|Year : 2017 | Volume
| Issue : 4 | Page : 125-131
Association of vitamin D deficiency with renal anemia and erythropoietin hyporesponsiveness in hemodialysis patients
Yaser A Ammar1, Yaser A Nienaa2, Salah S El-Banawy1, Thanaa F Moghazy3, Noha S Kandil3, Amira A El-Sayed1
1 Department of Internal Medicine, Medical Research Institute, Faculty of Medicine, Alexandria University, Alexandria, Egypt
2 Department of Internal Medicine, Faculty of Medicine, Alexandria University, Alexandria, Egypt
3 Department of Chemical Pathology, Medical Research Institute, Faculty of Medicine, Alexandria University, Alexandria, Egypt
|Date of Submission||11-Jul-2017|
|Date of Acceptance||12-Nov-2017|
|Date of Web Publication||17-Jan-2018|
Dr. Yaser A Ammar
Department of Internal Medicine, Medical Research Institute, Alexandria University, Alexandria, 21561
Source of Support: None, Conflict of Interest: None
Background Many maintenance hemodialysis (MHD) patients exhibit erythropoietin (EPO) hyporesponsiveness. An inverse association has been found between vitamin D levels and EPO requirements in patients with chronic kidney disease. Vitamin D supplementation may improve response to EPO by either suppression of the chronic inflammatory status, control of hyperparathyroidism, or direct stimulation of erythroid progenitors.
Aim A prospective clinical study is needed to assess the potential therapeutic role of vitamin D supplementation on EPO resistance index (ERI) in MHD patients.
Methods A total of 30 patients with anemia on MHD for more than 6 months were included. They were on standard anemia therapy with subcutaneous (SC) EPO 4000 U and intravenous iron sucrose 100 mg once or twice weekly. A total of 20 age- and sex-matched healthy individuals were included as controls. Baseline laboratory studies included complete blood picture, calculation of ERI [weekly EPO dose/body weight in kg/hemoglobin (Hb) level], serum iron, total iron-binding capacity, ferritin, hepcidin, calcium, phosphorus, alkaline phosphatase (ALP), intact parathyroid hormone, 25(OH) vitamin D (vitamin D3), C-reactive protein, and interleukin (IL)-6. The studies were repeated after 3 months of oral α-calcidol therapy (2 µg thrice weekly, with each dialysis session).
Results Hb increased significantly from 8.34±0.9 to 9.48±0.9 g/dl (P=0.000), and ERI decreased significantly from 7.39±1.13 to 6.61±1.2 IU/kg/g/dl (P=0.000). Inflammatory markers (serum C-reactive protein, IL-6, ferritin, and hepcidin) decreased significantly (P=0.000 for all). Serum intact parathyroid hormone and ALP decreased significantly (P=0.007 and 0.000, respectively). At the start of the study, there was a significant positive correlation between ERI and serum ferritin (P=0.026), and a significant negative correlation between serum vitamin D3 level and ALP (P=0.004). At the end of the study, there was a significant negative correlation between serum vitamin D3 level and each of serum ferritin (P=0.005) and IL-6 (P=0.019).
Conclusion A 3-month course of oral α-calcidol significantly ameliorates hyperparathyroidism and inflammatory markers, increases Hb, and decreases ERI in MHD patients.
Keywords: anemia, erythropoietin resistance, hemodialysis, inflammation, vitamin D
|How to cite this article:|
Ammar YA, Nienaa YA, El-Banawy SS, Moghazy TF, Kandil NS, El-Sayed AA. Association of vitamin D deficiency with renal anemia and erythropoietin hyporesponsiveness in hemodialysis patients. J Egypt Soc Nephrol Transplant 2017;17:125-31
|How to cite this URL:|
Ammar YA, Nienaa YA, El-Banawy SS, Moghazy TF, Kandil NS, El-Sayed AA. Association of vitamin D deficiency with renal anemia and erythropoietin hyporesponsiveness in hemodialysis patients. J Egypt Soc Nephrol Transplant [serial online] 2017 [cited 2018 May 23];17:125-31. Available from: http://www.jesnt.eg.net/text.asp?2017/17/4/125/223409
| Introduction|| |
Anemia affects nearly all patients with end-stage renal disease (ESRD) treated with maintenance hemodialysis (MHD). It is associated with reduced quality of life and increased cardiovascular disease, hospitalizations, cognitive impairment, and mortality . Anemia in patients with chronic kidney disease (CKD) is multifactorial, but its main mechanism is an erythropoietic hypoproliferative state owing to relative insufficiency of erythropoietin (EPO) production by the failing kidneys . The use of recombinant EPO has revolutionized the management of renal anemia by improving the patients’ debilitating symptoms and freeing them from dependence on blood transfusions with their attendant risks . Nevertheless, ∼5–10% of patients exhibit an inadequate response to EPO. The definition of EPO hyporesponsiveness/resistance has been introduced to identify the inability to achieve or maintain target hemoglobin levels despite higher than usual EPO doses . Identification of causes that enhance EPO responsiveness can optimize anemia management and improve financial costs and safety of EPO therapy .
Recently, an inverse association has been found between vitamin D levels and EPO requirements in patients with CKD . In the progressive course of CKD, vitamin D abnormalities not only include decreased renal activation but also deficiency of serum 25(OH)D3 owing to insufficient sunlight exposure, malnutrition, and increased urinary losses in proteinuric nephropathies . CKD is largely considered an inflammatory condition . Most studies concerning vitamin D deficiency or supplementation, and degree of renal anemia, point out the prevalent role of inflammation in the mechanisms underlying these associations . Proinflammatory cytokines such as interleukin (IL)-1, IL-6, tumor necrosis factor-α, and interferon-γ inhibit differentiation and proliferation of the marrow erythroid progenitors . Hepatic hepcidin synthesis increases because of IL-6 induction leading to iron retention in macrophages and enterocytes, with a consequent reduction in its availability for erythropoiesis .
Alternative hypotheses that were raised to explain the effects of vitamin D supplementation on anemia and EPO requirements in patients with CKD include its suppressive effect on parathyroid hormone (which may have direct inhibitory effects on early erythroid progenitors, EPO synthesis, and red cell survival)  and a direct stimulant action for vitamin D on bone marrow erythroid progenitors . These hypotheses remain a matter of debate. Further studies are needed to confirm the relationship between vitamin D deficiency and EPO hyporesponsiveness and to explore whether vitamin D supplementation exerts a therapeutic role in renal anemia.
| Patients and methods|| |
The study involved 50 participants enrolled in two groups:
- Group 1: 30 patients with ESRD who have been on MHD for more than or equal to 6 months and who fulfilled the following criteria:
- They have been maintained on treatment for renal anemia (intravenous iron and SC EPO) as per standard guidelines  for at least 3 months, and they still have some degree of anemia that warrants continuation of such therapy.
- They are indicated as well to receive vitamin D therapy for treatment of CKD-mineral bone disease according to standard guidelines .
- They do not have any acute inflammatory condition.
- Group 2: 20 healthy participants of comparable age and sex as controls.
Written informed consent was obtained from all participants. The study was approved by the ethics committee of the Faculty of Medicine, Alexandria University. It copes with the Helsinki Declaration of 1975, as revised in 2000.
For all participants (controls and patients), 10 ml of nonheparinized venous blood was withdrawn. A volume of 1 ml was put in EDTA tube for complete blood picture. Serum separated by centrifugation of the remaining sample was put in seven eppendorf tubes.
Two eppendorf tubes were immediately used to estimate the following:
- Serum iron, total iron-binding capacity (TIBC), and hence calculation of transferrin saturation (TSAT) (percentage of serum iron to TIBC) , using Cobas 6000 autoanalyzer (Roche Diagnostic International Ltd, Risch-Rotkreuz, Switzerland).
- Serum calcium and phosphorus and calculation of their product .
- Serum intact parathyroid hormone (iPTH) .
- Serum alkaline phosphatase (ALP) .
These investigations were used to identify patients eligible for EPO and vitamin D therapy, who were enrolled in the study.
The other five eppendorf tubes were stored at −20°C and used later on for estimation of the following:
- Serum ferritin .
- Serum hepcidin  using noncompetitive sandwich enzyme immunoassay (Human Hepcidin ELISA Kit, catalog number 95618; Glory Science Co. Ltd, Del Rio, Texas, USA).
- Serum C-reactive protein (CRP)  using Olympus AU 400 clinical chemistry autoanalyzer, Olympus, Corporate Parkway Center Valley, Pennsylvania, USA.
- Serum IL-6  using noncompetitive sandwich enzyme immunoassay principle (Assay Max Human IL-6 ELISA Kit, catalog number EI 1006-1, Assaypro, St. Charles, Missouri, USA).
- Serum 25(OH) vitamin D  using commercial kit from Dia Metra Company (ref. number DK0146; Italy).
Other studies for the 30 MHD were done as shown in [Figure 1]:
- Week (-1): Treatment with vitamin D and intravenous iron was temporarily held for 1 week, to minimize potential interference with laboratory results.
- Week (0): Fasting predialysis nonheparinized blood samples were withdrawn for the main panel of laboratory studies. In addition, 2 ml venous blood sample was taken in EDTA tubes for estimation of hemoglobin (Hb) level (Hb1).
- Week (2): Hb level was assessed once again (Hb2). The two Hb readings were averaged to provide mean Hb level (Hbpre), which was used as the denominator to calculate EPO resistance index (ERI) , before α-calcidol therapy (ERIpre), where:
The patients were given the following medications as per standard guidelines:
- Iron sucrose 100 mg intravenous slowly at the end of the dialysis session once or twice weekly.
- EPO 4000 IU SC once weekly.
- α-calcidol capsules: 2 µg orally with each dialysis session (thrice weekly).
- Week (12): Hb level was assessed (Hb3).
- Week (13): Treatment with vitamin D and intravenous iron was temporarily held for 1 week, to minimize potential interference with laboratory results.
- Week (14): Blood samples were withdrawn to repeat the complete panel of laboratory studies. Hb level (Hb4) was averaged with Hb3 to obtain Hbpost which was used to calculate ERI after α-calcidol therapy (ERIpost).
|Figure 1 Study design. Vitamin D and intravenous iron were temporarily held for 1 week at the start and at the end of the study to minimize potential interference with laboratory results.|
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Statistical methods 
Data were analyzed using SPSS software package version 20 (SPSS Inc., Chicago, Illinois, USA). Scale variables were tested for normality using Kolmogorov–Smirnov test. Parametric scale data were represented as mean±SD and compared by Student’s t-test (paired or unpaired as appropriate). Nonparametric scale data were represented as median (range) and compared by Wilcoxon’s signed rank test (paired data) or Mann–Whitney U-test (unpaired data). Categorical variables were compared using χ2-test. Correlation between different scale variables was tested by Spearman’s rank correlation coefficient, which suits comparisons involving nonparametric data. Significance of the obtained results was judged at the 5% level.
| Results|| |
[Table 1] shows that the control and MHD patient groups were comparable regarding sex and age. Following α-calcidol therapy of MHD patients, Hb level increased significantly from 8.34±0.9 to 9.48±0.9 g/dl (P=0.000) ([Figure 2]), whereas ERI decreased significantly from 7.39±1.13 to 6.61±1.2 IU/kg/g/dl (P=0.000) ([Figure 3]). Parameters of iron status (serum iron, TIBC, TSAT, ferritin, and hepcidin) had all decreased; the decrease was statistically significant only for serum ferritin and hepcidin (P=0.000, for both). The serum calcium×phosphorus product and vitamin D3 increased significantly whereas serum ALP, iPTH, CRP, and IL-6 had all significantly decreased. At the study start, there was a significant positive correlation between ERI and serum ferritin (r=0.407, P=0.026), and a significant negative correlation between serum vitamin D3 and ALP (r=−0.509, P=0.004). At the end of the study, there was a significant negative correlation between serum vitamin D3 level and each of serum ferritin (r=−0.496, P=0.005) and IL-6 (r=−0.428, P=0.019) ([Table 2] and [Figure 4]).
|Table 1 Comparison between controls and maintenance hemodialysis patients before α-calcidol therapy (P1) and comparison between maintenance hemodialysis patients before and after α-calcidol therapy (P2)|
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|Figure 2 Comparison of hemoglobin level in dialysis patients before and after α-calcidol therapy. Hemoglobin level increased significantly (P=0.000).|
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|Figure 3 Comparison of erythropoietin resistance index in dialysis patients before and after α-calcidol therapy. Erythropoietin resistance index decreased significantly (P=0.000).|
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|Table 2 Correlation between serum vitamin D level and different parameters in maintenance hemodialysis patients before and after α-calcidol therapy (n=30)|
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|Figure 4 Correlation between serum vitamin D level and interleukin-6 after α-calcidol therapy. There was a significant negative correlation.|
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| Discussion|| |
The hemopoietic response to EPO therapy shows wide interindividual and intraindividual variation owing to a multitude of factors . Improving response to EPO and other erythropoietic stimulating agents remains a challenging demand for the care of MHD patients . Such a response is usually assessed by two variables: the EPO dose required and the Hb level achieved . The ERI provides a single measure for straightforward assessment of response to EPO . The main finding of this study was that a 3-month course of standard guideline-based therapy with oral α-calcidol in MHD was associated with a significant increase in Hb level and a significant decrease in ERI.
Several studies have reported that administration of vitamin D to MHD patients leads to significant improvement of anemia and reduction of EPO requirements, particularly when the vitamin is administered in the active (hormonal) form, such as α-calcidol and calcitriol ,,. Studies employing the inactive (nutritional) forms, such as ergocalciferol and cholecalciferol, have been less consistent, with some studies reporting significant hemopoietic responses , and others failing to do so ,. Another important variable in vitamin D supplementation studies is the baseline vitamin D status. Improvements of hemopoietic parameters were found to be dependent on some degree of baseline vitamin D deficiency. These improvements cannot be detected in the subgroups of patients with normal baseline vitamin D level ,. Vitamin D deficiency is highly prevalent in patients with CKD, and particularly so in patients with ESRD treated with MHD . All patients included in the present study had initial serum vitamin D3 values less than 30 ng/ml, and were accordingly experiencing variable degrees of vitamin D insufficiency/deficiency . The beneficial erythropoietic responses to vitamin D supplementation could not be demonstrated in a group of hypertensive patients with preserved kidney function . The significant hematologic improvements revealed by the present study seem empowered by inclusion of only MHD with prevalent vitamin D deficiency and the use of a hormonally active vitamin D isoform.
Numerous mechanisms have been proposed to explain the potential benefits of vitamin D on erythropoiesis, including a direct effect on erythroid precursor cells  and/or an indirect one through amelioration of the erythropoietic inhibitory sequelae of the prevalent secondary hyperparathyroidism (HPTH)  and inflammation ,. Earlier studies have focused on the role of active vitamin D supplementation to control HPTH ,,. In these studies, the rising Hb level and declining EPO requirements were linked with a significant decline in serum iPTH and ALP levels. We found a significant decline of both parameters in the present study ([Table 1]). The significant negative correlation between serum vitamin D3 level and ALP at the study start (r=−0.509, P=0.004) may also indicate that the extent of initial changes attributed to HPTH were linked with the degree of vitamin D deficiency . Caution should be exercised to recognize and prevent undesirable increases of serum calcium, phosphorus, and their product; which are known to increase the risk of vascular calcification and augment the already high cardiovascular morbidity and mortality in these patients . We admit that our patients sustained a significant increase in these parameters, exceeding the recommended limits in some of them. A monthly check-up of serum calcium and phosphorus would have been warranted .
Recently, interest has been growing in the pleiotropic health benefits of vitamin D, including its immune-modulatory and anti-inflammatory actions ,. These actions can ameliorate the chronic inflammatory status that is highly prevalent in patients with CKD and particularly in those with ESRD on MHD . Several studies have demonstrated that the increasing Hb and decreasing ERI in response to vitamin D therapy of MHD patients is paralleled with, and possibly mediated through, a significant decrease in serum levels of several inflammatory markers including CRP , IL-6 , and hepcidin . Similarly in the present study, oral α-calcidol therapy resulted in a significant decrease of serum CRP, IL-6, and hepcidin, the latter being the major inhibitor of iron release from its storage sites  ([Table 2]). Ferritin, which has also sustained a significant decrease, may be regarded as both a marker of inflammation and a reflection of iron stores . The significant positive correlation between serum ferritin and ERI at the start of the study may denote inhibition of erythropoiesis by the prevalent inflammation leading to sequestration of iron into its storage sites in the reticulendothelial and hepatic cells ,,. On the contrary, the significant negative correlation between serum vitamin D3 level at the end of the study and each of serum ferritin and IL-6 lends further support to the anti-inflammatory effects of vitamin D as important contributors to its efficacy in improving anemia and reducing EPO resistance ,.
Potential strengths of this study include its interventional and prospective nature, the inclusion of several markers of iron homeostasis and inflammation, and the use of ERI as a simple numerical measure of EPO responsiveness. Study limitations include the relatively limited number of patients and short duration of follow-up and the inability to provide monthly checks of serum calcium and phosphorus, which would obviate an overly increased calcium×phosphorus product. Moreover, a direct effect of active vitamin D on erythroid progenitors has not been addressed.
We conclude that a 3-month course of oral α-calcidol therapy as per standard treatment guidelines of MHD patients leads to a significant increase in Hb level and a significant decrease in ERI. These beneficial effects are associated with, and potentially mediated through, control of changes of HPTH and amelioration of the prevalent chronic inflammatory status. Overshoots of serum calcium and/or phosphorus should be avoided by vigilant monthly follow-up. MHD patients with unexplained suboptimal erythropoietic stimulating agent response should be thoroughly evaluated for the presence of an underlying hidden infection or subtle chronic inflammation. Further vitamin D supplementation studies should include larger cohorts, extend for longer periods, and consider the use of vitamin D analogs with less calcemic potential.
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Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2]