Introduction

Vitamin D insufficiency, defined as a serum 25-hydroxyvitamin D (25D) level of less than the physiologic level of 30 ng/mL, indicates that body stores of vitamin D are inadequate [1, 2]. This condition is common in patients with chronic kidney disease (CKD), accounting for 80–90 % of patients on dialysis (CKD stage 5D) [3–5]. The clinical practice guidelines for the management of CKD-mineral bone disorder (CKD-MBD) by the National Kidney Foundation Disease Outcomes Quality Initiatives (KDOQI) and Kidney Disease: Improving Global Outcomes (KDIGO) indicate that serum 25D should be measured at the first encounter if serum parathyroid hormone (PTH) levels are above the target range for the stage of CKD [6, 7]. Nevertheless, vitamin D insufficiency is frequently overlooked as the majority of patients with CKD receive active vitamin D or its analogues [1,25-dihydroxyvitamin D3 (1,25D)] to treat CKD-MBD.

Beyond bone and mineral metabolism, vitamin D plays an important role in erythropoiesis by stimulating erythroid progenitor cells in a synergistic fashion with other hormones and cytokines, including erythropoietin. Adequacy of all components in the vitamin D axis is crucial for normal red blood cell production [8]. In the general population, the prevalence of anemia and the use of erythrocyte stimulating agents (ESA) have been found to be negatively correlated with serum 25D levels regardless of kidney function [9]. In patients with CKD, suboptimal serum levels of either 25D or 1,25D have been shown to be independently associated with an increased incidence of anemia [10, 11], indicating that vitamin D and 1,25D both play roles in erythropoiesis. Moreover, erythropoietin resistance has been observed in patients with CKD stage 5D who had low serum 25D levels [12].

There are two major forms of vitamin D: cholecalciferol and ergocalciferol. Cholecalciferol, a more potent form of vitamin D [13], is produced in the skin. Ergocalciferol, which is synthesized by plants, is the only form of high-dose vitamin D available in many countries, including the USA and Thailand [6, 14]. The KDOQI guidelines for ergocalciferol supplementation are only available for patients with CKD stages 2–4 [6, 14]. In patients with CKD stages 5 and 5D, repletion of body stores of vitamin D is theoretically dispensable as the renal 1α-hydroxylase enzyme is impaired and vitamin D toxicity is a main concern. Nevertheless, several recent studies have evaluated the safety of vitamin D supplementation in patients with CKD stages 5 and 5D. Supplementation of 200,000–350,000 IU of ergocalciferol to adult patients with CKD stage 5D over 4–6 months was not associated with any adverse effects [3, 15]. The data on efficacy and safety of vitamin D supplementation in children with CKD stages 5 and 5D are limited. The aim of our study was therefore to evaluate the effect of ergocalciferol on the dose of ESA utilized in children with CKD stage 5 and vitamin D insufficiency.

Patients and methods

Patients aged <18 years with CKD stages 5 or 5D and serum 25D levels of <30 ng/mL were enrolled into a prospective randomized controlled study. CKD stage 5 is defined as a glomerular filtration rate (GFR) of <15 mL/min/1.73 m2, and CKD stage 5D indicates that the patient requires chronic dialysis [16]. The GFR of the patients in our study was calculated using the formula of Schwartz et al. [17]. The inclusion criteria were hemoglobin levels of 10.0–12.5 g/dL, serum phosphorus levels of <6.5 mg/dL, corrected serum calcium levels of <10.5 mg/dL, and a calcium–phosphorus product of <65 mg2/dL2 for at least 1 month before the recruitment. The exclusion criteria were patients with thalassemia, chronic liver disease, gastrointestinal malabsorption, significant blood loss, serum PTH levels of >800 pg/mL, proteinuria of >2 mg/mg of urine creatinine, blood transfusion, chronic anticonvulsant therapy, prior ergocalciferol supplementation, and kidney transplantation. The study was approved by the Institutional Review Board Committee of Faculty of Medicine, Chulalongkorn University, and complied with the World Medical Association Declaration of Helsinki regarding ethical conduct of research involving human subjects. Informed consent was obtained, and patient anonymity was preserved.

Twenty patients were divided into two groups by simple randomization. Ten patients received oral ergocalciferol supplementation (treatment), whereas the other group did not (control). Demographic data were obtained by medical record review. Baseline and monthly laboratory determinants using standard automated methods included hemoglobin, iron, total iron binding capacity, ferritin, calcium, phosphorus, and albumin. Corrected serum calcium for low serum albumin was calculated using the formula: corrected serum calcium = 0.8 × (4 − serum albumin) + measured serum calcium. Transferrin saturation (%) was calculated using the ratio of serum iron to serum total iron binding capacity.

Management of CKD-MBD was directed by the KDOQI guidelines [6]. In addition to dietary phosphorus restriction, calcium carbonate was prescribed as a phosphate binder to patients with phosphorus levels of >5.5 mg/dL. For secondary hyperparathyroidism, patients received oral alfacalcidol (One-Alpha 0.25 mcg/capsule; LEO Pharma, Thornhill, ONT, Canada), and the dose was adjusted to keep serum PTH levels within the ranges recommended in the KDOQI guidelines [6].

For anemia management, ESA in the form of epoetin alfa (Hypercrit; Bio Sidus S.A., Buenos Aires, Argentina) was administered subcutaneously, and the dose of ESA was adjusted based on the KDOQI recommendations [18]. The target hemoglobin level is 11–12 g/dL. The ESA dose was generally adjusted on a monthly basis, but the adjustment may be performed biweekly for patients with a precipitous change in the hemoglobin levels. ESA treatment was withheld if hemoglobin levels were >13.9 g/dL and restarted when hemoglobin levels fell to <12.5 g/dL. The ESA dosage (units/kg of body weight/week) was recorded monthly. Iron deficiency was corrected by oral or intravenous iron supplementation with the target transferrin saturation of 20–50 % and target ferritin level of 300–500 ng/mL.

Measurement of serum 25D and PTH levels

Serum 25D levels (ng/mL) were measured at baseline and at the end of the study using the LIAISON® 25 OH Vitamin D TOTAL Assay (DiaSorin, Stillwater, MN), which is a chemiluminescent immunoassay (CLIA) able to detect both forms of 25D (25-hydroxycholecalciferol and 25-hydroxyergocalciferol).

Serum PTH levels (pg/mL) were measured at baseline and monthly until the completion of the study using the Elecsys PTH Immunoassay® (Roche Diagnostics, Indianapolis, IN), a electrochemiluminescence immunoassay (ECLIA).

Ergocalciferol supplementation

The only high-dose forms of vitamin D available in Thailand are capsules containing 20,000 IU of ergocalciferol (Calciferol 20,000 IU/cap; British Dispensary, Bangkok, Thailand). The degree of vitamin D insufficiency in our patients was classified according to the KDOQI guidelines into three categories based on serum 25D levels (<5, 5–15, 16–30 ng/mL) [6, 14]. In patients with severe 25D deficiency (serum 25D level <5 ng/mL), 40,000 IU of ergocalciferol was given weekly for 4 weeks followed by 40,000 IU biweekly for 8 weeks (total 320,000 IU of ergocalciferol). For mild 25D deficiency (25D level 5–15 ng/mL), 40,000 IU of ergocalciferol was given biweekly for 12 weeks (total 240,000 IU of ergocalciferol). For 25D insufficiency (25D level 16–30 ng/mL), 40,000 IU of ergocalciferol was given every 4 weeks for 12 weeks (total 120,000 IU of ergocalciferol).

Statistical analysis

The data were statistically analyzed using the Statistical Package for the Social Sciences ver. 17 (SPSS, Chicago, IL). Variables with a normal distribution were presented as mean values ± standard deviation (SD) and were evaluated using Student’s t test and the paired t test where appropriate. Serum 25D levels showed a nonparametric distribution, and the levels were presented as medians and interquartile range (IQR). Comparison of 25D levels between the groups was performed using the Mann–Whitney U test, whereas the Wilcoxon signed-rank test was used to compare baseline and post-treatment values within the group. The Fisher’s exact test was used to compare categorical variables between the two groups. The ESA dosages at different timepoints in the two groups were compared using repeated-measures analysis of variance (ANOVA) with the Bonferrini post-hoc test. The correlation between serum 25D levels and ESA dosages was estimated using the Pearson correlation coefficient r. A p value of <0.05 was considered to be statistically significant.

Results

Baseline characteristics of the patients in each group are shown in Table 1. The mean age of the patients was 8.5 ± 5.3 (range 2–17) years. The majority of patients (92 %) with CKD stage 5D received peritoneal dialysis as renal replacement therapy. Only one patient (treatment group) received hemodialysis. There were no significant differences in age, gender, and severity of CKD between both groups. Every patient in the study received alfacalcidol to control secondary hyperparathyroidism.

Table 1 Baseline characteristics of patients receiving ergocalciferol supplementation (treatment group) and controls
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Baseline laboratory parameters of the patients in each group are shown in Table 2. There was no significant difference in the levels of corrected calcium, phosphorus, PTH, hemoglobin, ferritin, and transferrin saturation between both groups at baseline. One patient (control group) had severe vitamin D deficiency (Table 1); however, the 25D levels in the treatment group did not significantly differ from those in the controls (Table 2). The required ESA dosage was similar in both groups (Fig. 1).

Table 2 Laboratory parameters at baseline and after the 12-week study period in patients receiving ergocalciferol supplementation (treatment group) and controls
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Fig. 1
figure 1

Erythrocyte-stimulating agent (ESA) dosages during the study period in patients receiving ergocalciferol supplementation (treatment group) and controls

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Laboratory parameters at the completion of the 12-week study are shown in Table 2. Serum 25D levels were higher in the treatment group than in the control group (p < 0.005). In the control group, serum 25D levels at the completion of the study were decreased from baseline, but the difference did not reach statistical significance. Serum 25D levels in the treatment group at the completion of the study are shown in Fig. 2. Serum 25D levels were significantly increased after the treatment when compared to the baseline values (p = 0.02). No patients in the treatment group had vitamin D deficiency at the end of 12 weeks. Three patients (30 %) had serum 25D levels above the physiologic level of 30 ng/mL and seven patients (70 %) had vitamin D insufficiency (serum 25D levels 16–30 ng/mL).

Fig. 2
figure 2

Serum 25-hydroxyvitamin D (25D) levels at baseline and after a 12-week course of ergocalciferol supplementation. The values are given as the median (interquartile range)

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The ESA dosages given to each group during the study are shown in Fig. 1. There was a significant difference in the pattern of changes in the ESA dosages over the 12-week study period (p = 0.02). Compared to baseline, there was a significant decrease in the ESA dosages at the end of the therapy in the treatment arm (p = 0.04). Although the overall ESA dosage over the 12-week study period was lower in the treatment arm compared to the control group, the difference did not reach statistical significance (p = 0.18). There was no significant correlation between serum 25D levels and ESA dosages (r = 0.21, p = 0.06).

All patients completed the 12-week study without any major adverse effects from ergocalciferol. No dialysis modality switching occurred in any patients. There were no significant differences in the laboratory parameters, including corrected calcium, phosphorus, PTH, hemoglobin, ferritin, and transferrin saturation levels, between the two groups at the end of the study (Table 2).

Discussion

Vitamin D has important pleiotropic biological functions in several organ systems, including bone, vascular smooth muscle, breast tissue, and bone marrow [19, 20]. The level of 1,25D is more than 500-fold higher in the bone marrow than in the serum, suggesting that 1,25D stimulates red blood cell production in the bone marrow by upregulating erythropoietin receptors in an autocrine or paracrine fashion [21]. The results of an in vitro study carried out by Alon et al. confirm that the proliferation of erythroid progenitor cells is under the stimulation of both 1,25D and erythropoietin in a synergistic manner [8].

In patients with CKD stage 5, the activity of renal 1α-hydroxylase activity is impaired, whereas the extra-renal 1α-hydroxylase enzyme remains active. Dusso et al. demonstrated that the local conversion of 25D to 1,25D can occur in the bone marrow when an adequate amount of 25D is provided as a substrate for the 1α-hydroxylase enzyme [22]. In this study, the requirement of ESA was significantly decreased when compared to baseline following ergocalciferol supplementation to children with CKD stage 5 and vitamin D insufficiency. This finding indicated that in children with vitamin D insufficiency who received only alfacalcidol to treat CKD-MBD, the levels of 1,25D in the bone marrow may be suboptimal to stimulate red blood cell production. Thus, ergocalciferol supplementation could replenish total body stores of vitamin D and increase the local production of 1,25D in the bone marrow which, in turn, would improve erythropoietin sensitivity in these patients.

Another important mechanism of erythropoietin resistance in patients with CKD is chronic systemic inflammation [23]. Holick recently demonstrated that the level of C-reactive protein, an inflammatory marker, is negatively correlated with the levels of 25D and hemoglobin in CKD [10]. Vitamin D is an immunomodulator with anti-inflammatory properties [24]. In patients with heart failure, treatment with vitamin D was found to significantly decrease the levels of tumor necrosis factor (TNF), a pro-inflammatory cytokine, and to increase the levels of interleukin-10 (IL-10), an anti-inflammatory marker [25]. Hence, ergocalciferol supplementation could be beneficial in improving erythropoietin sensitivity by decreasing the chronic inflammatory process in CKD. However, we did not observe any significant reduction of serum ferritin, an inflammatory cytokine, after ergocalciferol supplementation. Further investigations focusing on changes in the levels of cytokines known to associate with erythropoietin resistance, such as TNFα and IL-6 [26], in relation to body vitamin D status and erythropoietin sensitivity are therefore required.

Low serum 25D levels can exacerbate secondary hyperparathyroidism regardless of the 1,25D status, and vitamin D supplementation in conjunction with 1,25D therapy can improve lesions of osteitis fibrosa cystica [27]. Fibrosis in the bone marrow as a result of severe secondary hyperparathyroidism can decrease the bone marrow response to erythropoietin [28, 29]. As the measurement of serum 25D levels were not performed in these studies, it is unclear whether increased erythropoietin sensitivity following vitamin D supplementation was the direct effect of vitamin D repletion or the result of PTH reduction [27–29]. Although the PTH levels in our treatment group showed a trend towards being lower at both baseline and at the end of therapy when compared to the controls, the values did not reach statistical significance due to a large standard deviation. A larger sample size will be required to test whether lower PTH levels could contribute to the reduction in the ESA dose in the treatment arm. Nevertheless, the finding of similar PTH levels before and after ergocalciferol supplementation in the treatment group in our study suggests that the association between ergocalciferol and erythropoietin sensitivity in children with CKD stage 5 and vitamin D insufficiency may be partially independent of the regulation of PTH.

Vitamin D insufficiency occurs in 60–80 % of children with CKD stages 2–4 [30]. The actual prevalence of inadequate body stores of vitamin D in children with CKD stage 5 is not known, but the prevalence could be higher as a negative correlation between serum 25D levels and severity of CKD has been demonstrated [30]. Although Thailand, a tropical country located at latitude 13° north of the equator, receives sufficient year-round sun exposure, the prevalence of vitamin D insufficiency is high in children with CKD stage 5 at our center, with 95 % of such who were measured for serum 25D level demonstrating vitamin D deficiency (personal communication). Several factors contribute to suboptimal vitamin D status in these patients, including decreased sun exposure related to infirmity and inadequate cutaneous vitamin D production due to meticulous sun screen application. As their kidney function deteriorates, consumption of vitamin D-rich diets is more limited due to dietary restriction and reduction of appetite. Thus, correcting vitamin D insufficiency with vitamin D supplementation in children with CKD stage 5 is a rational approach.

The suggested doses of vitamin D supplementation in adults with CKD stage 5 differ depending on the study [3, 15]. In children with CKD stages 2–4, supplementation ranging from a single dose of 100,000 IU to 336,000 IU over 3 months of ergocalciferol was not associated with any adverse effects [31, 32]. Data on safety and dosage of ergocalciferol supplementation in pediatric CKD stage 5 are lacking. Although serum 25D levels had increased in the majority of our patients in the treatment group, the dose of ergocalciferol used in this study was suboptimal since only 30 % of the patients had a serum 25D level that was higher than the physiologic level of 30 ng/mL at the end of the study. Nevertheless, our findings demonstrate that the ESA dosage was significantly reduced after the treatment when compared to baseline. Thus, ergocalciferol, when administered in conjunction with alfacalcidol and ESA, is beneficial in managing anemia in patients with CKD.

One limitation to our study is a small number of subjects, which could reduce the power to detect the difference between the treatment and the control groups. Based on the results of our study, we have a 50 % power to detect a 30 % change in ESA dosages between the two groups at a two-sided significance level of 5 %. Further investigation with a larger number of patients and a higher dose of ergocalciferol is required. Manifestations of vitamin D toxicity, including hypercalcemia, hyperphosphatemia, and oversuppression of PTH, should be closely monitored when higher doses of ergocalciferol are used [33].

The mortality rate in patients with CKD is positively correlated with the ESA dose required to achieve hemoglobin levels within the target range [34]. Further studies should aim to evaluate whether repletion of total body stores of vitamin D in patients with CKD and vitamin D insufficiency could provide long-term clinical benefits by reducing the morbidity and mortality related to erythropoietin resistance. However, given the prohibitive cost of increased ESA dosing, it is more cost beneficial to treat erythropoietin resistance related to vitamin D insufficiency with vitamin D supplementation. In addition to the treatment of secondary hyperparathyroidism, chronic inflammation and iron deficiency, we recommend that serum 25D levels should be measured and body vitamin D status should be restored when erythropoietin resistance is suspected.

In conclusion, vitamin D deficiency should be routinely evaluated in children with CKD stages 5 and 5D. In addition to 1,25D therapy, ergocalciferol should be supplemented to these children with vitamin D insufficiency although the optimal dosage of ergocalciferol remains to be determined.