|Year : 2012 | Volume
| Issue : 3 | Page : 126-131
Serum prohepcidin level in children with chronic kidney disease in relation to iron markers
Doaa M Youssef1, Tarek A Desoki1, Doaa M Tawfeek1, Naglaa A Khalifa2
1 Pediatrics' Department, Zagazig University, Cairo, Egypt
2 Clinical Pathology Department, Zagazig University, Cairo, Egypt
|Date of Web Publication||15-Jan-2013|
Doaa M Youssef
Pediatrics' Department, Zagazig University, Cairo
Source of Support: None, Conflict of Interest: None
Introduction: It is possible that progressive renal insufficiency leads to altered hepcidin metabolism, subsequently affecting the enteric absorption of iron and the availability of iron stores. Because of the previous absence of an accurate serum assay, most studies of hepcidin in humans have been performed using a urinary assay. We conducted this study to evaluate the level of serum prohepcidin in anemia of chronic kidney disease (CKD) patients, with close analysis of data to different hematological parameters, especially iron markers. Subjects and Methods: This is an analytical cross-sectional case-control study involving: Group I - 20 patients of CKD on hemodialysis, nine males and 11 females, and their age ranged from 8.6 to 17 years; Group II - 10 patients of CKD stage 3 and 4, six females and four males, and their age ranged from 8.1 to 15 years on conservative treatment; and Group III, 10 control participants with normal kidney function, seven males and three females, and their with age ranged from 8 to 16 years. Diseased patients had six patients on intravenous iron supplementation. All participants met the following criteria at the time of sampling: A stable clinical state and no thrombosis or infection, with controlled blood pressure, no more than twice of the normal SGOT and SGPT. All participants were subjected to full medical history, thorough clinical examination and laboratory evaluation (kidney functions, liver functions, C-reactive protein, serum levels of hepcidin prohormone using ELISA, serum levels of iron, ferritin and total iron binding capacity (TIBC), hemoglobin, hematocrit, intact parathormon level (iPTH), serum albumin, serum cholesterol and triglycerides. Results : Our study demonstrated a significant rise in serum prohepcidin levels in the diseased groups, a significant rise in serum ferritin and no significant difference between TIBC and serum iron levels between the diseased groups and the control group. A significant positive correlation was demonstrated between serum prohepcidin levels and serum ferritin, while a significant negative correlation was revealed between serum prohepcidin and hemoglobin, hematocrit and TIBC. We did not find a significant correlation between serum prohepcidin and C reactive protein (CRP) as an inflammatory marker in our patients. No significant difference was detected in prohepcidin levels in the patients who were on intravenous iron therapy in comparison with who were not. Conclusion : We concluded that there is a positive association between serum prohepcidin level and serum ferritin and other conventional markers of iron metabolism in CKD patients.
Keywords: Chronic kidney disease, ferritin, prohepcidin
|How to cite this article:|
Youssef DM, Desoki TA, Tawfeek DM, Khalifa NA. Serum prohepcidin level in children with chronic kidney disease in relation to iron markers. Saudi J Health Sci 2012;1:126-31
|How to cite this URL:|
Youssef DM, Desoki TA, Tawfeek DM, Khalifa NA. Serum prohepcidin level in children with chronic kidney disease in relation to iron markers. Saudi J Health Sci [serial online] 2012 [cited 2020 Dec 4];1:126-31. Available from: https://www.saudijhealthsci.org/text.asp?2012/1/3/126/106081
| Introduction|| |
The low hemoglobin levels in patients with chronic kidney disease (CKD) are explained by many factors, including decreased production of erythropoietin by the kidneys,  decreased red blood cell survival,  chronic blood loss,  iron deficiency (which plays a major role necessitating iron administration when in need),  bone marrow suppression due to inadequate dialysis or specific inhibitory substances,  malnutrition with folate and vitamin B12 deficiency,  inflammation and infection suggested by increased inflammatory biomarkers such as interleukin-6 and C-reactive protein through increased oxidative stress,  hyperparathyroidism,  aluminum toxicity or as a consequence of medications such as angiotensin converting enzyme inhibitors. 
Recently, a complex regulatory network that governs iron traffic has emerged, and points to hepcidin as a major evolutionary conserved regulator of iron distribution.  This small hormone produced by the mammalian liver has been proposed as a central mediator of dietary iron absorption. Hepcidin was found to be associated with decreases in both iron uptake from the small intestine and release of iron from macrophages as well as decreased placental iron transport. 
It is demonstrated that erythropoietin downregulates liver hepcidin expression, acting, therefore, as a hepcidin-inhibitory hormone. Anemia/hypoxia seems to be the other factor that regulates hepcidin expression. In fact, anemia is associated with a decrease in hepcidin expression, resulting in an increase in intestinal iron absorption and iron release by the macrophages in order to augment iron availability for erythropoiesis. 
Because of the previous absence of an accurate serum assay, most studies of hepcidin in humans have been performed using a urinary assay. Because such an assay may not reliably reflect serum hepcidin levels in patients with CKD, previous studies have instead attempted to measure the serum levels of prohepcidin, the peptide precursor of hepcidin. 
However, these studies have been difficult to interpret because the relationship between prohepcidin, hepcidin and iron parameters remains unclear. Mass spectrometry is capable of measuring serum hepcidin, and was used to detect a positive correlation between serum hepcidin and ferritin levels in CKD, but this technique is limited by its semiquantitative nature and requirement for equipment that is not widely available. 
Because of its renal elimination and regulation by inflammation, it is possible that progressive renal insufficiency leads to altered hepcidin metabolism, subsequently affecting the enteric absorption of iron and the availability of iron stores. 
We conducted this study to evaluate the level of prohepcidin in anemia of CKD and to evaluate the relation between prohepcidin and other indices in those patients.
| Subjects and methods|| |
This analytical cross-sectional case-control study was carried out on CKD patients attending the Nephrology Unit of the pediatric department at Zagazig University Hospital. Participants were classified into three groups: Group I - 20 patients of end-stage kidney disease (ESKD) on hemodialysis (HD), nine males and 11 females, and their age ranged from 8.6 to 17 years. Dialysis was performed with Fresenius 2008K machines and hollow fiber polysulfone dialyzers (Fresenius, Bad Homburg, Germany) using standard citrate dialysate solution. The dialysis prescription was as follows: Three times a week, 3-5 hours per session, blood flow 300 mL\min, with urea reduction ratio URR >65%. Group II - 10 patients of chronic kidney disease stage 3 and 4, six females and four males, and their age ranged from 8.1 to 15 years on conservative treatment. Group III - 10 control participants with normal kidney function, seven males and three females, and their age ranged from 8 to 16 years. Of the diseased patients, six patients had intravenous iron supplementation.
All participants met the following criteria (inclusion criteria) at the time of sampling: A stable clinical state, no thrombosis or infection with controlled blood pressure and stable and no more than twice of the normal SGOT and SGPT.
All participants were subjected to full medical history regarding age of onset of ESKD, etiology of their disease, duration of disease, duration of dialysis and age at which dialysis began and history of drug administration either in the form of iron therapy or chelation and the need of repeated blood transfusion and the dose of administered recombinant human erythropoietin (rHEPO) either in normal or high dosage. Thorough clinical examination as regards pallor, organomegaly and recurrent infection, laboratory evaluation in the form of kidney functions, liver functions, C-reactive protein, serum levels of hepcidin prohormone using enzyme-linked immunosorbant assay (ELISA), serum levels of iron, ferritin and total iron binding capacity (TIBC), hemoglobin, hematocrit, intact parathormon level (iPTH), serum albumin, serum cholesterol and triglycerides.
Peripheral venous blood samples are withdrawn from all subjects after overnight fast and in HD patients before the session of dialysis. After centrifugation at 3500 rpm at 4°C for 15 min, sera were coded and stored at -80°C until being used for measurements.
Principle of the test
The DRG® Hepcidin Prohormone ELISA kit is a solid phase ELISA based on the principle of competitive binding.
The microtiter wells are coated with a polyclonal antibody directed toward an antigenic site on the Hepcidin Prohormone molecule (28-47 aa). Endogenous Hepcidin Prohormone of a patient sample competes with a Hepcidin Prohormone-biotin conjugate for binding to the coated antibody. After incubation, the unbound conjugate is washed off.
The amount of bound biotin conjugate is reverse-proportional to the concentration of Hepcidin Prohormone in the sample.
After addition of the substrate solution, the intensity of color developed is reverse-proportional to the concentration of Hepcidin Prohormone in the patient sample.
A standard curve is prepared from seven pro-hepcidin standard dilutions and the pro-hepcidin sample concentration is determined and expressed in ng/mL.
Data were checked, entered and analyzed using SPSS version 14. Data were expressed as mean±standard deviation for quantitative variables, number and percentage for qualitative ones. Unpaired t-test, ANOVA and Chi Square (χ2 ) were used when appropriate and least significant difference (LSD) was applied while using ANOVA to find the exact deference between each group and the other one [Table 1]. Correlation significant tests were done between prohepcidin and other parameters [Table 2] and linear regression analysis was done to demonstrate the most prognostic parameter in relation to prohepcidin [Table 3]. P-value <0.05 was considered to be statistically significant.
|Table 2: Correlation between prohepcidin and other parameters in the diseased group (I and II) |
Click here to view
|Table 3: Linear regression analysis of prohepcidin with other parameters |
Click here to view
| Results|| |
[Table 1] shows a significant decline in hemoglobin and hematocrit levels in the studied diseased groups with [Table 3] significant rise in serum urea and creatinine levels. A significant rise in serum ferritin and prohepcidin were detected while there was no significant difference between TIBC and serum iron levels between the diseased groups and the normal control group. There was a significant rise in iPTH levels in the diseased group [I and II] in comparison with the normal control group. A significant rise in serum cholesterol and triglycerides was detected while there was a significant decline in the level of albumin in our patients.
[Table 2] shows that a significant positive correlation was demonstrated between serum prohepcidin levels and serum ferritin, while a significant negative correlation was revealed between serum prohepcidin and hemoglobin, hematocrit and TIBC. Also, we did not find a significant correlation between serum prohepcidin and CRP as an inflammatory marker in our patients.
[Table 3] shows a linear regression analysis of prohepcidin with the other parameters, which identified a highly significant positive correlation with ferritin while there was no significant correlation with any of the other parameters.
| Discussion|| |
The synthesis of hepcidin is stimulated by anemia/hypoxia, inflammation and iron overload. It is synthesized as a pre-prohepcidin of 84 amino acids. The signal peptide is cleaved leading to the 60-amino acid prohepcidin, which is further processed giving rise to the 25-amino acid hepcidin. Hepcidin synthesis is regulated by inflammation, a common finding in HD patients, being enhanced in those that do not respond to rhEPO therapy.  The decreased availability of iron for erythropoiesis leads to the anemia of chronic disease; in HD patients, it aggravates an already existing anemia. HD is now widely considered an inflammatory state probably accounting for the increased serum hepcidin levels that have been associated with it. 
Our study was conducted to evaluate the level of prohepcidin in the serum of children with CKD and the correlation with hemoglobin level, serum iron, ferritin and other indices in those patients.
There was a highly significant reduction in hemoglobin to 9.7±1.1 gm/dL in Group I and 9.4±1.0 in Group II and the hematocrit levels in the studied diseased groups was highly significantly reduced to 29.9±3.1 in Group II and to 29.2±3.1 in Group II.
Concerning the albumin level in our patients, our study revealed that serum albumin is significantly decreased in the diseased groups (I and II) in comparison with the control group (3.4±0.5 gm/dL vs. 3.9±0.3 gm/dL), with a P-value of 0.02, which is significant. Similar results are shown in the study by Rajiv et al. (to demonstrate the serum albumin concentration), which is an important predictor of both baseline hemoglobin and EPO sensitivity in chronic HD patients. In this study, serum albumin was significantly lower (3.8±0.4 gm/dL). 
As regards serum cholesterol and triglycerides in our study, the results showed a significant rise of serum triglycerides in the diseased groups (186±84.5 mg/dL) with P = 0.02, which is significant while serum cholesterol was highly significant elevated (197.3±38.4 mg/dL) with P = 0.001, which is highly significant. The study done by Maheshwari et al. on 75 subjects, 50 on maintenance HD and 25 as controls, showed a highly significant rise in serum triglycerides in the HD patients than in the control group, with P < 0.001 while in contrary to our study, cholesterol showed no significant difference in the two groups.  Elevated serum triglycerides level in uremic patients appears to be caused primarily by the impaired catabolism of triglyceride-rich lipoproteins. This decreased catabolic rate leads to increased quantities of apo-B containing triglyceride-rich lipoproteins in Low density lipoprotein (LDL) and very low density lipoprotein (VLDL) and reduced concentrations of high density lipoprotein (HDL). 
While in our study there was a highly significant rise in serum ferritin in both diseased groups [Group I: 1235.9±1114 ng/mL and Group II: 430.8±431 ng/mL] in comparison with the control group (112±32.8 ng/mL), with a P-value of 0.001, which is highly significant. These data are consistent with the study by Costa et al., which showed a significant rise in serum ferritin levels in HD patients to 334.0 ng/mL (174.0-462.9).  Also, Kalantar-Zadeh et al., in a study on 83 patients, showed a significant rise in the serum ferritin levels with a mean concentration of 831 ng/mL and P = 0.03, which was significant.  Contrary to our study Rafi et al. showed lower levels of serum ferritin in the 19 HD patients (344±197 ng/mL) mostly because of proper iron chelation therapy and better patient compliance.  In addition to the usual rational of anemia in CKD patients, many of our patients are not on the proper dose of erythropiotin hormone due its high cost, which is not covered by the insurance system, and, therefore, they are exposed to frequent blood transfusions due to longer duration of dialysis because of lack of availability of kidney transplantation.
Our data also matched previous experiences in which serum prohepcidin level was significantly increased. In our study, there was a significant rise in serum prohepcidin to 305.7±75.4 ng/mL in Group I and to 271.2±105 ng/mL in Group II, with a P-value <0.01 [Figure 1], which is highly significant. In a study by Natascia et al., the serum hepcidin level in the HD patients was significantly higher as compared with the controls.  In another study by Peters et al., in 48 HD patients, the median hepcidin-25 levels were significantly high. 
In a correlation between prohepcidin and other parameters in our study, we found a significant negative correlation between prohepcidin and hemoglobin, hematocrit and TIBC [Figure 2] (P < 0.001) and significant positive correlation between serum prohepcidin and blood urea, significant positive correlation between serum prohepcidin and iPTH and serum ferritin [Figure 3], while there was no significant correlation between serum prohepcidin and other parameters.
|Figure 2: Significant negative correlation between serum prohepcidin and TIBC levels|
Click here to view
|Figure 3: Significant positive correlation between serum prohepcidin and ferritin level|
Click here to view
In our study, there was a highly significant negative correlation between serum prohepcidin level with the GFR of the studied CKD Group II with r = − 0.862 and P = 0.001, [Figure 4] which is highly significant. Our data matched with the study by Joshua et al., in which hepcidin was inversely correlated to GFR in patients with anemia of CKD grade 2-4. 
|Figure 4: Significant negative correlation between serum prohepcidin and GFR levels in group of CKD stage 3 and 4 on conservative treatment, Group II|
Click here to view
These results matched with the study by Joshua et al., in which they found, in their pediatric patients using univariate analysis, a positive correlation between hepcidin and percentage of iron saturation, ferritin, hsCRP, serum urea and phosphorus and a negative correlation with hemoglobin. 
The results in the study by Joshua et al. (2009) also matched with our results with a positive significant correlation between serum hepcidin and ferritin. 
In our study, there was no significant difference in prohepcidin levels concerning neither sex of the patients nor administration of intravenous iron preparations or iron chelation therapy nor their CRP positivity with a P-value of 0.16, which is not significant. Our data regarding CRP matched with data demonstrated in the study by Natascia et al., in which there was no significant correlation between serum prohepcidin and CRP with correlation coefficient = 0.016 and P = 0.9, which is non-significant.  But, in contrary to our study, Jolanta et al. found a significant positive correlation between serum hepcidin and hs-CRP, with r = 0.37 and P < 0.05. 
In a linear regression analysis of hepcidin with other parameters, we identified a highly significant relation with serum ferritin (0.007±0.025) with P < 0.01, which is highly significant while showing no correlation with albumin, hemoglobin, neither intact parathormon nor TIBC.
In a study by Joshua et al., multivariate regression models were developed to assess the relationship between serum hepcidin, hemoglobin, percentage of iron saturation, ferritin, hs-CRP, 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 (R 2 = 0.70). 
In another study by Roe et al. to emphasize the predictive capacity of hepcidin of interindividual variation in iron absorption in healthy men, a multilinear regression analysis in which the continuous variables plasma hepcidin, serum ferritin, serum iron, total iron binding capacity, transferrin saturation and plasma transferrin receptor significantly predicted log iron absorption. In a stepwise regression procedure, the reduced model that best predicted log iron absorption contained only serum ferritin and total iron binding capacity. Because serum ferritin was highly correlated with plasma hepcidin, multiple linear regression analysis was repeated using the same continuous variables but with serum ferritin excluded in a stepwise regression procedure; the reduced model that best predicted log iron absorption contained only plasma hepcidin and total iron binding capacity. 
We concluded that there is a positive association between serum prohepcidin and serum ferritin and other conventional markers of iron metabolism in CKD. Therefore, we recommend repeated evaluation of serum ferritin level in patients of CKD as it is expected to be elevated in correlation with elevated serum prohepcidin.
| References|| |
|1.||Locatelli F, Aljama P, Bárány P, Canaud B, Carrera F, Eckardt KU, et al. Revised European best practice guidelines for the management of anemia in patients with chronic renal failure. Nephrol Dial Transplant 2004;19, 2: ii1-47. |
|2.||Eknoyan G, Latos DL, Lindberg J. Practice recommendations for the use of L-carnitine in dialysis-related carnitine disorder. National Kidney Foundation Carnitine Consensus Conference. Am J Kidney Dis 2003;41:868-76. |
|3.||Tarif N, Mitwalli AH, Al-Wakeel JS, Aisha HA, Memon A, Sulaimani F, et al. Safety of intravenous ferrous saccharate administration in hemodialysis patients. A2136 Abstract from the Annual Meeting and Scientific Exposition of the American Society of Nephrology; 2001. |
|4.||Frankenfield DL, Neu AM, Warady BA, Fivush BA, Johnson CA, Brem AS. Anemia in pediatric hemodialysis patients: Results from the 2001 ESRD Clinical Performance Measures Project. Kidney Int 2003;64:1120-4. |
|5.||Schiffl H and Lang SM. Folic acid deficiency modifies the haematopoietic response to recombinant human erythropoietin in maintenance dialysis patients. Nephrol Dial Transplant 2006;21:133-7. |
|6.||Himmelfarb J, Stenvinkel P, Ikizler TA, Hakim RM. The elephant in uremia: Oxidant stress as a unifying concept of cardiovascular disease in uremia. Kidney Int 2002;62:1524-38. |
|7.||Duarte ME, Carvalho EF, Cruz EA, Lucena SB, Andress DL. Cytokine accumulation in osteitis fibrosa of renal osteodystrophy. Braz J Med Biol Res 2002;35:25-9. |
|8.||Arbeiter K, Greenbaum L, Balzar E, Müller T, Hofmeister F, Bidmon B. et al. Reproducible erythroid aplasia caused by mycophenolate mofetil. Pediatr Nephrol 2000;14:195-7. |
|9.||Kulaksiz H, Gehrke SG, Janetzko A, Rost D, Bruckner T, Kallinowski B, et al. Pro-hepcidin: Expression and cell specific localization in the liver and its regulation in hereditary hemochromatosis, chronic renal insufficiency, and renal anemia. Gut 2004;53:735-43. |
|10.||Nicolas G, Bennoun M, Porteu A, Mativet S, Beaumont C, Grandchamp B, et al. Severe iron deficiency anemia in transgenic mice expressing liver hepcidin. Proc Natl Acad Sci USA 2002;99:4596-601. |
|11.||Costa E, Swinkels DW, Laarakkers CM, Rocha-Pereira P, Rocha S, Reis F, et al. Hepcidin serum levels and resistance to recombinant human erythropoietin therapy in hemodialysis patients. Acta Haematol 2009;122:226-9. |
|12.||Halevy D. Relevance of aluminum screening in Hemodialysis patients. Am J Kidney Dis 2006;28:115-21. |
|13.||Kato A, Tsuji T, Luo J, Sakao Y, Yasuda H, Hishida A. Association of prohepcidin and hepcidin-25 with erythropoietin response and ferritin in hemodialysis patients. Am J Nephrol 2008;28:115-21. |
|14.||Zaritsky J, Young B, Wang HJ, Westerman M, Olbina G, Nemeth E, et al. Hepcidin-A potential novel biomarker for iron status in chronic kidney disease. Clin J Am Soc Nephrol 2009;4: 1051-6. |
|15.||Rajiv A, Davis J, and Smith L. Serum albumin is strongly associated with erythropoietin sensitivity in hemodialysis patients. Clin J Am Soc Nephrol 2008;3:98-104. |
|16.||Ansari MR, Maheshwari N, Shaikh MA, Laghari MS, Darshana, Lal K, et al. Pattern of lipid profile in patients on maintenance hemodialysis. Saudi J Kidney Dis Transpl 2010;21:565-70. |
|17.||de Gómez Dumm NT, Giammona AM, Touceda LA, Raimondi C. Lipid abnormalities in chronic renal failure patients undergoing hemodialysis. Medicina (B Aires) 2001;61:142-6. |
|18.||Kalantar-Zadeh K, McAllister CJ, Lehn RS, Liu E, Kopple JD. A low serum iron level is a predictor of poor outcome in hemodialysis patients. Am J Kidney Dis 2004;43:671-84. |
|19.||Rafi A, Karkar A, Abdelrahman M. Monitoring iron status in end-stage renal disease patients on hemodialysis. Saudi J Kidney Dis Transpl 2007;18:73-8. |
|20.||Natascia C, Castagna A, Zaninotto F. Evaluation of hepcidin isoforms in hemodialysis patients by a proteomic approach based on SELDI-TOF MS. J Biomed Biotechnol 2010;2010:329646. |
|21.||Peters HP, Laarakkers CM, Wetzels JF, Swinkels DW. Serum hepcidin-25 levels in patients with chronic kidney disease are independent of glomerular filtration rate. Nephrol Dial Transplant 2010;25:848-53. |
|22.||Zaritsky J, Young B, Gales B, Wang HJ, Rastogi A, Westerman M, et al. Reduction of serum hepcidin by hemodialysis in pediatric and adult patients. Clin J Am Soc Nephrol 2010;5: 1010-4. |
|23.||Malyszko J, Mysliwiec M. Hepcidin in anemia and inflammation in chronic kidney disease. Kidney Blood Press Res 2007;30:15-30. |
|24.||Roe MA, Spinks C, Heath AL, Harvey LJ, Foxall R, Wimperis J, et al. Serum prohepcidin concentration: No association with iron absorption in healthy men; and no relationship with iron status in men carrying HFE mutations, hereditary haemochromatosis patients undergoing phlebotomy treatment, or pregnant women. Br J Nutr 2007;97:544-9. |
[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2], [Table 3]