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Effect of vitamin K supplementation on serum calcification propensity and arterial stiffness in vitamin K-deficient kidney transplant recipients: A double-blind, randomized, placebo-controlled clinical trial
† These authors contributed equally: Coby Eelderink and Daan Kremer.
Daan Kremer
Correspondence
Corresponding author: Division of Nephrology, Department of Internal Medicine, University Medical Center Groningen, Hanzeplein 1, HPC: AA53, 9713GZ Groningen, The Netherlands.
Department of Internal Medicine, Division of Vascular Medicine, University of Groningen, University Medical Center Groningen, Groningen, the Netherlands
Vitamin K deficiency is common among kidney transplant recipients (KTRs) and likely contributes to progressive vascular calcification and stiffness. In this single-center, randomized, double-blind, placebo-controlled trial, we aimed to investigate the effects of vitamin K supplementation on the primary end point, serum calcification propensity (calciprotein particle maturation time, T50), and secondary end points arterial stiffness (pulse wave velocity [PWV]) and vitamin K status in 40 vitamin K-deficient KTRs (plasma dephosphorylated uncarboxylated matrix Gla protein [dp-ucMGP] ≥500 pmol/L). Participants (35% female; age, 57 ± 13 years) were randomized 1:1 to vitamin K2 (menaquinone-7, 360 μg/day) or placebo for 12 weeks. Vitamin K supplementation had no effect on calcification propensity (change in T50 vs baseline +2.3 ± 27.4 minutes) compared with placebo (+0.8 ± 34.4 minutes; Pbetween group = .88) but prevented progression of PWV (change vs baseline −0.06 ± 0.26 m/s) compared with placebo (+0.27 ± 0.43 m/s; Pbetween group = .010). Vitamin K supplementation strongly improved vitamin K status (change in dp-ucMGP vs baseline −385 [−631 to −269] pmol/L) compared with placebo (+39 [−188 to +183] pmol/L; Pbetween group < .001), although most patients remained vitamin K-deficient. In conclusion, vitamin K supplementation did not alter serum calcification propensity but prevented progression of arterial stiffness, suggesting that vitamin K has vascular effects independent of calciprotein particles. These results set the stage for longer-term intervention studies with vitamin K supplementation in KTRs.
Trial registry
EU Clinical Trials Register (EudraCT Number: 2019-004906-88) and the Dutch Trial Register (NTR number: NL7687).
Even after successful transplantation, kidney transplant recipients (KTRs) remain at high risk of premature death, which is largely attributable to a residual increased cardiovascular risk.
Progression of vascular calcification is increasingly regarded as an important contributor to adverse outcomes in patients with kidney disease, including KTRs.
Moreover, serum calcification propensity as measured by calciprotein particle maturation time (T50) is strongly associated with premature mortality in KTRs.
Unfortunately, treatment options that target vascular calcification are lacking, and progression of vascular calcification remains a major clinical challenge in KTRs. Vitamin K is a promising intervention that may, at least in part, prevent progression of vascular calcification.
Vitamin K is a cofactor for vitamin K-dependent proteins, including major regulators of calcification processes. For instance, carboxylated matrix Gla protein (MGP) is a potent inhibitor of vascular calcification.
Vitamin K deficiency: an emerging player in the pathogenesis of vascular calcification and an iatrogenic consequence of therapies in advanced renal disease.
Because MGP carboxylation is dependent on vitamin K, vitamin K deficiency or antagonism impairs carboxylation of MGP, reflected by increased concentrations of dephosphorylated uncarboxylated MGP (dp-ucMGP) and resulting in progression of vascular calcification.
Given the high prevalence of vitamin K deficiency in KTRs, vitamin K supplementation may be an easy, safe, and affordable therapy to alleviate vascular calcification among KTRs.
but evidence in the KTR population is sparse. Previous intervention studies with vitamin K supplementation in KTRs showed conflicting results regarding the effects of vitamin K supplementation on arterial stiffness.
The ViKTORIES trial: A randomized, double-blind, placebo-controlled trial of vitamin K supplementation to improve vascular health in kidney transplant recipients.
This discrepancy highlights the need for further research on the effect of vitamin K supplementation on vascular health in KTRs. Therefore, in this parallel-group, randomized, double-blind, placebo-controlled trial, we investigated the effect of vitamin K supplementation on serum calcification propensity and arterial stiffness in vitamin K-deficient KTRs.
2. Materials and methods
2.1 Trial design and study participants
We performed a single-center, parallel-group, randomized, double-blind, placebo-controlled trial in KTRs with a functioning kidney graft (estimated glomerular filtration rate [eGFR] >20 mL/min/1.73 m2). All participants were adult KTRs participating in the ongoing TransplantLines Biobank and Cohort Study
and signed additional informed consent for the current trial. Patients were only included if vitamin K deficiency was confirmed, based on a plasma dp-ucMGP concentration of ≥500 pmol/L determined prior to study initiation (November 2018). Detailed inclusion and exclusion criteria are provided in Supplementary Table 1. All participants had their first study visit between September 2020 and July 2021. All study visits were performed by the same researcher (CE) in the outpatient clinic of the University Medical Center Groningen (UMCG), Groningen, the Netherlands. The study was designed and reported in accordance with the CONSORT Statement on transparent reporting of trials (Supplementary Table 2).
The study protocol and statistical analysis plan are provided in Appendix A, Appendix A. There were no important changes to the trial design after commencement.
2.2 Intervention
Participants were randomized to vitamin K2 (menaquinone-7),
360 μg/day or placebo during 12 weeks. Vitamin K2 is listed on the European list of accepted food supplements. The capsules with vitamin K and placebo were produced specifically for this clinical trial by Vitals Voedingssupplementen BV with a provided ISO22000:2005 certificate. Apart from vitamin K or placebo, patients did not receive any treatment other than standard care and maintained their regular diet and prescribed medication.
2.3 Randomization and blinding procedures
Treatment allocation (vitamin K or placebo) was randomized for each patient by straight randomization, performed by Vitals Voedingssupplementen BV. Neither the patient nor the researchers/physicians were aware of treatment allocation. The UMCG research pharmacy provided the capsules to the patients and kept the randomization list until unblinding after database closure.
2.4 Drug accountability
Each study participant received 3 capsule containers with 60 capsules containing either 180 μg vitamin K or placebo. Participants were asked to bring their capsule containers to their second study visit, after which the number of remaining capsules was translated to a percentage compliance for that participant. Patients were considered compliant when consuming >85% of capsules.
2.5 Primary outcome
The primary outcome of this study was change in serum calcification propensity at 12 weeks after initiation of the treatment, which was compared between the vitamin K arm and the placebo arm. Serum calcification propensity was assessed using calciprotein particle maturation time (T50), which depends on the individual composition of the patient’s serum levels, and specifically on the balance between calcification promotors and calcification-inhibiting factors.
The measurement of all samples occurred prior to unblinding and is described in detail in Supplementary Table 3.
2.6 Secondary outcomes
The main secondary end point was change in arterial stiffness as measured by pulse wave velocity (PWV) at 12 weeks after the start of the intervention, which was compared between the vitamin K group and the placebo group. PWV was measured thrice during each study visit, at the outpatient clinic, using the Mobil-O-Graph device (IEM, Supplemental Table 3).
Accuracy of a newly-introduced oscillometric device for the estimation of arterial stiffness indices in patients on peritoneal dialysis: a preliminary validation study.
The mean intraindividual coefficient of variation assessed at baseline was 1.3%.
Additional secondary end points were changes in vitamin K status parameters, including dp-ucMGP, uncarboxylated osteocalcin (ucOC), and the ratio of uncarboxylated to carboxylated OC (ucOC/cOC ratio, Supplemental Table 3).
2.7 Exploratory outcomes defined after trial commencement
In exploratory analyses, we assessed potential treatment effects on kidney function (because of a recent scientific discussion on the potential effects of vitamin K on kidney function
Reply to Janssen et al. Comment on “Kremer et al. Kidney function-dependence of vitamin K-status parameters: results from the TransplantLines biobank and cohort studies.
) and blood pressure (to explore if blood pressure may explain the treatment effect on PWV).
2.8 Additional data collection
Transplant characteristics and medical history were collected using patient records. Body weight, height, hip and waist circumference, and blood pressure were assessed using strict protocols.
Additional laboratory parameters were collected using standard automated laboratory procedures.
2.9 Statistical methods
Baseline characteristics are summarized by treatment group, with continuous variables presented as mean ± standard deviation or median (interquartile range), depending on distribution. Categorical data are presented as number (percentage).
In intention-to-treat analyses to assess treatment effects on T50 (primary outcome), PWV and vitamin K status parameters (secondary outcomes), and blood pressure and kidney function (exploratory outcomes), changes in outcomes from baseline to end of follow-up were compared between the placebo group and the intervention group using t tests or Mann–Whitney U tests, depending on data distribution. For all outcomes, values at baseline and after intervention were presented to visualize individual trajectories of these outcomes. Distributions of intraindividual changes in T50 and PWV were visualized using density plots. In sensitivity analyses, analysis of covariance (ANCOVA) was used to assess treatment effects on T50 and PWV with adjustment for predefined covariables, including age, sex, eGFR, systolic blood pressure, hemoglobin, phosphate, magnesium, adjusted calcium, bicarbonate, and albumin concentrations, on top of the basic model. We additionally adjusted for ucOC, ucOC/cOC ratio, smoking, and calcineurin inhibitor and statin use, given imbalances in these parameters at baseline. Treatment effect estimates are presented alongside 95% confidence intervals (CI) and P values. In additional exploratory analyses, we assessed whether the degree of improvement of vitamin K status parameters is associated with the improvement in arterial stiffness using linear regression.
All statistical analyses were performed using IBM SPSS Statistics 23 or R (version 4.0.5), where P value of <.05 was considered to indicate statistical significance. There were no missing data, other than losses to follow-up and missing creatinine clearance for 1 participant. Missingness is reported in text and table footnotes, and cases were excluded only from the concerning analyses.
the expected mean serum calcification propensity was 220 ± 40 minutes and T50 increases of 44 minutes (ie, 20% of 220 minutes) were considered clinically relevant. With a probability of type 1 error of 5%, and a power of 90%, a sample size of 18 subjects per treatment group would be necessary. Assuming a dropout of 10%, we aimed to include 20 subjects in the vitamin K group and 20 subjects in the placebo group.
2.11 Follow-up
After 6 weeks, subjects were contacted by phone to verify compliance and potential adverse events. Patients were invited for a second study visit 12 weeks after baseline. All patients were allowed to leave the study at any time.
2.12 Safety
Adverse events were recorded from the time a participant consented until the last study visit. Serious adverse events were reported to the competent authority within 7 days after first knowledge of the event. In addition, gastrointestinal symptoms were recorded using a questionnaire, which was filled out before supplement intake and in the final week of the intervention period.
2.13 Registration
This clinical trial was prospectively registered in the EU Clinical Trials Register (EudraCT Number: 2019-004906-88) and the Dutch Trial Register (NTR number: NL7687).
2.14 Ethics
The study protocol was approved by the UMCG institutional review board (METc: 2019/249, March 24, 2020), adheres to the UMCG Biobank Regulations, and was performed in accordance with the WMA Declarations of Helsinki and Istanbul. The trial progress was monitored by the principal investigator (CAtVK), and one routine audit/monitoring visit was performed by an independent monitor.
The study was financially supported by the Dutch Kidney Foundation (Nierstichting), and the capsules with vitamin K or placebo were provided free of charge by Vitals Voedingssupplementen BV. Both supporting parties had no role in the design or conduction of the study or in the publication process.
3. Results
3.1 Study recruitment and participants
Data on dp-ucMGP concentrations were readily available in 799 patients from the TransplantLines Biobank and Cohort Study. From this cohort, 220 patients were selected based on dp-ucMGP and eGFR. In total, 94 participants (43%) fulfilled all eligibility criteria. Inclusion was stopped when 40 patients provided consent. These patients were randomized to vitamin K (n = 20) or placebo (n = 20).
Three patients withdrew from the study because of health-related issues unrelated to the intervention. A detailed overview of the flow of participants through the study is shown in Figure 1.
Figure 1Flow diagram of study participants. dp-ucMGP, dephosphorylated uncarboxylated matrix Gla protein; eGFR, estimated glomerular filtration rate.
Among the 40 included participants, 14 (35%) were women, age was 57 ± 13 years, time after transplantation was 8.8 (5.0-15.0) years, and eGFR was 40.0 ± 10.5 mL/min/1.73 m2 in the overall study population. Detailed baseline characteristics across the treatment groups are presented in Table 1. There was some imbalance between the treatment groups at baseline regarding systolic blood pressure, type of dialysis before transplantation, and plasma calcium concentrations.
Table 1Population characteristics per treatment group.
Serum calcification propensity (T50) at baseline was similar in both groups (vitamin K: 283 ± 52, P = .75 vs placebo: 278 ± 56 minutes). Individual changes in serum calcification propensity are visualized per treatment group in Figure 2A. No significant difference in change in serum calcification propensity over 12 weeks between the treatment groups was observed (vitamin K: +2.3 ± 27.4 vs placebo: +0.8 ± 34.4 minutes; Pttest = .88, Fig. 2B). Assessment of relative rather than absolute changes (%) in T50 showed similar results (Table 2).
Figure 2(A) Visualization of intraindividual changes in the primary end point of T50, along with (B) a density plot visualizing intraindividual changes in T50 from baseline to 12 weeks. P value indicates the level of statistical significance of difference in change from baseline to 12 weeks in the vitamin K group vs placebo group, as assessed using independent sample t tests. T50, calciprotein maturation time.
Sensitivity analyses using ANCOVA with adjustment for predefined parameters known to influence T50 and parameters that differed between treatment groups at baseline also yielded similar results (Supplementary Table 4).
3.4 Secondary outcomes–arterial stiffness
Baseline PWV values were 8.3 ± 1.8 m/s in the vitamin K group and 8.4 ± 1.7 m/s in the placebo group (P = .84). Individual changes in PWV are visualized per treatment group in Figure 3A. A significant treatment effect was observed regarding change of PWV between both groups (vitamin K: −0.06 ± 0.26 m/s vs placebo: +0.27 ± 0.43 m/s, Pttest = .010, Fig. 3B, Table 3).
Figure 3(A) Visualization of intraindividual changes in the secondary end point of PWV, along with (B) a density plot visualizing intraindividual changes in PWV from baseline to 12 weeks. P value indicates the level of statistical significance of difference in change from baseline to 12 weeks in the vitamin K group vs placebo group, as assessed using independent sample t tests. PWV, pulse wave velocity.
Sensitivity analyses using ANCOVA with adjustment for baseline PWV, age, systolic blood pressure, and other potential confounders yielded similar results. The between-group difference in PWV change was also independent of the change in blood pressure (Supplementary Table 4). In exploratory analyses, linear regression analyses showed no significant association of change in dp-ucMGP with change in PWV (adjusted R2 = 0.05; P = .2).
3.5 Secondary outcomes—vitamin K status parameters
As expected, there was a strong decrease in circulating dp-ucMGP in the vitamin K group compared with the placebo group (−385 [−631 to −269] pmol/L vs +39 [−188 to +183] pmol/L, respectively, P < .001). Strong decreases were also observed for ucOC and ucOC/cOC ratio in the vitamin K group (Table 4, Fig. 4), indicating that the intervention successfully improved vitamin K status, although the vitamin K status parameters did not reach physiological ranges.
Table 4Outcomes of secondary end points of dp-ucMGP, ucOC, and ucOC/cOC (ratio) per treatment group.
End point
Placebo (n = 19)
Vitamin K (n = 18)
P
dp-ucMGP (pmol/L)
Visit 1 (baseline)
1551 (1151-1898)
1318 (993-1504)
.11
Visit 2 (wk 12)
1580 (1296-1801)
856 (708-950)
<.001
Change (pmol/L)
39 (−188 to 183)
−385 (−631 to −269)
<.001
Change (%)
2.4 (−14.4 to 18.4)
−33.9 (−46.2 to −24.3)
<.001
ucOC (ng/mL)
Visit 1 (baseline)
13.1 (10.4-38.3)
5.6 (3.2-8.2)
.002
Visit 2 (wk 12)
15.2 (10.8-29.7)
3.0 (2.3-5.8)
<.001
Change (ng/mL)
0.1 (−1.4 to 2.6)
−1.9 (−4.2 to −0.3)
.009
Change (%)
0.5 (−20.8 to 11.1)
−39.1 (−57.4 to −8.5)
.001
ucOC/cOC (ratio)
Visit 1 (baseline)
0.48 (0.27-0.78)
0.21 (0.13-0.32)
.004
Visit 2 (wk 12)
0.41 (0.27-0.69)
0.12 (0.09-0.17)
<.001
Change (ratio)
0.00 (−0.05 to 0.08)
−0.09 (−0.20 to −0.04)
.001
Change (%)
1.6 (−12.8 to 13.7)
−49.4 (−65.1 to −20.8)
.001
P values represent statistical significance of differences between groups as assessed using Mann–Whitney U tests.
Figure 4Visualization of intraindividual changes in the secondary end point of vitamin K status parameters per treatment group. cOC, carboxylated osteocalcin; dp-ucMGP, dephosphorylated uncarboxylated matrix Gla protein; T50, calciprotein maturation time; PWV, pulse wave velocity; ucOC, uncarboxylated osteocalcin.
In both groups, 1 participant ingested <85% of the prescribed capsules and was labeled as noncompliant. In the other subjects (n = 35), mean compliance was 98%. Results of the aforementioned analyses repeated only with patients having ingested >85% of the prescribed intake yielded similar results regarding changes in serum calcification propensity, PWV, and circulating dp-ucMGP concentrations (data not shown).
3.7 Exploratory analyses
Additional analyses to explore other potential effects of vitamin K-supplementation
Reply to Janssen et al. Comment on “Kremer et al. Kidney function-dependence of vitamin K-status parameters: results from the TransplantLines biobank and cohort studies.
showed no treatment effects on kidney function (eGFR: between-group difference in change: +0.17 [95% CI, −2.25 to +2.59] mL/min/1.73 m2), yet a nonsignificant trend toward blood pressure-lowering treatment effects (eg, systolic blood pressure: mean between-group difference in change: −4.47 [95% CI, −11.95 to +3.02] mmHg, Supplementary Table 5).
3.8 Safety and adverse events
There were 3 serious adverse events (hospitalizations) unrelated to the study medication. Adverse events were diverse in both the vitamin K group and placebo group (12 adverse events and 11 adverse events, respectively, Supplementary Table 6). There were no notable (increases of) gastrointestinal symptoms (Supplementary Table 7).
4. Discussion
This trial showed no evidence that 12 weeks of treatment with vitamin K improves serum calcification propensity in KTRs with suggested vitamin K deficiency. Interestingly, there was a significant treatment effect of vitamin K on the main secondary outcome of arterial stiffness. The treatment also drastically lowered circulating concentrations of dp-ucMGP, ucOC, and ucOC/cOC ratio, indicating uptake and biological activity of vitamin K.
Vascular calcification and concomitant increased cardiovascular risk are increasingly recognized as important causes of premature mortality among KTRs.
The effect of phosphate binder therapy with sucroferric oxyhydroxide on calcification propensity in chronic haemodialysis patients: a randomized, controlled, crossover trial.
The effect of increasing dialysate magnesium on serum calcification propensity in subjects with end stage kidney disease: a randomized, controlled clinical trial.
Vitamin K deficiency: an emerging player in the pathogenesis of vascular calcification and an iatrogenic consequence of therapies in advanced renal disease.
Because the carboxylation of MGP is vitamin K-dependent, it appears biologically plausible that vitamin K supplementation inhibits vascular calcification in vitamin K-deficient KTRs. Indeed, a previous single-arm study in 60 KTRs showed improvements in vitamin K status and arterial stiffness after vitamin K supplementation.
These improvements were most pronounced in study participants with vitamin K deficiency. We therefore performed this randomized, placebo-controlled trial to study whether vascular calcification and arterial stiffness may be alleviated by vitamin K supplementation in KTRs with vitamin K deficiency.
In this study, we observed no treatment effect of vitamin K supplementation on the primary outcome of serum calcification propensity. Notably, actual serum calcification propensity results differed from those estimated for sample size calculations, for which we used data derived from a subgroup of a previous study.
Nevertheless, repeating sample size calculations based upon the actual measured T50 measurements resulted in a very similar sample size (ie, 19 patients per treatment group). Thus, the lack of observed treatment effects regarding serum calcification propensity appears not to be the result of lack of statistical power alone.
Moreover, these results are in line with the recently published Vitamin K in Transplanted kidney Organ Recipients: Investigating vEssel Stiffness (ViKTORIES) trial, which showed no effects of vitamin K supplementation on vascular calcification as measured using coronary artery calcium score on computed tomography.
The ViKTORIES trial: A randomized, double-blind, placebo-controlled trial of vitamin K supplementation to improve vascular health in kidney transplant recipients.
Unfortunately, that study was limited because the researchers were unable to assess vitamin K status prior to the trial, which may have led to inclusion of patients that were vitamin K-sufficient and may therefore have limited benefit from vitamin K supplementation. This limitation was not present in the current study, in which baseline dp-ucMGP concentrations were all >750 pmol/L, indicating that we only included KTRs with vitamin K deficiency. More importantly, in a subgroup of 72 participants in the vitamin K arm in the ViKTORIES trial, mean change in dp-ucMGP was only −186 (95%CI, −294 to −78) pmol/L. In our current study, a much larger dp-ucMGP reduction of −385 (95%CI, −631 to −269) pmol/L was achieved. This suggests that, despite the shorter duration of vitamin K supplementation, the intervention in the current study (ie, vitamin K2, 360 μg, once daily) more effectively improved vitamin K status compared with the intervention in the ViKTORIES trial (ie, menadiol diphosphate, not previously used for this indication).
The ViKTORIES trial: A randomized, double-blind, placebo-controlled trial of vitamin K supplementation to improve vascular health in kidney transplant recipients.
One explanation for the lack of treatment effects of vitamin K on measures of calcification is that the ViKTORIES trial and the current study included KTRs with advanced vascular calcification and stiffness. Vitamin K might be able to prevent but not reverse calcification, similar to effects of magnesium observed in preclinical studies.
In line with this notion, a post hoc analysis of the ViKCoVaC trial showed that supplementation of vitamin K prevented the development of newly calcifying lesions in coronary arteries and aorta in patients with diabetes mellitus.
The effect of vitamin K1 on arterial calcification activity in subjects with diabetes mellitus: a post hoc analysis of a double-blind, randomized, placebo-controlled trial.
Therefore, it is tempting to speculate that prevention of new calcifying lesions with longer-term follow-up may show effects of vitamin K on calcification progression that were currently not observed.
Another explanation for the lack of treatment effect on serum calcification propensity may lie in the chosen outcome measure of T50. This measure, reflecting calciprotein maturation time, is clearly prognostically important and reflects key components of calcification. Nevertheless, serum calcification propensity does not reflect all pathways involved in the complex process of vascular calcification. We cannot exclude the possibility that potential calcification-inhibiting effects of vitamin K supplementation may not directly be reflected by changes in T50 but may still have beneficial effects on newly calcifying lesions through other pathways.
Importantly, this notion is further supported by the observed treatment effect of vitamin K supplementation on the secondary end point of arterial stiffness in our study. The used method to assess arterial stiffness (PWV) has been extensively validated in multiple populations, but not KTRs specifically.
The used Mobil-O-Graph provides PWV estimates close to the gold standard of intraaortic readings and was superior to carotid-femoral PWV (measured using SphygmoCor) with respect to associations with cardiac load and hypertensive organ damage.
Cross-sectional comparison of office and ambulatory pulse wave velocity by two methods, and their changes after lifestyle or medical interventions in hypertension.
However, the low intraindividual coefficient of variation, the robustness of the association independent of (change in) blood pressure and age, and the use of intraindividual PWV change as an end point in our study provide convincing evidence that there is an actual treatment effect of vitamin K supplementation on PWV. This finding is in line with previous observational studies that consistently showed associations between arterial stiffness and dp-ucMGP in the general population
These associations were further substantiated in Mendelian randomization analyses and MGP knockout mice models, indicating a direct role for MGP in regulating the development of arterial stiffness.
The clinical relevance of PWV as assessed using this method has been shown in multiple populations. For example, PWV was independently associated with mortality in patients with chronic kidney disease.
Importantly, the observed mean treatment effect in our study was 0.33 m/s within 12 weeks of treatment. In a study among Greek dialysis patients, a PWV increment of 1 m/s (assessed using Mobil-O-Graph) was associated with a 58% increased risk of mortality.
Ambulatory pulse wave velocity is a stronger predictor of cardiovascular events and all-cause mortality than office and ambulatory blood pressure in hemodialysis patients.
A study among KTRs showed that a PWV increment of 1 m/s (assessed using a different measurement method) was associated with a 36% relative increased risk of mortality.
Clearly, the observed beneficial effects of vitamin K supplementation on PWV in our study cannot be extrapolated nor directly linked to concomitant risks observed in such observational studies. Nevertheless, the observed treatment effect size within 12 weeks of treatment suggests clinical relevance.
As previously mentioned, a treatment effect of vitamin K supplementation on arterial stiffness was also observed in a single-arm study by Mansour et al
in KTRs that were administered the same dosage as in the current study. It must be noted that that single-arm study observed intraindividual improvements in arterial stiffness, whereas in our placebo-controlled trial, we primarily observed prevention of worsening of arterial stiffness in the vitamin K group compared with the placebo group.
Nevertheless, the observed beneficial treatment effects on arterial stiffness without treatment effects on serum calcification propensity suggest that vitamin K may have local vascular effects in the absence of detectable systemic effects on serum calcification propensity as measured by T50. Hypothetically, these local vascular effects may be caused by direct effects on vascular smooth muscle cells.
Alternatively, inflammation may explain the observed treatment effects of vitamin K on arterial stiffness. Indeed, inflammation promotes endothelial-to-mesenchymal transition and vascular calcification in animal and human tissue.
Interestingly, vitamin K attenuates the severity of inflammation in mice, by inhibiting the NLRP3 inflammasome assembly. Thus, we may speculate that vitamin K supplementation inhibits inflammation, thus reducing inflammation-mediated calcification.
These mechanisms may explain the rapid treatment effects on arterial stiffness observed in the current study during the 12-week follow-up. In contrast, the occurrence or progression of vascular calcification is a multifactorial process developing over many years, which may not be captured in a study with short-term follow-up, thus explaining the absence of effects on calciprotein maturation time. Perhaps, accumulation of calcium in vessel walls, such as those observed in preclinical studies
may be able to capture these processes at an earlier time point than radiography or calciprotein maturation time, which was used in existing clinical studies.
The ViKTORIES trial: A randomized, double-blind, placebo-controlled trial of vitamin K supplementation to improve vascular health in kidney transplant recipients.
Either way, these studies set the stage for long-term studies with vitamin K supplementation to further substantiate the clinical value of vitamin K supplementation in KTR. Moreover, it is tempting to speculate to combine vitamin K supplementation with observed treatment effects on the vessel wall (as measured by vascular stiffness) with an intervention targeting the circulation (as measured by serum calcification propensity), such as magnesium.
The effect of increasing dialysate magnesium on serum calcification propensity in subjects with end stage kidney disease: a randomized, controlled clinical trial.
We observed no statistically significant beneficial treatment effects in the exploratory end points. Thus, the current study allows to reject the hypothesis that 12 weeks of vitamin K supplementation dramatically attenuates blood pressure or kidney function. However, notably, there was a trend toward a beneficial treatment effect on blood pressure (ie, mean between-group difference in change of −4.47 (95% CI, −11.95 to +3.02) mmHg. Despite the lack of a statistically significant treatment effect, these findings are promising, and the presented data may serve as sample size calculations in future trials.
Strengths of the current study include its randomized, controlled, double-blind design, and the selection of vitamin K-deficient KTRs. In addition, improvement in vitamin K status was confirmed by assessing intraindividual changes in multiple markers of vitamin K status. Several limitations of the current study should be acknowledged. First, the study population size was determined based on the primary end point of T50, and treatment groups were therefore relatively small. However, statistical power did not appear to be the reason for the null result for the primary outcome, and there was sufficient statistical power for assessment of the main secondary end point of arterial stiffness. Moreover, despite randomization of treatment allocation, we observed several imbalances between the study arms at baseline. However, all study findings regarding primary and secondary outcomes remained materially unchanged after adjustment for these imbalances. Moreover, because arterial stiffness was not the primary end point, the observed beneficial treatment effects should not be interpreted as confirmatory.
The European Agency for the Evaluation of Medicinal Products Committee for Proprietary Medical Products (CPMP). Points to consider on multiplicity issues in clinical trials.
Therefore, future phase 3 clinical trials, including large numbers of patients are warranted before clinical implementation of vitamin K supplementation among KTRs. Such trials should also have a longer intervention and follow-up time to achieve normalization of vitamin K status, rather than mere improvement. Potentially, a sustained vitamin K supplementation with longer follow-up and focus on prevention of new calcifying lesions may show improvements in vascular calcification that were not observed during the follow-up in the current study. Moreover, such large trials may also be able to detect potential smaller treatment effects of vitamin K supplementation on exploratory end points, particularly regarding the blood pressure-related parameters, that could not be detected in the current study due to the limited sample size.
In conclusion, in this randomized, double-blind, placebo-controlled clinical trial, vitamin K supplementation did not alter serum calcification propensity, but prevented progression of arterial stiffness in KTRs, suggesting that vitamin K may have vascular effects independent of serum calcification propensity. These results provide a basis for long-term intervention studies with vitamin K2 supplementation in KTRs.
Acknowledgments
The authors would like to thank the NTx-VitK participants. The study was supported by the Dutch Kidney Foundation (Nierstichting), and the capsules with vitamin K2 or placebo were provided free of charge by Vitals Voedingssupplementen BV. Both supporting parties had no role in the design or conduction of the study, in the collection, analyses, or interpretation of data, or in the writing and publication process.
Appendix A. Supplementary data
The following is the Supplementary data to this article:
The study was supported by the Dutch Kidney Foundation (Nierstichting), and the capsules with vitamin K2 or placebo were provided free of charge by Vitals Voedingssupplementen BV. Both supporting parties had no role in the design or conduction of the study, in the collection, analyses, or interpretation of data, or in the writing and publication process.
Disclosures
The authors of this manuscript have conflicts of interest to disclose as described by the American Journal of Transplantation. L. J. Schurgers received research funding from Boehringer Ingelheim, NattoPharma, Bayer, and IDS not related to this work, and is a stockholder in Coagulation Profile. A. Pasch holds stock in Calciscon, is an inventor of the T50 test, and founder and employee of Calciscon AG, which commercializes the T50 test. The other authors of this manuscript have no conflicts of interest to disclose. Public sharing of individual participant data was not included in the informed consent forms of the study, but data can be made available to interested researchers upon reasonable request by contacting the corresponding author (DK).
Vitamin K deficiency: an emerging player in the pathogenesis of vascular calcification and an iatrogenic consequence of therapies in advanced renal disease.
The ViKTORIES trial: A randomized, double-blind, placebo-controlled trial of vitamin K supplementation to improve vascular health in kidney transplant recipients.
Accuracy of a newly-introduced oscillometric device for the estimation of arterial stiffness indices in patients on peritoneal dialysis: a preliminary validation study.
Reply to Janssen et al. Comment on “Kremer et al. Kidney function-dependence of vitamin K-status parameters: results from the TransplantLines biobank and cohort studies.
The effect of phosphate binder therapy with sucroferric oxyhydroxide on calcification propensity in chronic haemodialysis patients: a randomized, controlled, crossover trial.
The effect of increasing dialysate magnesium on serum calcification propensity in subjects with end stage kidney disease: a randomized, controlled clinical trial.
The effect of vitamin K1 on arterial calcification activity in subjects with diabetes mellitus: a post hoc analysis of a double-blind, randomized, placebo-controlled trial.
Cross-sectional comparison of office and ambulatory pulse wave velocity by two methods, and their changes after lifestyle or medical interventions in hypertension.
Ambulatory pulse wave velocity is a stronger predictor of cardiovascular events and all-cause mortality than office and ambulatory blood pressure in hemodialysis patients.
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