European Journal of Cardiovascular Medicine

Hepcidin is a central regulator of systemic iron homeostasis. Serum iron levels are determined by the balance of iron entry from intestinal absorption, macrophage iron recycling, and mobilization of hepatocyte stores versus iron utilization, primarily by erythroid cells in the bone marrow. A peptide hormone secreted by the liver, hepcidin controls iron release into the plasma by downregulating cell-surface expression of the iron export protein ferroportin (FPN) on absorptive enterocytes, macrophages, and hepatocytes. Hepcidin production is inhibited by erythropoietic drive and hypoxia to ensure iron availability for erythropoiesis. Hepcidin production is stimulated by iron (through the hemochromatosis proteins HFE, hemojuvelin [HJV], and transferrin receptor 2 [TFR2]) as a negative feedback loop to maintain steady-state iron levels. Hepcidin production also is stimulated by inflammation, thereby sequestering iron from invading pathogens in the setting of infection, but also causing the hypoferremia of anemia of chronic disease (1).

Following transplantation, anemia corrects in an orderly manner with restoration of the normal biofeedback process between erythropoietin and red cell mass. As early as approximately 2 months after transplantation, a normal hematocrit might be expected in patients without graft failure and iron deficiency (2). This process is delayed by failure of graft to function initially and interrupted by acute early rejection (3,4,5). Hepcidin levels have also been found to have increased in many kidney transplant recipients with poor graft function (6,7). IL-6, a major regulator of hepcidin production, has been shown to be elevated immediately posttransplant and in the setting of acute rejection (8, 9).

Acute rejection results in a sharp decrease in erythropoietin production and anemia at the early stage of renal transplantation. Transplant rejection also causes systemic inflammatory response syndrome in renal transplant recipients. In pediatric renal allograft recipients with acute rejection, an “erythropoiesis cluster” of 11 genes involved in hemoglobin transcription and synthesis, iron and folate binding, and transport were found to be downregulated. Thrombotic micro-angiopathy (TMA) that may develop during episodes of severe vascular rejection, is another mechanism for the development of post-transplant anemia during rejection. Once acute rejection is controlled, the Erythropoietin secretion can be restored to normal level.

Episodes of acute rejection also have correlated with an average decrease of 0.5 g/dL in the Hemoglobin concentration, which may be due to decreased Erythropoietin levels (10). In a study by Vanrenterghem Y et al showed that transplant recipients who experienced rejection episodes or received more than one transplant have been reported to have a higher incidence of anemia (11). The underlying factors causing anemia in the setting of rejection may include suboptimal kidney function, more intensified immunosuppression, acute inflammation, or perhaps a chronic inflammatory state leading to Erythropoietin resistance. Patients returning to hemodialysis following failure of their kidney transplant suffer from a chronic inflammatory state that is associated with resistance to Erythropoietin stimulating agents (12). Hepcidin levels have also been found to be increased in many kidney transplant patients, and elevated levels correlate with poor transplant function, higher ferritin, and other inflammatory markers. Interleukin-6, a major regulator of hepcidin production, has been shown to be elevated immediately post-transplant and in the setting of acute rejection.

Hepcidin is synthesized in the liver as an 84–amino acid prepropeptide and processed by peptidase cleavage to a 60–amino acid propeptide (prohepcidin), followed by furin and related proprotein convertase cleavage to yield the mature carboxy terminal 25–amino acid hepcidin (hepcidin-25). Hepcidin-25 is a cationic peptide that forms a hairpin loop stabilized by four disulfide bonds. In addition to prohepcidin and hepcidin-25, 22 and 20 carboxy terminal amino acid forms of hepcidin are found in the circulation and/or urine, most likely because of N- terminal truncation of hepcidin-25, although the mechanism by which hepcidin-22 and hepcidin- 20 are generated is still poorly understood. Hepcidin-25 is the bioactive form of hepcidin, whereas the other forms of hepcidin have little or no biological activity. Fractional excretion of hepcidin is reported to be approximately 3 to 5% because it either is not freely filtered or it is reabsorbed, similar to other small peptides that are reabsorbed and degraded in proximal tubules. Hepcidin recently was reported to bind specifically to α2-macroglobulin, and it has been estimated that approximately 89% of circulating hepcidin is protein bound.

The development of immunochemical methods to detect mature hepcidin has been complicated by hepcidin’s small size and its conservation in animal species (13). The first described hepcidin assay was an immunodot assay to measure urinary hepcidin (8,14). However, this assay is semiquantitative, laborious, and not suitable for serum hepcidin measurements (13).

A commercially available immunoassay was developed to detect serum prohepcidin, but prohepcidin levels do not correlate with biological activity, iron status, or inflammation. Others have developed mass spectroscopic techniques to measure mature hepcidin in serum and urine. Although this technique has the potential advantage of being able to distinguish among hepcidin-25, hepcidin-22, and hepcidin-20, these assays depend on expensive equipment that is not widely available, and most are semiquantitative, although more recent refinements are improving the quantitative ability (13).

Recently, immunoassays to quantitate mature serum hepcidin have been developed, as well as an assay based on competition against hepcidin-25 labeled with iodine 125 binding to a peptide identical to the ferroportin hepcidin-binding site (15). Some of the problems with the current hepcidin assays are comparing different assay methods and the lack of standardization among methods. The most informative assays determine hepcidin-25 alone, and can be used on blood samples. Presently these assays include surface enhanced laser desorption / ionization time-of- flight mass spectrometry (SELDI-TOF MS), and enzyme-linked immunosorbent assays (ELISA).

The study was carried out to establish a correlation between serum Hepcidin level with renal transplant graft function. It was a Single Centre, Prospective Observational study. All patients with chronic kidney disease stage 5 who were going for renal transplant surgery were included in this study. Serum Hepcidin level was measured just before renal transplant surgery, 1-month after renal transplant surgery and 3-month after renal transplant surgery.

Statistical Analysis: After data collection, data entry was done in Excel. Data analysis was done with the help of SPSS Software version 15. Quantitative data was presented with the help of mean, standard deviation and median. For multiple group comparison of quantitative data, oneway RM ANOVA test was used with Holm-Sidak method for inter-group comparison. Comparison among study groups was done with Unpaired T test. Qualitative data was presented with the help of Frequency and Percentage table. Association among various study parameters were assessed by Chi-Square test (Fisher Exact test for 2 x 2 tables). Correlation between different parameters was analyzed using Pearson Correlation. The significance threshold of p-value was set at < 0.05.

Delayed graft function (DGF) defined as the requirement of dialysis within seven days of the transplant due to the poor function of allograft kidney.

Acute rejection episode (ARE) defined as an acute deterioration in allograft function associated with specific histopathologic changes in the allograft biopsy according to the Banff classification. It can be classified into 2 groups. Acute T-cell mediated rejection (TCMR) diagnosed by lymphocytic infiltration of the tubules, interstitium, and sometimes the arterial intima on kidney biopsy. Antibody-mediated rejection (ABMR) diagnosed by evidence of circulating donor-specific alloantibodies and immunological evidence of antibody-mediated injuries to the kidney (such as C4d deposition), like inflammation of glomeruli (Glomerulitis) or peritubular capillary (peritubular capillaritis).

Acute allograft dysfunction (AAD) defined as an increase in serum creatinine of ≥25 % from baseline within a one-to-three-month post-transplant period, failure of the serum creatinine to decrease following transplantation or proteinuria >1 g/day.

Principle of Hepcidin Test:

The Hepcidin 25 (bioactive) enzyme-linked immunosorbent assay (ELISA) Kit, is a solid phase enzyme-linked immunosorbent assay based on the principle of competitive binding. The microtiter wells are coated with a monoclonal (mouse) antibody directed towards an antigenic site of the Hepcidin-25 molecule. Endogenous Hepcidin-25 of a patient sample competes with a Hepcidin-25-biotin conjugate (Enzyme Conjugate) for binding to the coated antibody. After incubation the microtiter plate is washed to stop the competition reaction. In the following incubation the bound biotin molecules are detected with streptavidin peroxidase (Enzyme Complex). After incubation the plate is washed the second time. After addition of the substrate solution, the intensity of color developed is inversely proportional to the concentration of Hepcidin-25 in the patient sample.

In our study mean age was 41.90 ± 13.98 years with a range from 14 to 69 years. Maximum number of patients were from the age group of 41 to 50 years. Almost more than half of the study population were in the age group of 31 to 50 years. Male pre-dominance was seen among study group with 67.31 % males and 32.69 % females. Mean serum hepcidin level at transplant in males was 63.67 ± 2.06 ng/ml while in females it was 59.53 ± 2.01 ng/ml. There was no gender specific difference in the serum hepcidin level among males and females with functional iron deficiency anemia.

Among study groups, 24 (46.15 %) recipients received graft from living related donor, 18 (34.62 %) received graft from living unrelated donor and 10 (19.23 %) received graft from deceased donor. 41 (78.85 %) patients received graft from ABO compatible donor while 11 (21.15 %) patients received graft from ABO incompatible donor. Out of total 52 renal transplant recipients, ATG was used in 38 (73.08 %) recipients, ATLG was used in 10 (19.23 %), Basiliximab was used in 3 (5.77 %) recipients and no induction therapy was given in 1 (1.92 %) recipient. Maximum recipients in our study received ATG. In post renal transplant all patients were on triple immunosuppression with Tacrolimus, Mycophenolate mofetil and low dose prednisolone.

Delayed graft function was present in 11 (21.15 %) patients while 41 (78.85 %) patients had no delayed graft function. Acute rejection episode was present in 12 (23.08 %) patients while 40 (76.92 %) patients had no acute rejection episode. Among acute rejection episode, 10 patients had T cell mediated rejection while 2 patients had antibody mediated rejection. Acute allograft dysfunction was present in 13 (25 %) patients while 39 (75 %) patients had no acute allograft dysfunction.

Ashby et al showed that, chronic kidney disease patients on maintenance hemodialysis had significantly higher levels of hepcidin, with mean 58.5 ng/ml (16). In a study by Winnie Chan et al, mean serum hepcidin level was 43 ng/mL with a range 29-67 ng/mL (17). In our study, mean hepcidin level at transplantation was 52.09 ng/ml, which is similar to the study done by Ashby et al.

The hepcidin level at transplantation was 52.09 ± 2.72 ng/ml which reduced to 38.63 ± 2.54 ng/ml at 1-month post-transplant and 28.89 ± 2.21ng/ml at 3-month post-transplant. In our study, there was significant reduction in hepcidin level at 1 and 3-month post-transplant as compared to hepcidin level at transplant. The reduction of serum hepcidin level after renal transplant could be due to improvement in graft function and production of erythropoietin, reduction in the state of inflammation which is common in perioperative period, improvement in the nutritional status of the renal transplant recipients.

Hepcidin and renal graft function:

1. Delayed Graft Function (DGF):

In a study by Dirk R. J. Kuypers et al, delayed graft function was present in 8 % of patients while 92 % patients had no delayed graft function (18). In our study, delayed graft function was present in 21.15 % patients while 78.85 % of patients had no evidence of DGF. In present study, majority of patients with delayed graft function were deceased donor renal transplant recipients in whom delayed graft function could be due to increased cold ischemia time. Study by J. Malyszko et al, showed that Hepcidin may represent an early and predictive biomarker for delayed graft function and serum hepcidin level remained high in patients with DGF when compared with patients without DGF (7). Present study showed, mean hepcidin level of 43.82 ± 2.33 ng/ml in patients with delayed graft function as compared to 24.89 ± 2.02 ng/ml in patients without delayed graft function. Present study showed significant association between delayed graft function and higher hepcidin level at 3-month post-transplant, similar observation was seen in a study done by J. Malyszko et al (7).

2. Acute Rejection Episode (ARE):

In a study by Dirk R. J. Kuypers et al, 26 % patients had acute rejection episodes at the end of three months (18). In a study by Darshika Chhabra et al, showed that there was total 14 % acute rejection episodes at the end of 3 months post-transplant (19). In present study, acute rejection episode was present in 23.08 % patients while 76.92 % patients had no acute rejection episodes. Among acute rejection episode, 10 patients had T cell mediated rejection while 2 patients had antibody mediated rejection. Our study results of on percentage of acute rejection episode are similar with the study by Dirk R. J. Kuypers et al (18). Present study showed, mean hepcidin level in patients with acute rejection episode was 45.63 ± 2.48 ng/ml as compared to 23.88 ± 1.87 ng/ml in patients without acute rejection episode.

Hepcidin level correlated well with the renal graft function.  Renal transplant recipients with normal graft function had normal Hepcidin level while renal transplant recipient with acute allograft dysfunction (AAD), acute rejection episode (ARE) and delayed graft function (DGF) were associated with higher hepcidin levels. We propose that serum Hepcidin level could be a novel biomarker of renal graft function in early post-transplant period, further long-term studies are required to establish a role of Hepcidin as early marker of renal graft function.

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