Effect of Cholecalciferol Supplementation on Inflammation and Cellular Alloimmunity in Hemodialysis Patients: Data from a Randomized Controlled Pilot Trial

<div><p>Background</p><p>Memory T-cells are mediators of transplant injury, and no therapy is known to prevent the development of cross-reactive memory alloimmunity. Activated vitamin D is immunomodulatory, and vitamin D deficiency, common in hemodialysis patients awaiting transplantation, is associated with a heightened alloimmune response. Thus, we tested the hypothesis that vitamin D₃ supplementation would prevent alloreactive T-cell memory formation in vitamin D-deficient hemodialysis patients.</p><p>Methods and Findings</p><p>We performed a 12-month single-center pilot randomized, controlled trial of 50,000 IU/week of cholecalciferol (D₃) versus no supplementation in 96 hemodialysis patients with serum 25(OH)D<25 ng/mL, measuring effects on serum 25(OH)D and phenotypic and functional properties of T-cells. Participants were randomized 2∶1 to active treatment versus control. D₃ supplementation increased serum 25(OH)D at 6 weeks (13.5 [11.2] ng/mL to 42.5 [18.5] ng/mL, p<0.001) and for the duration of the study. No episodes of sustained hypercalcemia occurred in either group. Results of IFNγ ELISPOT-based panel of reactive T-cell assays (PRT), quantifying alloreactive memory, demonstrated greater increases in the controls over 1 year compared to the treatment group (delta PRT in treatment 104.8+/−330.8 vs 252.9+/−431.3 in control), but these changes in PRT between groups did not reach statistical significance (p = 0.25).</p><p>Conclusions</p><p>D₃ supplements are safe, effective at treating vitamin D deficiency, and may prevent time-dependent increases in T-cell alloimmunity in hemodialysis patients, but their effects on alloimmunity need to be confirmed in larger studies. These findings support the routine supplementation of vitamin D-deficient transplant candidates on hemodialysis and highlight the need for large-scale prospective studies of vitamin D supplementation in transplant candidates and recipients.</p><p>Trial Registration</p><p><a href="http://clinicaltrials.gov/show/NCT01175798" target="_blank">Clinicaltrials.gov NCT01175798</a></p></div

Laboratory parameters of bone and mineral metabolism and activated Vitamin D requirements.

<p>Mean (SD) unless otherwise specified.</p><p>*p-values for comparison of change from baseline to 12 months between groups (treatment vs control).</p><p><b>**</b>n = 41 in treatment group and 27 in control group.</p><p>Laboratory parameters of bone and mineral metabolism and activated Vitamin D requirements.</p

Vitamin D supplementation may prevent the time-dependent increase in PRT.

<p>(A) Representative ELISPOT PRT wells in duplicate at baseline and one year with no stimulation (media control) or response to allogeneic B cells. (B) Quantified results reveal a significant increase in the number of IFNγ ELISPOTs over time in the control group (517.4+/−280.8 to 797.8+/−542.3 spots, p = 0.03), but the comparison of “delta” PRT (1 year – baseline) in the treatment vs control group did not reach statistical significance (104.8+/−330.8 in treatment vs 252.9+/−431.3 in control, p = 0.25).</p

Gating strategy for enumeration of T cell and monocyte subsets.

<p>CD4 and CD8 memory (CD45RO+/CD45RA^neg) and naïve (CD45RO^neg/CD45RA+) T cells, and the Foxp3+/CD25+/CD4+ population containing regulatory cells were evaluated. Monocytes were identified by a CD19^neg/HLA-DR+ phenotype, and further characterized into the CD14++/CD16^neg classical subset, and the CD14^lo/CD16+ non-classical M-DC8+ and M-DC8^neg subsets.</p

Study flow diagram.

<p>A total of 116 hemodialysis patients were screened, and 96 were randomized in a 2∶1 ratio to receive oral cholecalciferol (n = 62) or no repletion (n = 34). There were no differences in dropout rates from transplantation (6.6% vs 5.9% at 1 year, p = .90) or death (16.4% vs 5.9% at 1 year, p = .14) between groups. A total of 68 subjects completed follow-up to 1 year. Of those 68 subjects, 51 (34 in the treatment group and 17 in the control group) had sufficient PBMC samples meeting predetermined quality-assurance criteria for immunologic assessment.</p

Effects of Vitamin D supplementation on T cell phenotypes^†.

<p>Values are expressed as median [IQR].</p>†<p>no statistically significant differences in baseline, 1 year, or change from baseline to 1 year between groups.</p><p>Effects of Vitamin D supplementation on T cell phenotypes^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109998#nt106" target="_blank">†</a>}.</p

Baseline serum 25(OH)D concentrations in the study cohort.

<p>Distribution of serum 25(OH)D in the entire study cohort (n = 116) is shown. Median (IQR) 25(OH)D was 14.7 (10.5–21.6) ng/mL. 17 subjects with a baseline vitamin D level >25 ng/mL were excluded from randomization.</p

Effects of Vitamin D supplementation on serum 25(OH)D concentrations.

<p>Values increased by six weeks in the treatment group (13.5 [11.2] ng/mL to 42.5 [18.5] ng/mL, p<0.001), but remained low in the control group throughout the study duration. Median (IQR) are displayed on the graph.</p

Vitamin D supplementation does not alter anti-HLA antibodies.

<p>Pie charts depicting the percentages of patients in each group that developed new anti-HLA antibodies between entry and 1 year (p = 0.393). A new reactivity was defined as having a MFI <1000 at baseline and >5000 at one year using the LuminexPRA assay.</p