Hyporesponsiveness to Erythropoietin Therapy in Hemodialyzed Patients: Potential Role of Prohepcidin, Hepcidin, and Inflammation

Abstract

Hepcidin is the key regulator of iron metabolism. Iron supplementation is often introduced in dialyzed patients to replete or to maintain iron stores, particularly in patients treated with erythropoietic-stimulating agents. The present study was aimed to assess possible relation between hepcidin and erythropoietin therapy, with particular attention being paid to erythropoietin-hyporesponsiveness in hemodialyzed patients. Prohepcidin and hepcidin were studied using commercially available kits from DRG Instruments GmbH, Germany (ELISA method) and Bachem, UK (RIA method). TNFα and IL-6 were studied using kits from and R&D (Abington, UK), and hsCRP was studied using kits from American Diagnostica, USA. Hyporesponsive patients to erythropoietin therapy had significantly lower serum albumin, cholesterol, LDL, hemoglobin, hematocrit, and residual renal function, and significantly higher serum ferritin, hsCRP, IL-6, TNFα, and erythropoietin dose. The difference in serum prohepcidin and hepcidin did not reach statistical significance; however, there was a tendency toward higher values of both prohepcidin and hepcidin in hyporesponsive patients. In conclusion, though hyporesponsiveness to erythropoietin therapy occur in dialyzed patients, it is mainly associated with subclinical inflammation than with hepcidin excess. Further studies are needed to develop a reliable and reproducible assay to elucidate the potential contribution of hepcidin to hyporesponsiveness during erythropoietin therapy.

Keywords:

• prohepcidin
• hepcidin
• hemodialyses
• hyporesponsiveness

INTRODUCTION

Anemia has remained one of the most characteristic and visible manifestations of chronic renal failure for over 150 years. Typically, it is a normocytic and normochromic anemia with bone marrow of normal cellularity. The pathogenesis of anemia of chronic kidney disease is multifactorial,[1] though inadequate production of erythropoietin is the most important factor in the pathogenesis of anemia in chronic kidney disease. Adequate iron stores are essential for achieving maximum benefit from the erythropoietin-stimulating agents' ESA therapy. Normally, three-quarters of a body's iron is present in circulating erythrocytes, and one-quarter is stored mainly in the liver and bone marrow. Because the demand for iron by the erythroid marrow frequently exceeds iron stores once ESA therapy has been initiated, iron supplementation is essential. More than 90% of patients respond adequately to erythropoietin-stimulating agents (erythropoietin alfa, beta, or darbepoetin-alfa). Patients with a chronic kidney disease have a chronic inflammatory state. This is due to many underlying factors, including an enhanced incidence of infections, the uremic milieu, elevated levels of proinflammatory cytokines, frequent presence of widespread arteriosclerosis, and others. There are additional factors on hemodialyses, which may contribute to this process (e.g., impure dialysate, bioincompatible dialysis membranes, intercurrent infections).[2–8] An increasing attention has focused on inflammation as a possible cause of the wasting syndrome in end-stage renal disease as well as anemia refractory to ESA therapy. Allen et al.[9] put forward a hypothesis that patients requiring higher doses of erythropoietin may have greater “inflammatory activity” and cytokine propagation. In addition, iron metabolism is disturbed in chronic inflammatory diseases. Our understanding of the molecular control of iron metabolism has increased dramatically over the past several years due to the discovery that hepcidin, a peptide hormone produced in the liver, has primary responsibility for modulating iron availability to meet iron needs.[10] Hepcidin is primarily made in hepatocytes and secreted into the circulation. The liver acts as a clearinghouse for a variety of signals affecting iron homeostasis. In 2002, Nicolas et al.[11] discovered that the gene encoding hepcidin was regulated in response to anemia, hypoxia, and inflammation. The link between hepcidin and anemia of chronic diseases was presented by Fleming and Sly.[12]

Taking all these data into account, the present study was aimed to assess possible relation between hepcidin and rHuEPO therapy, with particular attention being paid to erythropoietin hyporesponsiveness in hemodialyzed patients.

PATIENTS AND METHODS

The study was performed on 98 hemodialyzed patients (age range 26–82 years, 45F, 53M) who met the following criteria: a stable clinical state, no thrombosis, no inflammation/infection (C-reactive protein within normal range, below 6 mg/L using standard laboratory methods), no uncontrolled hypertension, stable, and no more than twice of the normal GOT and GPT activities (upper range 45 IU/L). All the patients required regular hemodialyses for 4–5 h a day three times a week for a mean time of 48 ± 40 months (range 8–179 months). Blood flow was usually 180–280 mL/min with a dialysate flow of 500 mL/min. Vascular access for HD was a native arterio-venous fistula on the forearm (n = 74) or arm (n = 10), PTFE graft (n = 10), or catheter (n = 4). Ultrafiltration varied according to patient's actual weight. All the patients were dialyzed using low-flux polysulfone membranes (Fresenius, Bad Homburg, Germany n = 75) or other low-flux modified cellulose dialyzers (n = 23) with bicarbonate-buffered dialysate. The causes of renal failure among HD patients varied between chronic glomerulonephritis (n = 31), chronic interstitial nephritis (n = 11), polycystic kidney disease (n = 11), amyloidosis (n = 8), or others/unknown (n = 21). Among hemodialyzed patients treated with rHuEPO or darbepoetin alfa (Eprex, Janssen-Cilag, Neorecormon, Roche, Aranesp, Amgen), a group with inadequate response to therapy was selected. Patients were enrolled on the basis of the fulfilling all three of the following criteria: hemoglobin <10 g/dL, ferritin ≥100 ng/mL, and weekly dose of rHuEPO of ≥9000 IU.

All patients were tested for the HBV and anti-HCV antibodies: 10 were anti-HCV-positive, 7 were HBV-positive, and 2 were positive for both HBV and anti-HCV.

All HD patients' blood was drawn in the morning between 8:00 and 9:00 AM, before the onset of a midweek dialysis session (and heparin administration), after hemodialysis from the arterial line of hemodialysis system, and immediately before discontinuation of the extracorporeal circulation (only for urea concentration necessary for Kt/V determination, according to Sargeant and Gotch, a marker of adequacy of dialysis). During hemodialysis, enoxaparin-Clexane (Aventis) was given as an anticoagulant, in a single intravenous injection at the beginning of each dialysis session. Samples were aliquotted and stored at −40°C before assay. All patients were informed about the aim of the study and gave their consent. The study was approved by the local Medical University Ethic Committee. The following biochemical parameters were assessed: complete blood count, serum iron, total iron binding capacity, ferritin, CRP, albumin, and serum lipids were assayed by means of standard laboratory methods in the central laboratory before and after the therapy. Prohepcidin was studied using commercially available kits from DRG Instruments GmbH (Marburg, Germany), and CRP was assayed by a high-sensitivity method using commercially available kits from American Diagnostica (Greenwich, Connecticut, USA). Hepcidin was assayed by RIA using commercially available kits form Bachem, UK. TNFα and IL-6 were studied using kits from and R&D (Abington, UK). Data were expressed as means ± SD. The data given were analyzed using Statistica 6.0 computer software. The examination of the distribution normality of variables was done using W Shapiro-Wilk test. The data were also logarithmically transformed to achieve normal distribution whenever possible. Measurements normally distributed are reported as mean ± SD; non-normally distributed data are expressed as a median and minimal-maximal value. The Mann-Whitney rank sum U test or Student's t-test were used in statistical analysis to compare differences between groups, with p < 0.05 considered statistically significant, when appropriate.

RESULTS

All the results are presented in Table 1. Hyporesponders to erythropoietin had significantly lower serum albumin, cholesterol, LDL, hemoglobin, hematocrit, and residual renal function; and significantly higher serum ferritin, hsCRP, IL-6, TNFα, and erythropoietin dose. The difference in serum prohepcidin and hepcidin did not reach statistical significance (p = 0.1); however, it was a tendency to higher values of both prohepcidin and hepcidin in hyporesponsive patients.

Table 1 Biochemical characteristics of hemodialyzed patients in regard to response to ESA therapy

	Responsive to ESA therapy (n = 89)	Hyporesponsive to ESA therapy (n = 9)
Age (years)	58.54 ± 14.41	59.33 ± 16.11
RRF (ml)	600 (0−3100)	80 (0−520)*
Time on HD (months)	46.81 ± 40.79	58.5 ± 32.77
Kt/V	1.247 ± 0.19	1.14 ± 0.23^‡
Hemoglobin (g/dL)	12.74 ± 3.39	9.71 ± 1.33^‡
Hematocrit (%)	34.80 ± 3.65	29.42 ± 1.41^‡
Cholesterol (mg/dL)	182.48 ± 48.37	140.5 ± 14.49*
HDL (mg/dL)	43.12 ± 12.38	39.83 ± 11.11
LDL (mg/dL)	109.24 ± 31.47	79.17 ± 11.21*
Triglycerides (mg/dL)	162.02 ± 71.53	130.83 ± 49.92
Albumin (g/dL)	4.00 ± 0.37	3.61 ± 0.23^†
TNFα (pg/dL)	21.84 ± 16.12	38.93 ± 18.29^‡
IL-6 (pg/mL)	9.21 ± 4.86	18.40 ± 7.4^‡
hsCRP (mg/L)	4.45 ± 3.12	9.81 ± 4.49^†
Serum iron (ng/mL)	81.74 ± 41.78	75.83 ± 31.52
Ferritin (ng/mL)	344.9 ± 244.9	657.83 ± 290.45^‡
TIBC (ng/mL)	251.91 ± 72.46	217.044.48 ± 17.54
Hepcidin (ng/mL)	135.35 ± 100.04	198.58 ± 116.33
Prohepcidin (ng/mL)	184.93 ± 53.85	243.55 ± 74.75

*p < 0.05, ^†p < 0.01 hyporesponsive vs responsive to ESA therapy; ^‡p < 0.001.

Data given are means ± SD.

Patients positive for HBV and/or anti-HCV were dialyzed significantly longer (108 ± 80 vs. 37 ± 35 months, p < 0.001) and had higher TIBC, whereas patients positive for anti-HCV had also higher activities of alanine and aspartate aminotransferases (alkaline phosphatase). There was no statistical significant difference in hepcidin and prohepcidin.

Patients with ADPKD had higher hemoglobin (12.27 ± 1.42 vs 10.85 ± 1.24 g/dL, p < 0.01), hematocrit, and red blood cell count, they required lower dose of erythropoietin-stimulating agents, and they had a tendency to lower hsCRP. They did not differ in regard to hepcidin and prohepcidin.

Patients dialyzed using a catheter had significantly higher ferritin (598.9 ± 81.3 vs. 318.9 ± 214.8 mg/dL), TNFα and IL-6, and hsCRP, and lower hemoglobin, hematocrit, and red blood cell count. They did not differ in regard to hepcidin and prohepcidin; however, it was a trend for their higher levels.

DISCUSSION

Some patients are relatively resistant to erythropoietin and require large doses. This may be an important clinical observation, as higher doses of erythropoietin have been associated with an increased mortality, an effect that persists after adjustment for the usually lower hematocrit in such patients.[13,14] A large erythropoietin requirement is defined as either the requirement of excessive doses during initiation of therapy or an inability to achieve or maintain target Hgb levels despite the large dose in the iron-replete patient. Although there is no universally accepted definition of hyporesponsiveness to erythropoietin, different guidelines have suggested different definitions.[15,16] Another approach is to define unresponsive patients as those who receive doses greater than 90 percent of patients in a given facility. We defined a HD patient as a nonresponder if the hemoglobin concentration was below 10 g/dL by administered weekly rHuEPO dose of ≥9000 IU and with serum ferritin ≥100 ng/mL. In the population studied, nonresponders made 10% of all of the patients studied. We found that patients hyporesponsive to rHuEPO had lower serum albumin, serum cholesterol, LDL, and residual renal function; and higher proinflammatory cytokines, hsCRP, and ferritin. In addition, there was a tendency to lower Kt/V and higher hepcidin and prohepcidin in hyporesponsive patients. Taken together, our findings indicated that serum prohepcidin or hepcidin measurement is only modestly suggestive of the response to rHuEPO therapy in hemodialyzed patients.

The most common cause of resistance to rHuEPO is absolute iron deficiency, most probably due to external blood losses and/or exhaustion of iron stores due to an increase in erythropoiesis caused by rHuEPO treatment. Patients with functional iron deficiency may also respond to supplemental iron administration with an increase in hemoglobin level and/or reduction in rHuEPO dose. To exclude these possibilities, we defined hyporesponsiveness on the basis of fulfilling of all the following criteria: hemoglobin less than 10 g/dL, ferritin ≥100 ng/mL, and weekly dose of rHuEPO ≥9000 IU. We looked very carefully at other additional causes of rHuEPO hyporesponsiveness, including bone disease due to secondary hyperparathyroidism, occult malignancy, and unsuspected hematologic disorder (vitamin B12 and folic acid deficiency), multiple myeloma/myelofibrosis/myelodysplastic syndrome, and the administration of angiotensin-converting enzyme inhibitors and/or angiotensin II receptor antagonists. All of these possible causes were excluded in hyporesponsive patients. In well-dialyzed patients who are iron replete, the acute-phase response is the most important predictor of rHuEPO resistance.[17–20] Therefore, we looked at chronic inflammation (with inhibition possibly due to enhanced cytokine production).[1] In our patients, both proinflammatory cytokines (IL-6 and TNFα) and hsCRP were significantly elevated in hyporesponsive patients. Chronic renal failure has been associated with the impaired immunity and subclinical inflammation. In humans, declining renal function may also affect the levels of inflammatory molecules, as serum C-reactive protein (CRP) and interleukin-6 levels are inversely correlated with creatinine clearance.[22] Deteriorating renal function may enhance overall inflammatory responses because of the decreased renal clearance of factors that are directly or indirectly involved in inflammation. In hyporesponsive patients, residual renal function was significantly lower than in responders. Occult infection of old nonfunctioning a-v fistula is a common cause of erythropoietin resistance and chronic inflammatory state among hemodialysis patients.[20] Higher rHuEPO doses may be required to maintain similar or slightly lower mean hemoglobin values among chronic hemodialysis patients with higher numbers of catheter insertions and a-v fistula infections, compared to patients without any.[21] In our hyporesponsive group, only 2 patients had proximal a-v fistula; they were dialyzed for more than five years without any residual renal function. None of them had a catheter. No hyporesponsive patients showed any signs of occult infections. In the recent paper of Bradbury et al.,[23] CRP appeared to be an independent predictor of greater erythropoietin requirements. In our study, hyporesponsive patients had significantly higher hsCRP than responders as in the study of Kato et al.[24] We also found lower serum cholesterol, LDL, and albumin, as well as higher hsCRP and proinflammatory cytokines, suggesting MIA syndrome, a finding not confirmed by Kato et al.[24] They observed no difference in hepcidin-25 (measured using SELDI-TOF), whereas in our study, a tendency to higher hepcidin levels (measured by RIA) were found. Similarly, in the study of Shinzato et al.,[25] no significant difference in serum prohepcidin was found between HD patients with rHuEPO-resistant anemia and HD patients without anemia and iron deficiency. It has been hypothesized that hyperhepcidinemia may explain reduced sensitivity to rHuEPO in dialyzed patients.[26,27] In a recently published study, Ashby et al.[28] reported an association between high hepcidin and low hemoglobin, but were also strongly and independently related to low erythropoietin dose, regardless of whether erythropoietin dose was measured as an absolute value or normalized by body weight or by hemoglobin. The correlation was consistent across each tertile of hemoglobin in dialysis patients. Moreover, they measured hepcidin levels in seven CKD patients as they were starting darbepoetin alfa therapy for the first time and found that hepcidin fell during the first few days and remained at similar levels after 2–4 weeks of continued therapy. They concluded that hepcidin was rather not a potential marker of erythropoietin resistance. They suggested that erythropoietin therapy could suppress hepcidin, ameliorate disordered iron transport, and treat erythropoietin deficiency. They also suggested that altered hemoglobin levels in renal disease were not due to changes in growth differentiation factor 15, which might function as signal from active bone marrow to suppress hepcidin expression.[29] In the study of Vokurka et al.,[30] a different approach was chosen: erythropoiesis was inhibited by irradiation or posttransfusion polycythemia and stimulated by phenylhydrazine administration and erythropoietin in animal model. They found that hepcidin expression decrease was caused by exogenous erythropoietin and was blocked by postirradiation bone marrow suppression. They proposed that hepcidin was exclusively sensitive to iron utilization for erythropoiesis and hepatocyte iron balance, and these changes were not sensed by other genes involved in the control of iron metabolism in the liver. It may explain the lack of differences in hepcidin and prohepcidin between HBV and/or antiHCV-positive patients and subjects with ADPKD when compared to the population without this conditions.

In concluding, however, that hyporesponsiveness to erythropoietin therapy occurs in dialyzed patients, it should be noted that it is mainly associated rather with subclinical inflammation than with hepcidin excess. Further studies are needed to develop a reliable and reproducible assay to elucidate the potential contribution of hepcidin to hyporesponsiveness to erythropoietin therapy.