Abstract
Background and objectives: Hepcidin, the principal regulator of iron homeostasis, may play a critical role in the response of patients with anemia to iron and erythropoiesis-stimulating agent therapy; however, the contribution of hepcidin to iron maldistribution and anemia in hemodialysis (HD) patients and the ability of HD to lower serum hepcidin levels have not been fully characterized.
Design, setting, participants, & measurements: We measured serum hepcidin using a competitive ELISA in 30 pediatric and 33 adult HD patients. In addition, we determined serum hepcidin kinetics and calculated hepcidin clearance by measuring serum hepcidin before, during, and after HD in eight pediatric and six adult patients.
Results: Hepcidin was significantly increased in pediatric (median 240.5 ng/ml) and adult HD patients (690.2 ng/ml) when compared with their respective control subjects (pediatric 25.3 ng/ml, adult 72.9 ng/ml). Multivariate regression analysis showed that serum hepcidin was independently predicted by ferritin and high-sensitivity C-reactive protein in the pediatric group and ferritin, percentage of iron saturation, and high-sensitivity C-reactive protein in the adult group. Hepcidin levels decreased after dialysis from 532 ± 297 to 292 ± 171 ng/ml. Hepcidin clearance by HD was 141 ± 40 and 128 ± 44 ml/min in pediatric and adult patients, respectively (NS).
Conclusions: These findings suggest that hepcidin may mediate the negative effects of inflammation on both disordered iron metabolism and erythropoiesis in HD patients and that intensification of HD could be used therapeutically to reduce hepcidin concentrations and thereby improve erythropoiesis-stimulating agent responsiveness.
The peptide hepcidin has emerged as a key regulator of iron metabolism in both health and disease. Hepcidin is secreted by the liver and binds to the major cellular iron exporter ferroportin, causing its internalization (1). This in turn prevents enteral iron absorption as well as iron release from the liver and the reticuloendothelial system. Hepcidin production is induced by iron loading and inflammation, and it is suppressed by erythropoietic activity and iron deficiency (2–5).
Serum hepcidin levels are greatly elevated across the spectrum of chronic kidney disease (CKD). In previous studies, we (6) and Ashby et al. (7) both demonstrated an inverse correlation between serum hepcidin and GFR in adults with CKD, with serum hepcidin levels being highest in dialysis-dependent patients. In addition, using multivariate analysis, we found that hepcidin levels correlated with markers of iron status and inflammation (6). Thus, in the setting of CKD, increased serum hepcidin and the resulting iron restriction could play a major role in disordered iron homeostasis and resistance to erythropoiesis-stimulating agents (ESA).
The removal of hepcidin via hemodialysis (HD) has been demonstrated in adult patients using mass spectrometry (MS)-based assays, with varying degrees of efficacy seen (8,9). Definitive resolution of this issue is needed, because increased removal of hepcidin by intensified HD could provide a much-needed therapeutic intervention in cases of functional iron deficiency by relieving reticuloendothelial blockade as a result of inflammation-induced hepcidin overproduction. The improvement in ESA responsiveness reported in prolonged dialysis regimens supports the potential utility of this intervention (10,11). In this study, using a competitive ELISA assay, we investigated bioactive serum hepcidin levels in both pediatric and adult HD patients, assessed the effect of HD on circulating hepcidin levels, and measured hepcidin clearance by HD.
Materials and Methods
Patient Criteria
The patient population was composed of children and adults who were receiving outpatient HD care at the Davita/UCLA Dialysis Program. This study was approved by the UCLA institutional review board, and all patients/parents gave informed consent to participate. All patients had received at least 3 months of maintenance HD.
Patients who were receiving recombinant erythropoietin (rhEPO) and iron supplementation were enrolled provided that the dosages of each had been stable for at least 4 weeks. All rhEPO supplements were in the form of recombinant epoetin alfa (Amgen). Parenteral iron supplementation was withheld for 1 week before the measurement of hepcidin.
Exclusion criteria were (1) previously diagnosed nonrenal cause of anemia other than iron deficiency; (2) evidence of active or occult bleeding; (3) blood transfusion within the past 4 months; (4) history of malignancy, end-stage liver disease, or chronic hypoxia; and (5) recent hospitalization or infection that required antibiotics within the past 4 weeks. The control groups consisted of 20 healthy children (eight male, 12 female) aged 14.4 ± 4.0 years and 24 healthy adults (12 male, 12 female) aged 28.4 ± 6.6 years.
Data Collection and Biochemical Measurements
Demographic data obtained included age, gender, cause of CKD, medical history, and medication history. A blood sample was collected for markers of erythropoiesis (hemoglobin [Hb]), iron status (percentage of iron saturation and ferritin), inflammation (high-sensitivity C-reactive protein [hsCRP; CardioPhase hsCRP; Dade Behring]), renal osteodystrophy (intact parathyroid hormone and phosphorus), and dialysis adequacy (single-pool Kt/V [spKt/V] and urea). Quantitative measurement of bioactive hepcidin in serum was carried out as described previously using a sensitive competitive ELISA (12).
HD Clearance Measurements
Serum hepcidin measurements were obtained immediately before HD initiation and then at 30-minute time points from both the arterial and the venous access points during the dialysis treatment. A final sample was obtained immediately after dialysis was completed. The average blood flow (Qb) was 320 ± 52 and 375 ± 32 ml/min in pediatric and adult patients, respectively (NS), and the average spKt/V was 1.51 ± 0.48 and 1.61 ± 0.36 in pediatric and adult patients, respectively (NS). All patients underwent dialysis with a Polyflux Revaclear dialyzer (Gambro) with a dialysate flow (Qd) of 800 ml/min for an average of 3.2 ± 0.2 and 3.0 ± 0.4 hours in pediatric and adult patients, respectively (NS). Average ultrafiltration was 1.6 ± 1.1 and 2.1 ± 1.7 L in pediatric and adult patients, respectively (NS). Clearance was then calculated as Qb × (arterial hepcidin − venous hepcidin)/arterial hepcidin. The hepcidin reduction ratio was calculated as (hepcidin at start of dialysis − hepcidin at end of dialysis)/hepcidin at start of dialysis. The observed elimination rate (λ) for each patient was estimated via linear regression of time versus log hepcidin concentration. The plasma disappearance half-life (t½) of hepcidin was calculated as Ln(2)/λ.
Statistical Analysis
Study variables were summarized using mean and SD. Because of its non-normal distribution, hepcidin values were presented as medians. The differences in biochemical and HD clearance measurements between patient groups were compared using the Mann-Whitney rank sum test for nonparametric continuous data or independent t test for parametric data. For univariate and multivariate analysis, log transformation was applied to variables with non-normal distribution. Univariate correlations between biochemical measurements and hepcidin were calculated using the Pearson correlation test. In multiple linear regression models used to investigate the association of biochemical parameters with hepcidin, variable selection was done by the stepwise method. All tests are two-sided with significance level of 0.05, and all analyses were performed using SAS statistical software (SAS Institute).
Results
Patient Demographics and Biochemical Characteristics
Demographic data for the 30 pediatric and 33 adult HD patients are listed in Table 1. The major cause of CKD was renal dysplasia in the pediatric group and diabetes in the adult group.
Table 1.
Patient demographics
| Parameter | Pediatric (n = 30) | Adult (n = 33) |
|---|---|---|
| Male/female | 11/19 | 22/11 |
| Age (years; mean ± SD) | 16.0 ± 4.6 | 60.3 ± 20.7 |
| Diagnosis | ||
| renal dysplasia | 11 | 0 |
| FSGS | 4 | 0 |
| glomerulonephritis | 8 | 7 |
| polycystic kidney disease | 2 | 2 |
| hypertension | 0 | 7 |
| diabetes | 0 | 11 |
| other | 5 | 6 |
Biochemical markers of erythropoiesis (Hb), iron status (percentage of iron saturation and ferritin), and inflammation (hsCRP), in addition to intact parathyroid hormone, serum phosphorus, and serum urea, are presented in Table 2. The only difference seen between the two groups was a higher ferritin level in the adult group. The average ± SD weekly dosage of rhEPO was 302 ± 233 and 257 ± 348 U/kg in pediatric and adult patients, respectively (NS). Similarly, no difference was seen in the spKt/V between the two groups: 1.6 ± 0.3 and 1.5 ± 0.3 in pediatric and adult patients, respectively (NS).
Table 2.
Biochemical markers
| Parameter | Pediatric (Mean ± SD) | Adult (Mean ± SD) | P |
|---|---|---|---|
| % Iron saturation | 32.2 ± 12.1 | 35.8 ± 13.7 | NS |
| Ferritin (ng/ml) | 578 ± 572 | 740 ± 410 | <0.02 |
| Hb (g/dl) | 12.1 ± 1.7 | 11.9 ± 1.3 | NS |
| hsCRP (mg/L) | 10.2 ± 21.4 | 11.8 ± 20.9 | NS |
| iPTH (pg/ml) | 529 ± 678 | 527 ± 714 | NS |
| Serum urea | 53.3 ± 21.7 | 54.0 ± 15.8 | NS |
| Serum phosphorus | 5.4 ± 1.6 | 5.2 ± 1.4 | NS |
iPTH, intact parathyroid hormone.
Serum Hepcidin Levels in HD Patients
Serum hepcidin levels in pediatric and adults HD patients were higher than in their respective age-matched control subjects (P < 0.001; Figure 1), and values were higher in adult versus pediatric HD patients (P < 0.01).
Figure 1.
Hepcidin levels in healthy control subjects and HD patients. Box plots represent second quartile, median, and third quartile of each group. Error bars denote the 10th and 90th percentiles. Hepcidin levels in each group of HD patients were significantly elevated compared with respective age-matched control subjects. In addition, pediatric and adult HD patients had significantly different hepcidin levels (see text).
In pediatric patients, univariate analysis revealed a positive correlation between hepcidin and percentage of iron saturation, ferritin, hsCRP, serum urea, and phosphorus and a negative correlation with Hb (Table 3). In adult patients, both percentage of iron saturation and ferritin showed positive correlations with hepcidin. In both groups, there was no correlation between hepcidin and patient age, weekly rhEPO dosage, or spKt/V.
Table 3.
Univariate correlation with serum hepcidin
| Parameter | Pediatric |
Adult |
||
|---|---|---|---|---|
| r | P | r | P | |
| % Iron saturationa | 0.52 | <0.01 | 0.64 | <0.001 |
| Ferritina | 0.80 | <0.001 | 0.68 | <0.001 |
| Hb | −0.42 | <0.03 | – | – |
| hsCRPa | 0.58 | <0.001 | – | – |
| Serum urea | 0.60 | <0.001 | – | – |
| Serum phosphorus | 0.40 | <0.04 | – | – |
Log transformation applied to variables with non-normal distribution.
Multivariate regression models were developed to assess the relationship between serum hepcidin, Hb, percentage of iron saturation, ferritin, hsCRP, phosphorus, and urea in each group of HD patients. Using these models, the only independent predictors of serum hepcidin were ferritin and hs-CRP in the pediatric group (R2 = 0.70) and ferritin, percentage of iron saturation, and hs-CRP in the adult group (R2 = 0.63; Table 4).
Table 4.
Multivariate correlation with serum hepcidin
| Parameter | Pediatric |
Adult |
||
|---|---|---|---|---|
| β ± SE | P | β ± SE | P | |
| % Iron saturationa | – | – | 388 ± 102 | <0.001 |
| Ferritina | 5.48 ± 1.01 | <0.001 | 0.50 ± 0.10 | <0.001 |
| hsCRPa | 1.76 ± 0.80 | <0.03 | 61.10 ± 28.20 | <0.039 |
Log transformation applied to variables with non-normal distribution.
Clearance of Hepcidin by HD
Serum hepcidin clearance measurements were made in eight pediatric and six adult patients who were undergoing their standard dialysis regimens. Hepcidin levels measured at the arterial side decreased with time in all patients during their dialysis treatment (Figure 2A) with a difference before (532 ± 297 ng/ml) and after (292 ± 171 ng/ml) dialysis (P < 0.01; Figure 2B). There was a higher mean hepcidin reduction ratio of 0.53 ± 0.12 in pediatric patients versus 0.33 ± 0.14 in adults (P < 0.01). Although we were able to detect hepcidin in effluent samples, we were unable to quantify it reliably to account fully for the observed arterial-venous difference. Clearance measurements demonstrated a mean hepcidin clearance of 141 ± 40 and 128 ± 44 ml/min in pediatric and adult patients, respectively (NS). The mean plasma disappearance half-life of hepcidin was 5.3 ± 2.7 hours.
Figure 2.
(A) Hepcidin reduction from baseline during HD. Mean serum hepcidin levels as a fraction of their pre-HD values are shown at 30-minute intervals during HD. Error bars denote SD. (B) Hepcidin levels before and after HD. Serum hepcidin levels before the initiation and after termination of HD are shown in eight pediatric and six adult patients. Average pre-HD hepcidin levels were significantly higher than post-HD measurements (see text).
Discussion
In this study, we report detailed quantitative measurements of serum hepcidin levels along with HD clearance measurements of hepcidin in both pediatric and adult patients. Similar to previous studies (7,13), hepcidin levels were elevated severalfold in HD patients, suggesting that hepcidin production and/or clearance is severely altered in the setting of CKD. As expected, the elevation of serum hepcidin seems multifactorial in this particular population, with hepcidin correlating with markers of both iron storage and inflammation. Furthermore, this study demonstrates that hepcidin is decreased by nearly 50% during standard-duration HD treatment.
Consistent with our previous findings in patients with stages 2 through 4 CKD and peritoneal dialysis patients, we demonstrated a strong correlation between current day markers of iron storage and serum hepcidin levels in HD patients. This likely reflects the known regulation of hepcidin by iron stores, which was previously demonstrated in populations without CKD (14). Previous studies that used MS to measure hepcidin also demonstrated a correlation between ferritin and hepcidin in HD patients (13,15). In contrast, Ashby et al. (7), using a radioimmunoassay, did not observe this correlation in adult HD patients, although target-driven intravenous iron therapy may have confounded those results.
Previous studies of humans with chronic infections and severe inflammatory disease have shown markedly increased levels of hepcidin, strongly suggesting that elevated hepcidin levels play a key role in the anemia of inflammation and reticuloendothial blockade (2,16). The correlation between hs-CRP and hepcidin in this study and our previous studies of adult and pediatric patients provides further evidence in support of the relationship between both variables through all stages of CKD (6). Thus, hepcidin may mediate the deleterious effects of inflammation on both iron homeostasis and erythropoiesis across the spectrum of CKD. It is important to note that this study protocol excluded patients with active illness or infection; therefore, a more robust correlation between inflammation and hepcidin may exist in the general population with CKD. Alternatively, the study protocol of holding parenteral iron 1 week before hepcidin measurements may underestimate the effect of iron status on hepcidin levels, and, therefore, the relationships observed between inflammation and hepcidin may be obscured with more frequent iron administration.
The findings of this study clearly demonstrated that hepcidin itself is removed by HD, confirming the results of two previous studies that used an MS-based hepcidin assay (8,9). The results were not unexpected, because hepcidin is a small molecule (2.8 kD), similar in size to vitamin B12. The published in vitro clearance of vitamin B12 using the Revaclear dialyzer (Gambro) is approximately 200 ml/min using a Qb/Qd of 400/800 ml/min. The decreased clearance of hepcidin compared with vitamin B12 may reflect the larger molecular weight of hepcidin and possible binding of hepcidin to plasma proteins in vivo. In addition, ultrafiltration may have reduced our clearance measurements by causing hemoconcentration of hepcidin during dialysis. Our inability to quantify hepcidin reliably in the effluent was likely due to a severalfold dilution of hepcidin in the effluent compared with plasma and the low protein content of the effluent, which likely caused extensive binding of hepcidin to the large surface area of the dialyzer and tubing. The other study that quantified hepcidin clearance by HD using MS determinations showed a somewhat lower clearance of 82 ml/min (8). The two assay methods yield different absolute values of serum hepcidin but are very highly correlated (17) and so should yield similar clearance values. Thus, the different clearance measurements are likely due to different dialysis conditions, including that median blood (300 ml/min) and dialysis (500 ml/min) flows in the MS study were lower than in this study. Finally, we did not observe an inverse correlation between hepcidin and Kt/V, but this again may simply reflect the much larger size of hepcidin when compared with urea. Future studies will need to address the kinetics of hepcidin clearance during dialysis, including studies in which ultrafiltration is not performed and hepcidin levels are measured at several time points after dialysis to assess for rebound. Formal kinetic modeling of hepcidin clearance will allow for estimation of generation rate and volume of distribution of hepcidin and would be valuable for predicting hepcidin removal via various dialysis regimens.
It remains unclear why hepcidin levels were higher in adult than in pediatric HD patients. One possible explanation is that the adults had higher iron stores and ferritin levels in part because of the preponderance of male patients in the adult compared with pediatric groups. In addition, the higher iron requirements as a result of increased tissue and muscle growth and expanding blood volume may account for the differences seen not only in the pediatric and adult HD patients but also between the two control groups. In general, higher iron stores and ferritin levels correlate with higher hepcidin levels (12). An alternative explanation is that dialysis provides more effective hepcidin removal in the pediatric setting. Indeed, the pediatric patients had a higher hepcidin reduction ratio than the adult patients. Whether this may be related to a smaller volume of distribution of hepcidin in pediatric patients or perhaps higher hepcidin production rates in adults remains to be determined.
Conclusions
The increased serum hepcidin levels found in pediatric and adult HD patients may have important diagnostic and potentially therapeutic implications. The lack of sensitivity and specificity of current day markers of iron storage often forces empiric iron dosing in HD patients with anemia, with possible increased iron loading of nonerythropoietic tissues. Hepcidin may in the future improve the targeting and timing of iron therapy by identifying patients during periods of reticuloendothelial blockage of iron transport, when they would likely not benefit from iron therapy. In addition, the clearance of hepcidin via HD suggests a therapeutic intervention in which patients with anemia and high hepcidin levels are changed to a more intensive dialysis regimen to lower circulating hepcidin concentrations, release iron from stores, and increase enteral iron absorption. The end result of lowering hepcidin levels would be improved erythropoiesis and potentially decreased use and safer dosing of ESA and iron therapies.
Disclosures
T.G. and E.N. are co-founders and officers and M.W. is a shareholder and an officer of Intrinsic LifeSciences, LLC.
Acknowledgments
This work was supported in part by National Institutes of Health grants 5RO1DK067563-04 and 1K08DK074284-01 and funds from the Casey Lee Ball Foundation.
Preliminary results were presented in abstract form at the annual meeting of the American Society of Nephrology; October 27 through November 1, 2009; San Diego, CA.
We thank Dr. Larry Froch, Georgina Ramos, and Yolanda Tejedor for help in the recruitment of patients and Dr. Martin Roberts for help in HD clearance measurements.
Footnotes
Published online ahead of print. Publication date available at www.cjasn.org.
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