Effect of Vitamin D Supplementation on Serum Hepcidin Levels in Non-Diabetic Chronic Kidney Disease Patients

Introduction

Iron deficiency is commonly associated with anemia in patients with chronic kidney disease (CKD). Hepcidin, a small cysteine-rich cationic peptide and an important regulator of iron metabolism, was elevated in patients with CKD. Encoded by the hepcidin antimicrobial peptide (HAMP) gene, hepcidin is produced in the liver and negatively regulates iron uptake by suppressing the post-translational expression of cellular iron transporter ferroportin by causing its internalization. Elevated levels of hepcidin block iron absorption in the gut and iron efflux from macrophages and hepatocyte stores, leading to reduced iron bioavailability for erythropoiesis. Given its importance, new treatment strategies[1] that target the hepcidin–ferroportin axis are being developed for the treatment of anemia in CKD.

Vitamin D deficiency has been postulated to play a role in CKD-associated anemia.[2] Experimental data suggest that insufficient 25-hydroxyvitamin D₃ [25(OH)D] levels leads to decreased production of local calcitriol in the bone marrow, limiting erythropoiesis. Vitamin D may also directly affect the proliferation of burst forming unit–erythroid cells. The immunomodulatory effect of vitamin D is key to the systemic production of cytokines that suppress the anemia-specific inflammatory pathways.[3–7] Vitamin D has been shown to reduce hepcidin levels in healthy adults and in people with CKD.[8,9] This mechanism involves regulation of hepcidin and ferroportin expression, as well as pro-hepcidin cytokines, interleukin-6 (IL-6), and interleukin-1 beta (IL-1β) release by vitamin D.[10] Hence, treatment with vitamin D may reduce serum hepcidin levels, and thereby have a salutary effect on the anemia in CKD. Studies till date, however, have shown that vitamin D does not reduce serum hepcidin concentrations in participants with CKD.[11,12] Based on this background, we investigated the effect of vitamin D (cholecalciferol) on serum hepcidin levels in vitamin D–deficient, non-diabetic individuals with CKD in a randomized, double-blind, placebo-controlled trial.

Methods

Results

A total of 120 CKD patients were enrolled in the clinical trial. There were no between-group differences in the baseline characteristics with respect to demographic details and causes of CKD [Supplementary Table 1].[13] Serum 25(OH) D levels were similar in the placebo and the cholecalciferol group at baseline (13.21 ± 4.78 ng/ml and 13.40 ± 4.42 ng/ml; P = 0.88). After 16 weeks, the serum 25(OH) D levels increased in the cholecalciferol group but not in the placebo group (between-group difference in mean change was 23.40 ng/ml (95% CI: 19.76 to 27.06; P < 0.001) [Table 1]. Serum hepcidin levels were similar at baseline (median [IQR]: 33.6 (8.6–77.8) ng/ml vs. 24.6 [9.3–70.7] ng/ml, P = 0.903) and did not vary between groups after 16 weeks (median [IQR]: 41.5 (10.9–75.0) ng/ml vs. 34.8 [12.3–63.75] ng/ml, P = 0.703) [Figure 1a]. The between-group difference in mean change at 16 weeks was 2.71 ng/ml (95% CI: −17.06 to 11.64; P = 0.709) [Table 1]. Hemoglobin levels were similar at baseline (12.02 ± 1.94 mg/dl vs. 11.97 ± 1.69 mg/dl, P = 0.947) and did not change after 16 weeks in either group (between-group difference in mean change; 0.21 mg/dl, 95% CI: −0.22 to 0.63, P = 0.340) [Figure 1b]. Within-group difference and between-group difference of other biochemical parameters are shown in Table 1.

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Figure 1

Line plots showing serum level of (a) hepcidin and (b) hemoglobin at baseline and at 16 weeks in placebo and cholecalciferol for individual patients

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Discussion

In the current study, cholecalciferol supplementation did not lead to any significant change in serum levels of hepcidin and hemoglobin in non-diabetic patients with stage III–IV CKD and vitamin D deficiency despite there being a significant elevation in circulating vitamin D levels. As shown previously,[16] the increase was physiologically relevant and corrected the markers of bone metabolism.

Hepcidin is a potent regulator of iron–ferroportin axis as it suppresses the expression of ferroportin post translationally, which is the only exporter of intracellular iron, thus leading to anemia by making iron unavailable for erythropoiesis.[17] Several studies have highlighted the effect of oral vitamin D supplementation on iron homeostasis by lowering the levels of serum hepcidin.[8,18,19] This is due to anti-inflammatory effects of vitamin D on reduction of serum IL-6.[20] Although there was a reduction in IL-6 after vitamin D supplementation,[13] there was no associated reduction in hepcidin levels. In a study of 28 healthy adults, a high dose vitamin D significantly reduced plasma hepcidin levels one week post dosing.[8] Bacchetta et al.,[15] in a pilot study of seven healthy individuals, showed that a single oral dose of 100,000 IU vitamin D₂, decreased the levels of hepcidin by 34% after 24 hours and by 33% after 72 hours. In vitro and in vivo studies have shown the inverse relation between vitamin D and hepcidin stimulatory cytokines and the direct effect of vitamin D on lowering hepcidin mRNA expression through the HAMP gene. Studies on animal models have also shown the positive effect of anti-hepcidin therapy on inflammation-induced anemia. Treatment with 25(OH) D or 1,25-dihydroxyvitamin D led to a 0.5-fold reduction in expression of hepcidin mRNA in cultured hepatocytes or monocytes.[15]

Our study showed no effect of high-dose cholecalciferol on serum hepcidin and blood hemoglobin levels. A similar randomized, placebo-controlled, double -blinded trial of 40 participants with mild-to-moderate kidney disease failed to find a significant effect of oral calcitriol on hepcidin levels after six weeks of administering 0.25 mcg oral calcitriol daily or matching placebo.[11] A pilot study on the effect of cholecalciferol on hepcidin in children with CKD also suggested no correlation between these two parameters.[12] Similarly, recent studies involving pregnant women[21] and patients with digestive tumors[22] also did not show any association between vitamin D and hepcidin levels. In a recent placebo-controlled, double-blind randomized trial on 96 hemodialysis patients, cholecalciferol supplementation did not change the serum hepcidin levels after three and six months.[14] The mixed results were likely due to differences in dosage, the form of vitamin D administered, and the type of study population [Table 2].

There may be several reasons for a lack of effect of high-dose cholecalciferol on serum hepcidin in the present study. While other studies have shown the effect of 25(OH) D on participants with early-stage CKD or on healthy individuals, we enrolled patients with stage III–IV CKD. In later CKD stages, factors like reduced GFR, treatment with iron, increased inflammation may collectively contribute to increased serum hepcidin levels, which may override the effect of cholecalciferol supplementation.

An important strength of this study is that it was a randomized, placebo-controlled, double-blind trial. However, this was a secondary analysis. Therefore, the limited sample size of the study population could also have affected the outcome parameters. Furthermore, we did not collect data on iron, ferritin, and iron therapy.

To conclude, our study provides preliminary data based on which a larger, adequately powered clinical trial can be conducted to conclusively assess the impact of vitamin D supplementation on hepcidin and anemia in patients with CKD and vitamin D deficiency.