Abstract
Increasing evidence describes a possible interplay between vitamin D insufficiency with increased aldosterone. The authors sought to evaluate the effect of vitamin D supplementation on plasma aldosterone concentration (PAC) in patients with hypertension and 25‐hydroxyvitamin D[25(OH)D] insufficiency. The Styrian Vitamin D Hypertension Trial was a single‐center, double‐blind, placebo‐controlled randomized clinical trial conducted from 2011 to 2014. Two hundred patients with arterial hypertension and 25(OH)D levels <30 ng/mL were enrolled. Study participants were randomized to receive either 2800 IU of vitamin D3 or placebo. The present investigation is a post hoc analysis using analysis of covariance adjusting for baseline differences. A total of 188 participants (mean±standard deviation age, 60.1±11.3 years; 47% women; 25(OH)D, 21.2±5.6 ng/mL) completed the trial. Mean differences between baseline and follow‐up PAC in the control and intervention arm were +3.3 ng/dL and +0.9 ng/dL, respectively (P=.04). The findings indicate that vitamin D3 supplementation significantly decreases PAC in patients with arterial hypertension and 25(OH)D insufficiency.
Vitamin D insufficiency is an established risk factor for cardiovascular (CV) events and mortality.1, 2, 3, 4, 5 However, it remains unclear whether low 25‐hydroxyvitamin D (25[OH]D) concentrations are a causal risk factor or simply an epiphenomenon6 related to adverse outcomes that reflect confounding factors.1, 2, 3, 4, 5 The physiological effects of vitamin D receptor (VDR) activation in experimental studies include the suppression of neurohumoral activation and improvements in endothelial and vascular function.1, 2, 3, 4, 5 These factors have been linked to high blood pressure (BP), myocardial infarction, and CV mortality.1, 2, 7, 8, 9 It has been suggested that an interplay between the renin‐angiotensin‐aldosterone system (RAAS) and VDR activation could mediate the link of 25(OH)D insufficiency with arterial hypertension and CV disease.10, 11, 12, 13 In 1986, Resnick and colleagues14 described a continuous relationship between calcitriol (1‐alpha,25(OH)2 vitamin D3) levels and plasma renin activity. Zhang and colleagues15 demonstrated a marked increase in angiotensin II and renin in VDR knockout mice. These results were in line with findings from a cohort study in patients with high CV risk, showing that lower 25(OH)D and calcitriol levels are related to an upregulated circulating RAAS.16 So far, interventional data in humans on the effects of vitamin D supplementation on plasma aldosterone concentration (PAC) are missing. In our previous publication of a vitamin D randomized controlled trial (RCT), we adhered to a prespecified analysis plan and observed a moderate yet nonsignificant trend for a PAC‐lowering effect of vitamin D supplementation.17 In that work, we did not adjust for differences in the placebo vs the treatment group with regard to parameters with relevance for the RAAS, ie, the use of ACE inhibitors. Therefore, we performed a post hoc analysis of this RCT in hypertensive patients with 25(OH)D insufficiency to address the question of whether oral vitamin D supplementation for 8 weeks lowers PAC. We additionally report plasma renin concentration and aldosterone to renin ratio as they are part of interacting regulatory circuits using the same adjustments.18, 19
Methods
Study Design
The Styrian Vitamin D Hypertension Trial results have been previously published.17 The study was a double‐blind, placebo‐controlled, parallel‐group single‐center study performed at the Medical University of Graz, Graz, Austria. The publication of this trial adheres to the Consolidated Standards of Reporting Trials (CONSORT) 2010 statement.20 The RCT was registered at both www.clinicaltrialsregister.eu (EudraCT number 2009‐018125‐70) and clinicaltrials.gov (ClinicalTrials.gov identifier NCT02136771).
Participants
Eligible study participants were 18 years or older with a diagnosis of arterial hypertension and a 25(OH)D serum concentration <30 ng/mL. Arterial hypertension was classified in patients adhering to published guidelines21 or when patients were taking ongoing antihypertensive treatment. The main exclusion criteria were pregnancy or lactating women, hypercalcemia, drug intake as part of another clinical study, an acute coronary syndrome, or a cerebrovascular event in the previous 2 weeks, as published previously.17 All study participants gave written informed consent and the study was approved by the ethics committee at the Medical University of Graz, Graz, Austria. The study was designed to comply with the Declaration of Helsinki.
Intervention
Study medication was placed into numbered bottles according to a computer‐generated randomization list. Randomization procedures were conducted using Web‐based software (http://www.randomizer.at/), with good clinical practice‐compliance as confirmed by the Austrian Agency for Health and Food Safety. Eligible study participants were randomly allocated in a 1:1 ratio to receive 2800 IU of vitamin D3 as seven oily drops per day (Oleovit D3, Fresenius Kabi Austria, Austria) or a matching placebo as seven oily drops per day for 8 weeks. We performed a permuted block randomization with a block size of 10 and stratification according to sex. All investigators/authors who enrolled participants, collected data, and handed out the study medication were blinded to participant allocation.
Outcome Measure
PAC was predefined as the secondary outcome in the trial.17 The present investigation reanalyzes the dataset adhering to a strict protocol and investigates the change of aldosterone between baseline and follow‐up visit, adjusting for baseline differences in angiotensin‐converting enzyme (ACE) inhibitor intake and 25(OH)D levels.19
Measurements
Physical examinations, blood sampling, and patient interviews (medication intake and medical history) were performed at study visits between 7 am and 11 am after an overnight fast. The patients left the hospital for ambulatory BP measurements and 24‐hour urine collections before returning to the outpatient clinic the next day. On the second day, eligible study participants were randomized and started intake of the study medication. PAC (in ng/dL) was determined by means of a radioimmunoassay (RIA), active aldosterone RIA DSL‐8600 (DiagnosticSystems Laboratories, Inc, Webster, TX). Plasma renin concentrations were measured in EDTA plasma by a Renin III Generation RIA (Renin IRMA RIA‐4541; DRG Instruments GmbH, Marburg, Germany). Parathyroid hormone (PTH) (in pg/mL) was measured from plasma with a sandwich electrochemiluminescence immunoassay on an Elecsys 2010 (Roche Diagnostics, Mannheim, Germany). Intra‐assay and interassay coefficients of variation (CVs) were 1.6% and 3.9%, respectively, and reference ranges were 15 pg/mL to 65 pg/mL. Plasma 25(OH)D (in ng/mL) was determined using a chemiluminescence assay (IDS‐iSYS 25‐hydroxyvitamin D assay; Immunodiagnostic Systems Ltd., Boldon, UK) on an IDS‐iSYS multidiscipline automated analyzer with an intra‐assay and interassay CV of 6.2% and 11.6%, respectively. The other measurements were performed by routine laboratory procedures. Further details on different laboratory measurements have been published previously.22, 23, 24
Data Analyses
Continuous data following a normal distribution are shown as means with standard deviations, and variables with a skewed distribution are shown as medians with interquartile ranges. Categorical data are presented as percentages. When appropriate, skewed variables were log(e) transformed before use in parametric statistical analyses. Group comparisons at baseline were performed by unpaired Student t test or chi‐square test. Analyses of all variables were performed according to the intention‐to‐treat principle but with no data imputation and thus only inclusion of all participants with baseline and follow‐up values of the respective outcome variable. Analyses of covariance with adjustments for differences in baseline values, ie, use of ACE inhibitors and baseline 25(OH)D levels, were used to test for differences between the treatment and the placebo groups. Selection process was based on a strict protocol, where we first selected parameters that (based on available literature) are thought to be highly relevant for the RAAS and then tested for significant differences between placebo and intervention groups at the baseline visit. In addition, plasma renin concentration and aldosterone to active renin ratio was assessed using the identical adjustments as for aldosterone. In sensitivity analysis, we excluded participants undergoing treatment with a mineralocorticoid receptor antagonist and used multiple data imputation to assess reliability of the results.25 A P value <.05 was considered statistically significant.
All statistical analyses were performed using SPSS version 21.0 software (IBM Corp, Armonk, NY) and STATA version 13 (StataCorp LP, College Station, TX).
Results
A total of 518 invited study participants gave written informed consent and were assessed for eligibility. The entire trial was performed between 2011 and August 2014. Baseline characteristics of all randomized study participants are shown in Table 1. In the vitamin D group, ACE inhibitor use was significantly lower than that in the placebo group (Table 1). Despite strict monitoring, one study participant with an elevated serum calcium concentration of 2.69 mmol/L was randomized, thus violating the exclusion criterion of hypercalcemia. By adhering to the intention‐to‐treat principle, we did not exclude this study participant from our final analyses. A total of 187 study participants (mean±standard deivation age: 60.1±11.3 years; 47% women; baseline 25[OH]D: 21.2±5.6 ng/mL) completed the trial. The overall treatment period was 54±10 days in the vitamin D group and 54±9 days in the placebo group. There was a significant increase in 25(OH)D (mean treatment effect, +11.5; 95% confidence interval [CI], 9.4–13.7 ng/mL; P<.001) and a significant decrease in PTH (mean treatment effect, −5.7; CI, −9.3 to –2.1 pg/mL; P=.003) with no effect on total calcium, as published previously.17 Changes in PAC were significantly different between the placebo and intervention arm (+3.24 ng/dL vs +0.89 ng/dL; P=.04) (Table 2, Figure). When further adjusting for plasma PTH levels at the follow‐up visit, the difference in PAC was no longer statistically significant (P=.10), suggesting an interaction. No effect was seen for plasma renin concentration (P=.41) or aldosterone to renin ratio (P=.98).
Table 1.
Baseline Characteristics of Patients in the Placebo and Vitamin D Groups at Randomization
| Placebo (n=95) | Vitamin D (n=92) | |
|---|---|---|
| Mean±SD Median (IQR) | Mean±SD Median (IQR) | |
| Age, y | 59.7±11.4 | 60.5±10.9 |
| Women, % | 51.1 | 48.9 |
| Aldosterone, ng/dL | 16.8±10.5 | 16.8±10.6 |
| Renin, μU/mL | 16.1 (9.5–51.6) | 16.3 (10.2–38.7) |
| AARR, ng/dL/μU/mL | 0.88 (0.26–1.55) | 0.81 (0.38–1.58) |
| Different blood pressure–lowering drugs, No. | 2.14 (1–3) | 1 (1–3) |
| Parathyroid hormone, pg/mL | 53.8 (39.6–65.6) | 53.5 (40.3–61.6) |
| 25‐Hydroxyvitamin D, ng/mL | 20.4±5.7 | 21.8±5.4 |
| Serum total calcium, mmol/L | 2.37±0.11 | 2.37±0.1 |
| Serum potassium, mmol/L | 4.03±0.36 | 4.03±0.38 |
| 24‐h sodium excretion, mmol/24 h | 169.8±79.6 | 163.5±74.5 |
| eGFR by MDRD‐6, mL/min/1.73 m2 | 70.2±15.7 | 73.3±16.7 |
| ACE inhibitor (yes), % | 38 | 25 |
| Angiotensin type 1 receptor blocker (yes), % | 31 | 33 |
| Mineralocorticoid receptor antagonist (yes), % | 2.0 | 1.0 |
| Thiazide or loop diuretics (yes), % | 48 | 42 |
Abbreviations: AARR, aldosterone to active renin ratio; eGFR, estimated glomerular filtration rate; IQR, interquartile range; MDRD‐6, Modification of Diet in Renal Disease based on 6 variables; SD, standard deviation. Baseline characteristics of the whole cohort including all participants who attended the follow‐up visit. P values are not routinely reported based on statistical guidelines.20
Table 2.
Treatment Effects and Group Comparisons Including Subgroup Analysis
| Characterisitics | Placebo | Vitamin D | Mean Change From Baseline | Treatment Effect (95%CI) | P Value | |||
|---|---|---|---|---|---|---|---|---|
| Baseline | Follow‐Up | Baseline | Follow‐Up | Placebo | Vitamin D | |||
| All patients randomized (intention to treat) | ||||||||
| No. (control vs intervention: 95 vs 92) | ||||||||
| Aldosterone, ng/dL | 16.8±10.5 | 19.4±10.5 | 16.8±10.6 | 17.3±8.3 | +3.24 | +0.88 | −2.93 (−5.67 to −0.18) | .04 |
| Renin, μUmL | 16.1 (9.5–51.6) | 20.9 (9.9–54.7) | 16.3 (10.2–38.7) | 16.3 (10.3–34.7) | +28.79 | −1.54 | −0.13 (−0.44 to 0.18)a | .41 |
| AARR, ng/dL/μU/mL | 0.88 (0.26–1.55) | 0.87 (0.28–1.75) | 0.81 (0.38–1.58) | 0.88 (0.50–1.77) | +0.6 | +0.7 | −0.00 (−0.32 to 0.31)a | .98 |
| Vitamin D <20 ng/mL (n=45 vs n=33) | ||||||||
| Aldosterone, ng/dL | 16.0±11.7 | 17.2±7.0 | 17.4±11.8 | 16.4±8.1 | +2.34 | −0.39 | −0.99 (−4.6 to 2.63)a | .59 |
| Renin, μU/mL | 16.6 (11.3–50.9) | 21.5 (12.7–44.2) | 19.3 (12.4–41.6) | 15.8 (10.0–32.4) | +5.51 | −10.90 | −0.20 (−0.67 to 0.27)a | .41 |
| AARR, ng/dL/μU/mL | 0.9 (0.25–1.37) | 0.74 (0.25–1.46) | 0.8 (0.4–1.6) | 0.85 (0.56–1.70) | −0.06 | +0.26 | 0.17 (−0.33 to 0.68)a | .50 |
| Vitamin D <12 ng/mL (n=9 vs n=6) | ||||||||
| Aldosterone, ng/dL | 13.0±5.6 | 13.8±6.7 | 12.1±8.7 | 12.5±5.3 | +0.89 | +0.42 | 0.11 (−0.51 to 0.73)a | .70 |
| Renin, μU/mL | 26.6 (11.3–75.7) | 25.9 (13.0–73.8) | 69.1 (14.7–121.2) | 24.8 (14.8–44.1) | −6.48 | −8.86 | −0.13 (−1.55 to 1.3)a | .85 |
| AARR, ng/dL/μU/mL | 0.24 (0.11–1.70) | 0.3 (0.14–1.22) | 0.30 (0.10–0.52) | 0.79 (0.30–0.83) | 0.00 | +0.10 | 0.31 (−1.04 to 1.66)b | .62 |
| ACE inhibitor– and angiotensin type 1 receptor blocker treatment–naive patientsb (n=31 vs n=42) | ||||||||
| Aldosterone, ng/dL | 17.2±10.4 | 22.1±14.7 | 17.4±11.3 | 17.2±8.3 | +5.29 | +0.76 | −0.21 (0.46 to 0.04)a | .10 |
| Renin, μU/mL | 13.3 (9.6–17.4) | 12.9 (9.0–21.4) | 11.8 (6.9–1.98) | 12.4 (7.9–16.8) | −1.1 | −3.3 | −0.17 (−0.49 to 0.15)a | .29 |
| AARR, ng/dL/μU/mL | 1.23 (0.69–1.85) | 1.46 (0.88–2.02) | 1.38 (0.69–1.98) | 1.27 (0.87–2.01) | +0.17 | +0.9 | −0.05 (−0.37 to 0.27)a | .77 |
Abbreviations: AARR, aldosterone to active renin ratio; CI, confidence interval. aLog(n) transformed values. bExcluding patients treated with angiotensin‐converting enzyme (ACE) inhibitors or angiotensin type 1 receptor blockers. Analysis of covariance adjusted for baseline differences in ACE inhibitor intake and 25(OH)D status. P values <.050 were considered statistically significant. Bold value indicates significance.
Figure 1.
Changes in plasma aldosterone concentration (PAC) were significantly different between the intervention and placebo arm and relative to placebo vitamin D supplementation resulted in a lesser increase in PAC compared with placebo (+3.24 ng/dL and +0.89 ng/dL; adjusted P=.035).
No patient died during the study and there was no excess of adverse events (ie, hypercalcemia or hospitalizations) in the vitamin D intervention group.
Discussion
The present investigation is the first RCT to describe a significant reduction of plasma aldosterone by vitamin D supplementation compared with placebo in patients with arterial hypertension. The current investigation has some important caveats, intrinsically linked with post hoc analysis.20 We adjusted the statistical analysis model based on differences in the proportion of ACE inhibitor intake and baseline 25(OH)D levels between the placebo and vitamin D groups, as both are important confounding factors that may have altered aldosterone response to vitamin D supplementation.15, 19, 26, 27, 28 Our findings are in contrast to results from Schrotten and colleagues,29 who published a single‐center, open‐label trial in 101 stable heart failure patients demonstrating a significant reduction in plasma renin activity and concentration by supplementing 2000 IU oral vitamin D3 daily for 6 weeks compared with a control population. However, they were unable to show an effect on PAC concentration in patients with arterial hypertension. This may be caused by differences in the cohorts studied, as we recruited patients with arterial hypertension in contrast to patients with heart failure.18, 30, 31 The conflicting results might also be related to independent regulatory circuits or the result of interactions with antihypertensive medication.18 Nevertheless, our findings are in line with another study demonstrating that treatment with cholecalciferol in spontaneously hypertensive rats significantly reduced plasma aldosterone levels.27 The treatment effect might be based on the interaction between PTH and aldosterone.32
PTH was markedly suppressed in our study, which might be the physiologic explanation of the reduction in PAC.
Strengths and Limitations
Because the main outcome (reduction of 24‐hour mean systolic BP) was missed, the question of whether the significant reduction of PAC is of clinical relevance arises.17 It should also be noted that the difference is modest and not necessarily of clinical relevance.7, 8, 9, 18 The low prevalence of patients with (severe) vitamin D deficiency (<20 ng/mL or <12 ng/mL, respectively) and therefore the small sample sizes in these subgroups are drawbacks of our study. We thus cannot exclude more pronounced effects of vitamin D in populations with very low vitamin D levels or without antihypertensive treatment.18 Future clinical trials should therefore follow Heaney's guidelines and in particular include participants with lower 25(OH)D concentration.32 Furthermore, the before‐mentioned interaction with RAAS‐modifying treatment in the majority of our patients hampers the interpretation of changes in aldosterone and renin plasma levels, although per protocol, all randomized patients had to be on a stable treatment. Nevertheless, the strengths of the study are clearly the randomized design and the double‐blinded placebo‐controlled intervention.
Conclusions
The findings show a small, albeit significant, effect of vitamin D supplementation on PAC but no effect on renin or aldosterone to renin ratio. This finding warrants additional investigations on the potential interaction between vitamin D and the RAAS and its clinical significance. Therefore, we advocate further and larger RCTs powered to detect hard clinical endpoints to answer this critical question.
Sources of Funding
The Styrian Hypertension Study was supported by funding from the Austrian National Bank (Jubilaeumsfond: project no.: 13878 and 13905).
Disclosures
None.
Acknowledgments
We thank all study participants. We also thank Fresenius Kabi for providing the study medication and BioPersMed (COMET K‐project 825329, funded by the Austrian Federal Ministry of Transport, Innovation and Technology [BMVIT], the Austrian Federal Ministry of Economics and Labour/Federal Ministry of Economy, Family and Youth [BMWA/BMWFJ], and by the Styrian Business Promotion Agency [SFG]) for analysis equipment.
J Clin Hypertens (Greenwich). 2016;18:608–613. DOI: 10.1111/jch.12825 © 2016 Wiley Periodicals, Inc.
Clinical Trials registration: EudraCT Number 2009‐018125‐70 (additional registration at ClinicalTrials.gov Identifier NCT02136771).
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