The authors have declared that no competing interests exist.
Conceived and designed the experiments: GS GND AM GKK EP. Performed the experiments: AL GC EK EP. Analyzed the data: NKG KZ GKK. Contributed reagents/materials/analysis tools: PE SG AS. Wrote the paper: GS GND AL NKG.
Hepcidin, a liver hormone, is important for both innate immunity and iron metabolism regulation. As dysfunction of the hepcidin pathway may contribute to liver pathology, we analysed liver hepcidin mRNA and serum hepcidin in patients with chronic liver diseases. Hepcidin mRNA levels were determined in liver biopsies obtained from 126 patients with HCV (n = 21), HBV (n = 23), autoimmune cholestatic disease (primary biliary cirrhosis and primary sclerosing cholangitis; PBC/PSC; n = 34), autoimmune hepatitis (AIH; n = 16) and non-alcoholic fatty liver disease (NAFLD; n = 32). Sera sampled on the biopsy day from the same patients were investigated for serum hepcidin levels. Hepatic hepcidin mRNA levels correlated positively with ferritin and negatively with serum γ-GT levels. However, no correlation was found between serum hepcidin and either ferritin or liver hepcidin mRNA. Both serum hepcidin and the serum hepcidin/ferritin ratio were significantly lower in AIH and PBC/PSC patients’ sera compared to HBV, HCV or NAFLD (P<0.001 for each comparison) and correlated negatively with serum ALP levels. PBC/PSC and AIH patients maintained low serum hepcidin during the course of their two-year long treatment. In summary, parallel determination of liver hepcidin mRNA and serum hepcidin in patients with chronic liver diseases shows that circulating hepcidin and its respective ratio to ferritin are significantly diminished in patients with autoimmune liver diseases. These novel findings, once confirmed by follow-up studies involving bigger size and better-matched disease subgroups, should be taken into consideration during diagnosis and treatment of autoimmune liver diseases.
Hepcidin is a hormone predominantly synthesized and secreted by hepatocytes. It was first identified in human blood and urine as an antimicrobial peptide of innate immunity, named LEAP-1 (liver-expressed antimicrobial peptide-1) and involved in host defense [
Hepcidin production can be regulated at the transcriptional level by different signaling pathways. Up-regulation of hepcidin under iron-replete hepatic conditions involves the BMP/SMAD-signaling pathway [
Although there are many studies demonstrating altered hepcidin levels in iron disorders, data on hepcidin regulation in chronic liver diseases and in particular autoimmune liver diseases are scarce. Chronic liver diseases can lead to disorders in iron homeostasis [
In older studies, liver hepcidin mRNA levels in biopsies were considered as an indicator of hepcidin expression while more recent ones analyze circulating serum hepcidin levels. In both types of studies the hepcidin/ferritin ratio is often used as a variable in combination with patients data. Ferritin, a marker of iron stores, and serum hepcidin correlate positively [
In this work, we evaluated liver hepcidin gene expression, serum hepcidin levels and hepcidin/ferritin ratios in patients with diverse liver diseases, including for the first time patients with autoimmune liver disorders, and subsequently correlated these measurements with the clinical, histological and laboratory data of the patients. Our aim was also to investigate whether alterations of hepcidin expression in liver tissue or in the circulation can be observed and compared between groups of patients at the onset of chronic liver diseases of different etiology.
One hundred and twenty-six consecutive patients with chronic liver diseases [52 males, 74 females; age (mean±SD) 46.8±15.5 years] who attended and followed at the outpatient Hepatology clinic of the Department of Medicine, Larissa Medical School, University of Thessaly, Larissa, Greece were included in the study. Disease etiology included HCV (n = 21), HBV (n = 23), AIH (n = 16), PBC/PSC (n = 34; PBC n = 30, PSC n = 4) and NAFLD (n = 32). The demographic, clinical, laboratory and histological characteristics of the patients are shown in
Variables | Total patients (n = 126) | HCV (n = 21) | HBV (n = 23) | AIH (n = 16) | PBC/PSC (n = 34) | NAFLD (n = 32) | Healthy (n = 17) | |
---|---|---|---|---|---|---|---|---|
Age (years) | 46.8±15.5 | 40.6±13.6 | 42.6±14.6 | 53.5±16.3 | 53±13.5 | 43.9±16.1 | 45.2±15 | 0.009 |
Sex, male / female | 52 / 74 | 10 / 11 | 15 / 8 | 4 / 12 | 7 / 27 | 16 / 16 | 10 / 7 | 0.006 |
ALT (IU/L) | 60 (73) | 49 (59) | 71 (97) | 95 (241) | 32 (57) | 65 (56) | 32 (5) | <0.001 |
AST (IU/L) | 35 (34) | 32 (27) | 45 (60) | 58 (83) | 31 (20) | 30 (21) | 31 (4) | 0.027 |
γGT (IU/L) | 45 (69) | 27 (34) | 28 (50) | 30 (88) | 71 (77) | 57 (66) | 29 (8) | <0.001 |
ALP (IU/L) | 76 (42) | 63 (30) | 68 (41) | 71 (39) | 105 (100) | 81 (34) | 62 (12) | <0.001 |
Bilirubin (mg/dL) | 0.66 (0.51) | 0.72 (0.35) | 0.95 (0.66) | 0.65 (1.32) | 0.66 (0.48) | 0.55 (0.32) | 0.65 (0.15) | 0.008 |
Hemoglobin (g/L) | 13.8 (2.5) | 14.2 (2.3) | 14.3 (2.8) | 13.4 (1.6) | 13 (1.6) | 14.5 (2.3) | 14.7 (1) | 0.003 |
Ferritin (ng/ml) | 119 (124) | 91 (114) | 143 (207) | 126 (213) | 88 (120) | 123 (99) | NA | 0.109 |
Albumin (g/L) | 4.4±0.4 | 4.5±0.3 | 4.4±0.3 | 4.2±0.4 | 4.3±0.4 | 4.6±0.3 | 4.5±0.2 | 0.001 |
INR | 0.96±0.11 | 0.96±0.1 | 0.99±0.12 | 1±0.12 | 0.95±0.13 | 0.92±0.09 | 0.92±0.1 | 0.065 |
Inflammation | 0.05 | |||||||
89 (70.6) | 12 (57.1) | 14 (60.8) | 9 (56.3) | 26 (76.5) | 28 (87.5) | NA | ||
37 (29.4) | 9 (42.9) | 9 (39.2) | 7 (43.8) | 8 (23.5) | 4 (12.5) | NA | ||
Fibrosis | <0.001 | |||||||
101(80.2) | 15 (71.4) | 13 (56.5) | 11 (68.8) | 31 (91.1) | 31 (96.8) | NA | ||
25 (19.8) | 6 (28.6) | 10 (43.5) | 5 (31.2) | 3 (8.9) | 1 (3.2) | NA | ||
Cirrhosis | 0.439 | |||||||
120 (95.2) | 19 (90.5) | 21 (91.3) | 15 (93.7) | 33 (97.1) | 32 (100) | NA | ||
6 (4.8) | 2 (9.5) | 2 (8.7) | 1 (6.3) | 1 (2.9) | 0 (0) | NA | ||
Liver hepcidin mRNA | 0.74 (1.22) | 0.72 (1.21) | 1.32 (1.08) | 0.47 (1.10) | 0.74 (1.12) | 0.65 (1.18) | NA | 0.267 |
hepcidin mRNA / log ferritin | 0.35 (0.61) | 0.44 (0.54) | 0.61 (0.47) | 0.26 (0.53) | 0.35 (0.64) | 0.29 (0.65) | NA | 0.315 |
Serum hepcidin (ng/ml) | 35 (100) | 56 (101) | 109 (165) | 9 (6) | 8 (14) | 85 (116) | 61 (39) | <0.001 |
Serum hepcidin/ log ferritin | 16.9 (50) | 26.4 (49.7) | 45.9 (72.1) | 4.3 (2.9) | 4.5 (6.9) | 39.4 (60.1) | NA | <0.001 |
The diagnosis of HCV and HBV was based on the EASL criteria [
Fine needle liver biopsy was performed for diagnostic purposes. Whenever the material was sufficient, a small sample of the hepatic specimen was immediately immersed in RNA Later buffer (Qiagen, Hilden, Germany) and incubated overnight at 4°C. Then the tissue was removed from the reagent and stored into cryogenic vials at -80°C until further use. Serum samples were obtained from patients on the day of liver biopsy and were immediately frozen and stored at -80°C until used. The study was approved by the ethical committee of the University of Thessaly Medical School. Verbal informed consent was obtained from all participants in the study instead of written consent because the data were analyzed anonymously. The method of obtaining verbal consent was approved by the ethical committee. More specifically, the routine for every patient followed-up in our outpatient Hepatology clinic includes requesting the informed oral consent of the patients for the anonymous use of their samples for potential future retrospective research studies, always provided that there is sufficient sample (e.g. serum, tissue) for definite diagnosis. For this particular study, the required elements of consent were presented orally to the subjects at the day of the liver biopsy, which was always performed for diagnostic purposes. Sufficient time was provided to explain all pertinent information and every patient was free to ask questions. A patient’s relative always witnessed the process. All the procedure of obtaining verbal consent was documented in the source data file of each patient and signed by the treating physician. The procedures followed were in accordance with the Helsinki Declaration of 1975, as revised in 2000.
Total RNA from liver tissues was isolated using TRIZOL Reagent (Invitrogen, Carlsbad, CA, USA) along with 10μg RNase-free glucogen (Invitrogen), as carrier to the aqueous phase, according to the manufacturer’s instructions.
One microgram of total RNA was reversed transcribed using random primers and 50 Units of Multiscribe Reverse Transcriptase according to the cDNA reverse transcription protocol (Applied Biosystems, Foster City, CA, USA) in a 20μl reaction for 10 min at 25°C, 120 min at 37°C and 5 sec at 85°C. qRT-PCR was carried out using SYBR Green I dye (KAPA Biosystems, Wilmington, MA, USA) in a total volume of 20μl containing 0.5 μM specific forward and reverse primers. Amplification and detection were performed in a MiniOpticon Real-Time PCR Detector (Bio-Rad Laboratories Inc., Hercules, CA, USA) system under the following conditions: 1 cycle at 50°C for 2min and 95°C for 10 min and 40 cycles at 95°C for 15 sec and 60°C for 1 min. The mRNA of human
Serum hepcidin-25 determination was performed using a validated ELISA, as already previously described [
Liver biopsy specimens were evaluated by a single experienced liver immunopathologist (GKK) using the Knodell histological/activity index score [
Kolmogorov-Smirnov test was used to assess the normality of distribution. Normally distributed values are expressed as mean±SD, while non-normally as median (IQR). Data were analysed by x2 test, Fisher’s exact test, ANOVA, Kruskal-Wallis or Mann-Whitney U test, Spearman’s coefficient of correlation (r) and Wilcoxon Signed-Rank test where applicable. Multivariate analysis was performed in the total group of our patients (n = 126) by a stepwise linear regression with hepcidin levels as the dependent variable, while factors found to be significantly associated with serum hepcidin levels at univariate analysis were included as independent variables. We also included ferritin levels in our multivariate model to assess their impact as a confounding factor. Two-sided P-values <0.05 were considered as statistically significant.
Clinical characteristics of the 126 patients, with different liver diseases that underwent liver biopsy, are summarized in
Histological analysis was performed on the biopsy specimens of our patients evaluating levels of inflammation (minimal/mild or moderate/severe), the extent of fibrosis (none/mild/moderate or severe/cirrhosis) and appearance of cirrhosis in liver tissue (
Samples of liver biopsies were analysed for hepcidin gene expression by quantifying the levels of hepcidin mRNA using reverse transcription and quantitative real-time PCR. Serum samples drawn on the same day as the biopsy were also analysed using a homemade validated ELISA to quantify the levels of circulating mature hepcidin peptide. Liver hepcidin mRNA, serum ferritin levels and liver hepcidin mRNA/log ferritin ratio did not significantly differ among disease groups (
Liver hepcidin mRNA (A), serum hepcidin levels (ng/ml) (B), liver hepcidin mRNA/log ratio (C) and serum hepcidin/log ferritin ratio (D) in the studied groups. Graphs depict the median (line within the box), 25th to 70th percentiles (upper and lower border of the box), and 10th to 90th percentiles (whiskers).
Serum hepcidin levels and serum hepcidin/log ferritin ratio negatively correlated with age (
Variables | Liver hepcidinmRNA | Serum hepcidin(ng/ml) | Ferritin(ng/ml) | Hepcidin mRNA/log ferritin | Serum hepcidin/log ferritin | |||||
---|---|---|---|---|---|---|---|---|---|---|
Statistics | ||||||||||
Age | 0.11 | 0.202 | -0.23 | 0.011 | 0.05 | 0.598 | 0.12 | 0.185 | -0.23 | 0.009 |
ALT (IU/L) | -0.09 | 0.313 | 0.12 | 0.168 | 0.35 | <0.001 | -0.16 | 0.081 | 0.07 | 0.428 |
AST (IU/L) | -0.09 | 0.326 | 0.07 | 0.406 | 0.23 | 0.010 | -0.14 | 0.114 | 0.04 | 0.658 |
γGT (IU/L) | -0.19 | 0.028 | -0.14 | 0.124 | 0.11 | 0.236 | -0.21 | 0.017 | -0.16 | 0.069 |
ALP (IU/L) | -0.14 | 0.126 | -0.21 | 0.020 | -0.06 | 0.509 | -0.15 | 0.094 | -0.20 | 0.022 |
Billirubin (mg/dL) | 0.14 | 0.125 | -0.01 | 0.896 | 0.27 | 0.002 | 0.08 | 0.391 | -0.05 | 0.562 |
Hemoglobin (g/L) | 0.07 | 0.456 | 0.27 | 0.002 | 0.23 | 0.009 | 0.03 | 0.759 | 0.23 | 0.010 |
Ferritin | 0.25 | 0.006 | 0.12 | 0.192 | NA | NA | 0.08 | 0.389 | -0.03 | 0.716 |
Albumin (g/L) | 0.09 | 0.309 | 0.20 | 0.029 | 0.14 | 0.126 | 0.07 | 0.466 | 0.18 | 0.043 |
INR | 0.07 | 0.420 | 0.01 | 0.865 | 0.15 | 0.092 | 0.05 | 0.555 | -0.01 | 0.918 |
Liver hepcidin mRNA | NA | NA | 0.08 | 0.396 | 0.25 | 0.006 | NA | NA | 0.05 | 0.585 |
Serum hepcidin | 0.08 | 0.396 | NA | NA | 0.12 | 0.192 | 0.06 | 0.505 | NA | NA |
NA: not applicable.
As expected by the fact that iron overload is a stimulus for hepcidin transcriptional up-regulation, hepatic hepcidin mRNA expression correlated positively with ferritin (
Parameters | Liver hepcidin mRNA | Serum hepcidin (ng/ml) | Ferritin (ng/ml) | |||
---|---|---|---|---|---|---|
0.716 | 0.176 | 0.007 | ||||
0.745 (1.05) | 40.6 (115.6) | 107 (114) | ||||
0.727 (1.45) | 13.5 (60.4) | 162 (180) | ||||
0.495 | 0.067 | 0.847 | ||||
0.744 (1.23) | 25.6 (97.6) | 120 (141) | ||||
0.839 (1.31) | 56.2 (120.6) | 106 (106) |
Values are expressed as the median and IQR.
That low serum hepcidin is a true characteristic of patients with PBC/PSC and AIH was confirmed by multivariate analysis. More specifically, predictor factors for serum hepcidin levels were assessed through a stepwise linear regression model in the total group of the patients (n = 126). In this model, serum hepcidin was the dependent variable, while the presence of AIH or PBC/PSC along with factors found to be significant in the univariate analysis (age, gender, hemoglobin, albumin, ALP levels and fibrosis stage) were included as independent variables. Ferritin levels were also included in order to assess their impact as a confounding factor. By using this analysis (
β-coefficient | ||
---|---|---|
Age | -0.119 | 0.181 |
Gender | -0.114 | 0.203 |
Hemoglobin | 0.081 | 0.362 |
Albumin | 0.138 | 0.120 |
ALP | -0.173 | 0.055 |
Fibrosis stage | 0.116 | 0.195 |
Ferritin levels | -0.067 | 0.451 |
Presence of AIH or PBC/PSC | -15.211 | 0.020 |
In the subgroup of patients with AIH or PBC/PSC (n = 50), liver hepcidin mRNA was significantly lower in females [0.43 (1.1)] compared to males [1.29 (0.98); p = 0.036] and associated positively with ferritin levels (P = 0.035). The analysis in patients with AIH (n = 16) also showed that females are characterized by decreased hepatic hepcidin mRNA expression [0.3 (0.44) versus 1.56 (0.87); P = 0.004] and, in the same group, serum hepcidin levels associated positively with INR (P = 0.003). In the patients with PBC/PSC (n = 34), liver hepcidin mRNA correlated positively with ferritin levels (P = 0.022). Regarding the fibrosis stage, serum hepcidin levels and serum hepcidin/log ferritin ratio were significantly increased in patients with severe fibrosis/cirrhosis compared to patients with none/mild/moderate fibrosis either in the combined group of AIH and PBC/PSC patients [16.6 (19.1) versus 8.4 (7.2); P = 0.017 and 9.7 (11.8) versus 4 (3.8); P = 0.018, respectively] or in AIH patients separately [12.9 (71.8) versus 8.3 (5.2); P = 0.027 and 6.2 (41.05) versus 3.4 (2.19); P = 0.038, respectively], while no association was revealed in PBC/PSC patients.
To gain an insight of the etiology behind low serum hepcidin concentration in patients with autoimmune liver diseases, we investigated the sera of ten non-cirrhotic patients in the course of their subsequent two-year conventional treatment. Five patients with PBC having low serum hepcidin at the time of first biopsy [9.9 (4.2)] were re-analysed after one and two years of treatment. As shown in
Serum hepcidin levels in patients with PBC (
In the present study, we assessed liver hepcidin mRNA expression and serum hepcidin levels as well as their corresponding ratios to ferritin in patients with non-iron related liver disorders including for the first time patients with autoimmune liver diseases. The most important point arising from the present investigation is that serum hepcidin concentrations alone, as well as their corresponding ratios to serum ferritin, were extremely reduced in patients with AIH or PBC/PSC compared to any of the study groups and these levels remained low despite adequate treatment during a 2-years follow-up period. It should be stressed that the serum hepcidin levels observed in the AIH and PBC/PSC patients are not lower only when compared to the other patients of our cohort but they are also lower when compared to the “normal” concentrations of serum hepcidin as determined in healthy controls. Although absolute values given by in-house ELISA methods are not directly comparable (because of the use of different antibodies and peptide preparations), the values obtained for our healthy controls, median: 75 and 41 ng/ml for men and women, respectively, lie between those reported in the literature by previous bigger studies with healthy volunteers or general population, e.g. median: 112 (men) and 65 (women) ng/ml [
In addition to the novel finding of low serum hepcidin in patients with autoimmune hepatitis, our study provides several interesting pieces of data that complement or confirm those from previously published works. Both liver hepcidin mRNA and serum hepcidin were significantly lower in female patients of our cohort, which, at least partially, can be due to the lower iron stores in the same patients as depicted by the strong negative correlation of ferritin with the female gender. This has been previously reported, for serum hepcidin, in patients with chronic hepatitis C [
Hepatic hepcidin mRNA expression correlated with serum ferritin depicting the direct effect of iron concentration on the transcriptional activation of hepcidin. Serum hepcidin positively correlated with hematological parameters (albumin, hemoglobin) supporting a possible role of reduced erythropoiesis and anemia on circulating hepcidin levels whereas liver hepcidin mRNA and serum hepcidin correlated negatively with cholestatic markers in agreement with previous findings [
The HCV patient group as well as the AIH group had the highest percentage of patients with moderate to severe inflammation (43–44%) while the HBV group the highest proportion (43.5%) of severe fibrosis as judged by the histological analysis. Although, no significant association could be shown between liver hepcidin mRNA or serum hepcidin and inflammation severity or fibrosis, patients with higher grading score for inflammation had lower serum hepcidin while patients with severe fibrosis had a tendency for higher serum hepcidin. On the other hand, inflammation and fibrosis did not have an effect on liver hepcidin mRNA. These findings are unexpected as they contrast with a previous study showing negative correlation between fibrosis status and liver hepcidin mRNA or urinary hepcidin [
The lower serum hepcidin levels in PBC/PSC and AIH are probably not the result of inhibition of liver hepcidin mRNA expression levels, as these levels did not differ significantly among the study groups, with a single exception (lower hepcidin mRNA in AIH when compared to HBV). This may be surprising but it is in agreement with the fact that hepcidin gene expression levels in the liver (the principal site of hepcidin production) do not correlate with levels of hepcidin mature peptide in the serum of our patients and it is not without precedent. Previous studies that have determined liver hepcidin mRNA as well as urinary or serum hepcidin in patients reported different results concerning the correlation between these values. A moderate correlation was observed between urinary hepcidin concentrations and hepatic hepcidin mRNA values in patients with liver cancer or cirrhosis [
There are several possible explanations for theses inconsistencies. First, changes in hepcidin mRNA expression may be subtler than changes in secreted peptide amounts and, therefore, more difficult to become statistically significant with a relatively small group of subjects or due to restrictions in the accuracy and the detection limits of the measurement methods. In this respect, it should be noted that AIH patients, the smallest group in our study, show both the lowest serum hepcidin (compared to all other non-autoimmune groups) and the lowest liver hepcidin mRNA, but the latter differs significantly only when compared to the HBV group. Second, extrahepatic sources can also contribute to the secretion of hepcidin [
Although AIH and PBC/PSC are complex autoimmune liver diseases that differ in autoimmune injury and clinical phenotype, the diminished serum hepcidin levels found in our study seem to be a common characteristic, which may either might be associated with the pathogenicity or be a specific consequence of liver autoimmunity. The answer to these questions clearly necessitates much additional investigation but one speculation that could explain our data is the involvement of hepcidin in autoimmunity. Despite being critical for iron homeostasis, hepcidin is an acute phase peptide of the innate immunity with antimicrobial properties. Both roles of hepcidin are tightly related to each other, since iron homeostasis affects immune defense and vice versa. Hepatocyte production of hepcidin is responsive to body iron stores, while as a member of the β-defensin family [
In conclusion, parallel determination of hepcidin expression levels in liver biopsies and sera of patients with different hepatic disorders has revealed that serum hepcidin concentrations and their corresponding ratios to ferritin are dramatically low in patients with autoimmune liver diseases in comparison to other liver diseases. This striking and novel finding necessitates further studies to evaluate the role of serum hepcidin as a sensitive biomarker for liver autoimmunity and its pathogenic involvement along with the underlying molecular mechanisms.
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