Effect of Cholecalciferol Supplementation on CVD, Inflammation, and Bone Metabolism Markers in CKD: A Randomized Double Blind Placebo Controlled Feasibility Trial

Introduction

Vitamin D deficiency has been linked with cardiovascular disease (CVD) and mortality in both the general population and in individuals with CKD. The impact of vitamin D supplementation on surrogate endpoints for CVD in pre-dialysis CKD has been investigated in several recent randomized controlled trials (RCTs).^1-7 Overall, the findings from these clinical trials suggest that vitamin D supplementation favorably modifies endothelial and vascular function in CKD.^3,8 Additionally, vitamin D is effective in decreasing the circulating interleukin-6 (IL-6), serum C-terminal cross-linked collagen type I telopeptides (CTX-1), serum total and bone-specific alkaline phosphatase (SAP, BAP).⁸ KDIGO has recommended BAP as a bone biomarker for the diagnosis and management of renal osteodystrophy.⁹ Also, it has been shown to be an effective indicator for distinguishing low bone turnover.¹⁰

In CKD, the impact of native vitamin D forms (cholecalciferol) might be mediated through conversion to 1,25(OH)₂D, but limited data suggest that different effector mechanisms might be present that can be inferred from our work, suggesting opposing effects on fibroblast growth factor 23 (FGF-23) when compared to calcitriol.¹¹ The native forms might be important for paracrine and autocrine effects, and higher circulating levels might be required for extra-skeletal effects, including favorable modulation of vascular function and inflammatory state.¹² These data suggest that cholecalciferol supplementation targeted to higher circulating vitamin D levels would be the most appropriate intervention when extraskeletal effects, like modulation of vascular function, are the primary endpoint of a clinical trial. In this feasibility study, we evaluated the effect of cholecalciferol supplementation on CVD, inflammation, and bone metabolism markers in CKD in a randomized double-blind placebo-controlled trial.

Materials and Methods

The study was a feasibility phase clinical trial conducted at the Postgraduate Institute of Medical Education and Research (PGIMER), Chandigarh, India. It was a prospective, randomized, placebo-controlled, double blind clinical trial. The study was approved by the Institute Ethics Committee of PGIMER, Chandigarh (No. PGI/IEC/2019/000042 dated: 07- 01- 2019) and registered at the Clinical Trials Registry of India (www.ctri.nic.in, CTRI/2019/05/019211, dated 20-05-2019).

Adult patients (≥18 years) with pre-dialysis CKD attending the Department of Nephrology outpatient clinic were screened. Inclusion criteria were ages between 18–75 years, estimated glomerular filtration rate (eGFR) ranging from 10 to 45 mL/min/1.73m² by the CKD-EPIcr equation 2009, serum 25(OH)D levels 20-50 ng/mL, and a clinically stable course for the last 3 months. Patients receiving long-term maintenance dialysis or immunosuppressive therapy, diagnosis of chronic liver disease, primary hyperparathyroidism, sarcoidosis, malignancy, history of having been treated for hypercalcemia due to any cause, anticipated need of long-term kidney replacement therapy within 6 months, poor functional status, life expectancy <1 year, pregnancy in case of females, blood hemoglobin <8 g/dL, serum calcium >9.5 mg/dL, history of having received any type of organ transplant, history of allergy to the interventional drug (cholecalciferol), having received vitamin D supplementation in last 30 days or participation in another interventional clinical trial, were excluded. Patients were enrolled after obtaining informed consent.

The study population included patients with mild to moderately advanced CKD at high risk of the development of CVD. As fatal and non-fatal MACE is the primary outcome parameter of our proposed study, the selection of patients with CKD and eGFR 10-45 mL/min/1.73m2 will ensure inclusion of patients who are at high risk and at least 10% event rate for the primary outcome parameter by 3 years of follow-up in the control group that receives placebos. Including patients with circulating 25(OH)D levels between 20-50 ng/mL will ensure that patients with true vitamin D deficiency would be excluded, which would take care of ethical concerns, as we would not be putting patients with documented true deficiency under the placebo arm and also not subjecting those with higher levels to the risk of hypervitaminosis. We expect the levels to rise by 10-20 ng/mL, which would maintain levels of 70 ng/mL even in those with baseline levels around 50 ng/mL.

Results

A total of 692 patients were screened between August 2019 and October 2021, out of which 126 were enrolled. 37 enrolled participants dropped out before randomization, and 89 participants were randomized till end of October 2021 [Figure 1]. The following data analysis pertains to baseline measurements from 89 participants and follow-up comparative measurements from 80 participants who completed 1 year of follow-up.

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Figure 1: CONSORT flow diagram of the study.

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A total of 89 participants were randomized,46 in the cholecalciferol group and 43 in the placebo group. Table 1 shows the baseline characteristics of the study population. The mean age of the overall participants was 50.84 ± 12.68 years, with 38% being female. At baseline, both groups were similar with respect to demographic characteristics, distribution of CKD causes, risk factors, presence of CVD, and medication use. Similarly, there were no significant differences between the two groups in baseline biochemical and biomarker measurements [Table 2].

Biochemical and biomarkers at follow-up

The biochemical and biomarker characteristics of these participants were compared at the annual follow-up visit [Figure 2]. Table 4 shows the within-group and between-group differences with respect to biochemical and biomarker characteristics.

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Figure 2: Individual levels of biomarkers at baseline and 12-month follow-up. IL-6: Interleukin-6, FGF-23: Fibroblast growth factor-23, CRP: C-reactive protein, CTX: Carboxy terminal telopeptide, BAP: Bone alkaline phosphatase, iPTH: intact parathyroid hormone.

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Table 4: Within-group and between-group differences in biochemical and biomarker measurements at 12 months in the study population who completed at least one year of follow-up.

Parameters	Cholecalciferol (n=Mean difference (95%CI)	p value	N (number of patients)	Placebo Mean difference (95% CI)	p value	N (number of patients)	Between-group difference	p value
Serum 25(OH)D (ng/mL)	31.15 (21.37 to 40.9S2)	<0.001	31	-0.75 (-7.43 to 5.94)	0.510	34	31.89 (20.46 to 43.32)	<0.001
Serum iPTH (pg/mL)	35.51 (-24.19 to 95.94)	0.329	30	58.39 (-17.92 to 134.69)	0.081	33	-22.87 (-119.56 to 73.81)	0.660
Hemoglobin (g/dL)	-0.21 (-0.89 to 0.48)	0.642	34	-0.37 (-0.75 to 0.020)	0.07	35	0.160 (-0.61 to 0.93)	0.442
Serum Cr (mg/dL)	0.46 (0.12 to 0.81)	0.029	34	0.44 (0.071 to 0.81)	0.010	35	0.022 (-0.47 to 0.52)	0.871
eGFR (CKD-EPIcr)	-1.72 (-3.80 to 0.37)	0.07	34	-2.54 (-4.71 to -0.38)	0.021	35	0.82 (-2.13 to 3.78)	0.848
Serum calcium (mg/dL)	0.07 (-0.14 to 0.29)	0.817	34	-0.49 (-0.82 to -0.15)	0.001	33	0.56 (0.17 to 0.95)	0.024
Serum inorganic phosphorous (mg/dL)	0.80 (0.006 to 1.59)	0.010	34	0.29 (-0.088 to 0.658)	0.238	33	0.517 (-0.35 to 1.39)	0.270
Serum alkaline phosphatase (U/L)	8.26 (-12.11 to 28.62)	0.648	27	15.28 (1.19 to 29.37)	0.023	25	-7.02 (-31.56 to 17.52)	0.197
UPCR (mg/g)	215.11 (-878.61 to 1308.83	0.690	28	-49.12 (-523.19 to 425.67)	0.264	25	264.23 (-950.39 to 1478.85)	0.664
Serum IL-6 (pg/mL)	-0.244 (-22.75 to 22.26)	0.694	35	-4.56 (-14.69 to 5.57)	0.081	39	4.315 (-19.08 to 27.72)	0.221
Serum intact FGF-23 (pg/mL)	101.74 (21.72 to 181.76)	0.001	35	82.08 (15.74 to 148.43)	0.015	39	19.66 (-81.75 to 121.06)	0.335
Serum CRP (ng/mL)	-0.105 (-1.65 to 1.44)	0.909	35	1.86 (0.51 to 3.23)	0.004	39	-1.97 (-3.99 to 0.04)	0.042
Serum CTX (ng/mL)	-0.395 (-0.68 to -0.112)	0.020	35	-0.185 (-0.53 to 0.16)	0.288	39	-0.215 (-0.655 to 0.23)	0.452
Serum BAP (U/L)	-4.61 (-9.90 to 0.68)	0.074	35	0.471 (-5.25 to 6.20)	0.085	39	-5.08 (-12.80 to 2.64)	0.019

Data presented as mean difference (95% confidence interval). 25(OH)D: 25-hydroxyvitamin D3, BAP: Bone alkaline phosphatase, CRP: C-reactive protein, CTX: Carboxy terminal telopeptide, eGFR: Estimated glomerular filtration rate, FGF-23: Fibroblast growth factor-23, iPTH: Intact parathyroid hormone, IL-6: Interleukin-6, UPCR: Urine protein creatinine ratio

Discussion

In this study, cholecalciferol supplementation significantly increased 25(OH)D levels. Most randomized controlled clinical trials have shown an effective increase in 25(OH)D levels after supplementation with the most common forms of vitamin D.^1,14,15 We did not observe any significant effect of cholecalciferol treatment on MACE, need for RRT, and mortality; however, markers of inflammation and bone metabolism were favorably changed.

The data from this study, indicating no effect of vitamin D on CVD outcomes or CKD progression, are consistent with previously reported data. Studies including a low daily dose of Vitamin D (VITAL) or a monthly high-dose regimen (ViDA) did not show any effect on cardiovascular outcomes.^16,17 In the PRIMO and OPERA randomized controlled trials, paricalcitol treatment did not improve cardiovascular outcomes, specifically left ventricular structure or function, in stage ¾ CKD.^18,19 A recent meta-analysis of RCTs, including the VITAL and ViDA studies, confirms these findings and suggests that vitamin D supplementation does not prevent cardiovascular events, CVD mortality, or all-cause mortality.²⁰ Similarly, a meta-analysis of RCTs of different vitamin D supplements, with or without calcium, did not show a positive impact on preventing CVD risk.²¹ Additionally, vitamin D supplementation in patients receiving hemodialysis has been found to be ineffective in improving CVD.²² D-Health randomized trial, however, showed a reduction in the incidence of major cardiovascular events like myocardial infarction and coronary revascularization when given a monthly dose of 60000IU of vitamin D₃ for five years.²³ These variations in outcomes across all these studies could be attributed to the observed heterogeneity among them in various aspects, including differences in study design, vitamin D analogues and dosage, baseline vitamin D levels, and variations in study duration, among other factors.

We also analyzed the biomarkers of bone turnover, as vitamin D has been reported to play a beneficial role in bone mineral metabolism. Among these, FGF-23 significantly increased in the cholecalciferol as well as in the placebo group, while serum CTX levels significantly decreased in the cholecalciferol group at 12 months follow-up. FGF-23 levels gradually rise, starting in the early stages of CKD, and are believed to be a physiological adaptation aimed at preserving normal serum phosphate levels and overall phosphorus balance.²⁴ It suppresses calcitriol synthesis in the kidney and promotes the breakdown of active vitamin D sterols. Conversely, calcitriol promotes FGF-23 expression.²⁵ Our findings suggest that vitamin D therapy may boost FGF-23 levels. In a recent randomized controlled trial of 127 patients with CKD stage 3 and type 2 diabetes mellitus, calcitriol was shown to significantly decrease CTX levels and increase FGF-23 levels.²⁶ Although vitamin D plays an important role, its supplementation in CKD should be carefully monitored to balance correction of deficiency with the potential risk of elevating FGF-23 levels, which are independently associated with increased CVD risk and mortality.^27-29 CTX is a bone turnover marker that is directly released during bone resorption. Hence, decreased levels of CTX are an indicator of a decrease in bone resorption due to cholecalciferol. We observed a significant between-group difference in BAP, a specific marker of bone formation and remodeling. Similar results were reported in a study by Lerch et al.³⁰ on children with CKD. These data indicate the effectiveness of Vitamin D in markers of bone metabolism.

Continuous accumulation of inflammatory factors like IL-6 and CRP leads to microinflammation in the kidneys.³¹ In this context, our study showed no significant effect of vitamin D on IL-6 levels, whereas CRP levels decreased significantly. Although a recent meta-analysis of randomized controlled trials in CKD indicates no effect of vitamin D on inframammary markers (IL-6 and CRP),³² however, our data suggest that vitamin D is effective in reducing the inflammation in CKD.

Vitamin D deficiency is nearly ubiquitous in CKD. It is of utmost importance to evaluate whether interventions aimed at intermediate outcomes, such as vitamin D supplementation in CKD, lead to enhancements in clinical outcomes like CVD, mortality, and need for RRT. The precise procedure for addressing vitamin D deficiency is not clearly defined. We administered 60,000 IU of cholecalciferol once every 2 weeks, which successfully restored 25(OH)D levels and improved markers of bone turnover. However, this approach did not impact the occurrence of CVD, all-cause mortality, or the necessity of RRT. This data is consistent with the recent meta-analysis, which concluded that widespread vitamin D supplementation does not confer clinical benefits on all-cause or cause-specific mortality, cardiovascular events, cancer, or numerous other clinically relevant end points across several large clinical trials.³³ Our study, however, was not specifically structured to explore the long-term effects of cholecalciferol on bone turnover in these patients. Hence, it is essential to monitor the long-term effects of this strategy.

There are a few limitations of our study. A shorter study duration may not be appropriate to achieve the MACE events. We studied the protective effect of vitamin D among patients with CKD with insufficient levels of vitamin D at baseline, which may vary in patients with deficient levels. We did not assess intermediate cardiovascular outcomes like pulse wave velocity, bone health markers like bone biopsy, and dual energy X-ray absorptiometry (DXA). We did not consider cardiovascular health at baseline. We exclusively examined a single form of vitamin D, which may not always be the most efficient option. A small proportion of participants in both groups received activated forms of vitamin D during follow-up, which could have influenced the observed biochemical outcomes. Activated vitamin D analogues are known to directly suppress parathyroid hormone levels and may also impact bone turnover markers and FGF-23 concentrations. Although their use in an equal number of participants across both arms likely minimized systematic bias; however, even limited exposure could have attenuated between-group differences.

In conclusion, this study’s findings suggest that although cholecalciferol is biologically active, its role in reducing cardiovascular risk in CKD remains unproven. Larger, adequately powered studies with longer follow-up are required to determine whether vitamin D supplementation can meaningfully impact MACE and other hard clinical outcomes in this population.