Levels in patients with MHD The above detailed study demonstrates the following” – Higher and relatively stable serum hepcidin levels in MHD patients – Decreased renal removal does not explain the high level of hepcidin in MHD patients – Hepcidine levels are principally dependent on iron store index and serum ferritin levels – Hypercytokinemia is not linked to serum hepcidin levels – Erythropoietic activity is not closely related with hepcidin levels in dialysis patients

Predictors of Hepcidin Levels Ferritin and transferrin are signifi cant predictors of serum hepcidin levels. In vitro studies have identifi ed the regulation of hepcidin mRNA expression by holotransferrin concentration via a hemojuvelin/ bone morphogenetic protein 2/4-dependent pathway. Positive correlation of ferritin and hepatic hepcidin mRNA expression is also demonstrated. Moreover, Serum ferritin is an acute phase protein and marker of iron stores in liver and RES. Association of hepcidin and ferritin level is also observed in studies reporting patients with several liver diseases. In a recent study, hepcidin levels have shown signifi cant elevation in MHD patients when radioimmunoassay were used. But this study did not demonstrate the correlation with ferritin levels.

Strong erythropoietic regulation of hepcidin has been evident in several conditions like iron-defi ciency anemia and thalessemia. However, higher sTfR levels decrease the hepcidin levels in thalassemia, despite high iron storage levels. Further, in MHD patients, sTfR level or weekly rHuEPO dose was not identifi ed as a signifi cant determinant. Further, the hepcidin levels in MHD patients were not affected by IL-6 or TNF-α level. However, the expression of hepcidin was associated with serum IL-6 in condition of microinfl ammation. In MHD patients without infl ammation, hepcidin might be involved in ACD. Through iron sequestration in the RES in patients with higher hCRP levels, hepcidin could exclusively cause ACD.

Conclusion In MHD patients without infl ammation, serum hepcidin levels faithfully refl ect on to serum ferritin levels. Impairment of renal function or accumulation of uremic substances might not affect the relation between hepcidin and iron storage. IL-6 might infl uence hepcidin expression in case of microinfl ammation. Hence, intravenous iron administration could be a major cause for the rise in serum hepcidin levels in MHD patients.

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