Association of Vitamin D Metabolites With Arterial Function in the ...

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Mar 27, 2017 - the Hemodialysis Fistula Maturation Study. Adriana J. van Ballegooijen, PhD,1,2 Leila Zelnick, PhD,1. Andrew N. Hoofnagle, MD, PhD,3 Naomi ...
Original Investigation Association of Vitamin D Metabolites With Arterial Function in the Hemodialysis Fistula Maturation Study Adriana J. van Ballegooijen, PhD,1,2 Leila Zelnick, PhD,1 Andrew N. Hoofnagle, MD, PhD,3 Naomi M. Hamburg, MD,4 Cassiane Robinson-Cohen, PhD,1 Prabir Roy-Chaudhury, MD, PhD,5 Alfred K. Cheung, MD,6 Yan-Ting Shiu, PhD,6 Ian H. de Boer, MD, MS,1 Jonathan Himmelfarb, MD,1 Gerald Beck, PhD,7,8 Peter B. Imrey, PhD,7,8 John W. Kusek, PhD,9 and Bryan Kestenbaum, MD, MS,1 on behalf of the Hemodialysis Fistula Maturation (HFM) Study Group* Background: Disturbances in vitamin D metabolism are common in patients with end-stage renal disease and may contribute to vascular dysfunction. Study Design: Cross-sectional. Setting & Participants: We evaluated 558 of 602 participants at baseline of the Hemodialysis Fistula Maturation (HFM) Study, a 7-center prospective cohort study of a cohort of patients with chronic kidney disease awaiting arteriovenous fistula (AVF) creation surgery. Factor: 4 vitamin D metabolites measured with liquid chromatography–tandem mass spectroscopy from samples obtained within 4 weeks prior to AVF surgery. Outcomes: Vasodilator functions and measurements of arterial stiffness. Measurements: Trained HFM Study personnel measured brachial artery flow-mediated dilation, nitroglycerinmediated dilation, and carotid-femoral and carotid-radial pulse wave velocities (PWVs) prior to AVF creation. We evaluated associations after basic adjustment for sex, age, and clinical site and more fully adjusted additionally for baseline education, smoking, body mass index, diabetes, dialysis status, and medication use. Results: Mean participant age was 55 6 13 (SD) years and 65% were receiving maintenance dialysis. None of the vitamin D metabolites were significantly associated with flow-mediated dilation, carotid-femoral PWV, or carotid-radial PWV in basic or fully adjusted analyses. Higher serum concentrations of bioavailable vitamin D and 1,25-dihydroxyvitamin D were associated with 0.62% and 0.58% greater nitroglycerin-mediated dilation values, respectively, in basic models; however, these associations were no longer statistically significant with full adjustment. There were no significant associations of vitamin D metabolites with carotid-femoral or carotid-radial PWV in fully adjusted analyses. Limitations: Cross-sectional ascertainment of vitamin D metabolites and vascular functions late during the course of kidney disease. Conclusions: Serum concentrations of vitamin D metabolites are not associated with vasodilator functions or vascular stiffness at baseline in a cohort study of patients with chronic kidney disease awaiting AVF creation surgery. Laboratory measurements of vitamin D metabolites are unlikely to provide useful information regarding vascular functions in this setting. Am J Kidney Dis. 69(6):805-814. ª 2017 by the National Kidney Foundation, Inc. Published by Elsevier Inc. All rights reserved. INDEX WORDS: Vascular function; endothelial function; vitamin D metabolites; calcitriol; vasodilation; stiffness; arterial function; epidemiology; hemodialysis; arteriovenous fistula (AVF); flow mediated dilation (FMD); nitroglycerine-mediated dilation (NMD); pulse wave velocities (PWV); end-stage renal disease (ESRD).

From the 1Kidney Research Institute, University of Washington, Seattle, WA; 2VU University Amsterdam, Amsterdam, the Netherlands; 3Department of Laboratory Medicine, University of Washington, Seattle, WA; 4Whitaker Cardiovascular Institute, Boston University School of Medicine, Boston, MA; 5Division of Nephrology, University of Arizona, Tucson, AZ; 6Division of Nephrology, University of Utah School of Medicine, Salt Lake City, UT; 7Department of Quantitative Health Sciences, Cleveland Clinic Lerner Research Institute; 8Cleveland Clinic Lerner College of Medicine of Case Western Reserve University, Cleveland, OH; and 9National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD. * A list of the members of the HFM Study Group has been previously published.14 Received November 8, 2016. Accepted in revised form January 16, 2017. Originally published online March 27, 2017. Am J Kidney Dis. 2017;69(6):805-814

Because Editor-in-Chief Feldman and Deputy Editor Dember recused themselves from consideration of this article, the editor who handled the peer-review and decision-making processes (Roy D. Bloom, MD) also served as Acting Editor-in-Chief. Details of the journal’s procedures for potential editor conflicts are given in the Information for Authors & Journal Policies. Address correspondence to Adriana J. van Ballegooijen, PhD, VU University Amsterdam, De Boelelaan 1085, 1081 HV Amsterdam, the Netherlands. E-mail: [email protected]  2017 by the National Kidney Foundation, Inc. Published by Elsevier Inc. All rights reserved. 0272-6386 http://dx.doi.org/10.1053/j.ajkd.2017.01.049

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itamin D exerts a broad range of potentially favorable effects on vascular function. Calcitriol (1,25-dihydroxyvitamin D3 [1,25(OH)2D3]), the biologically potent form of vitamin D, binds to its intracellular target receptor to regulate the transcription of target genes that play important roles in vasoregulation, inflammation, and thrombosis.1,2 Calcitriol directly stimulates the production of nitric oxide in cultured endothelial cells and downregulates cyclooxygenase 1 expression.3,4 In animal models, calcitriol suppresses renin expression, leading to subsequent reductions in angiotensin and aldosterone synthesis.5 Calcitriol also exhibits broad immunomodulatory effects, including suppression of major inflammatory cytokines and differentiation of T-helper cell subsets.6,7 Chronic kidney disease (CKD) leads to multiple inter-related disturbances in vitamin D metabolism.8 The kidneys are the primary site for converting 25-hydroxyvitamin D (25[OH]D) to its biologically potent 1,25-dihydroxylated form: 1,25-dihydroxyvitamin D (1,25[OH]2D).9 A decline in serum 1,25(OH)2D concentrations and secondary hyperparathyroidism are among the earliest detectable metabolic disturbances of CKD.10 A loss of functioning nephrons also leads to stagnant vitamin D catabolism, evidenced by a decline in serum concentrations of 24,25-dihydroxyvitamin D3, (24,25 [OH]2D3), the most abundant vitamin D metabolite.11 Moreover, 25(OH)D substrate deficiency is common in CKD and other chronic disease populations. These aggregate disturbances in vitamin D metabolism may contribute to the high prevalence of vascular dysfunction and cardiovascular disease in the setting of end-stage renal disease (ESRD) and earlier stages of CKD. Previous studies have suggested associations of vitamin D metabolites with endothelial function and arterial stiffness in ESRD populations with ESRD or earlier stages of CKD.12,13 However, these studies are limited by small sample sizes, differing procedures for measuring vascular function, and evaluation of limited numbers of vitamin D metabolites. We addressed these shortcomings by measuring a comprehensive set of inter-related vitamin D metabolites, including vitamin D2binding globulin, for estimation of bioavailable vitamin D, and determining associations with arterial vasodilator functions and vascular stiffness in the Hemodialysis Fistula Maturation (HFM) Study, a prospective cohort study designed to identify predictors of arteriovenous fistula (AVF) maturation failure outcomes.14 We hypothesized that higher serum concentrations of vitamin D metabolites would be associated with greater vasodilatory responses and reduced arterial stiffness.

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METHODS Study Population The HFM Study recruited 602 patients with CKD prior to their undergoing AVF creation surgery at one of 7 clinical sites across the United States: Boston University, University of Cincinnati, University of Alabama2Birmingham, University of Florida, University of Texas Southwestern, University of Utah, and University of Washington. Patients were eligible if they were either currently receiving maintenance hemodialysis or expected to initiate dialysis therapy within 3 months. Study participants were recruited from nephrology and vascular surgery clinics, dialysis units, interventional radiology and nephrology practices that perform vascular access procedures, and hospitals that provide CKD care. Exclusion criteria were age younger than 18 years, age older than 80 years if not yet on maintenance hemodialysis therapy, inability to provide informed consent, anticipated life expectancy less than 9 months, or inability to meet protocol requirements in the judgment of the study investigators. All participants gave written informed consent, and the protocol was approved by the institutional review boards/ethics committees of the participating sites and is in accordance with the Declaration of Helsinki. We excluded 44 HFM participants from our analysis; 40 did not provide a baseline blood sample and 4 had indeterminate values of vitamin D2binding globulin. Among the remaining 558 participants, technically acceptable measurements of flow-mediated dilation, nitroglycerin-mediated dilation, and pulse wave velocity (PWV) were obtained for 510, 423, and 422 participants, respectively.

Vitamin D Metabolites The HFM Study coordinators collected blood samples typically within 3 weeks prior to vascular function testing. Coordinators shipped samples to the National Institute of Diabetes and Digestive and Kidney Diseases Biosample Repository, where they were stored until shipment to the University of Washington Nutrition Obesity Research Center. Personnel from this research center measured serum concentrations of 1,25-dihydroxyvitamin D2 (1,25[OH]2D2), 1,25(OH)2D3, 24,25(OH)2D3, 25-hydroxyvitamin D2 (25[OH]D2], 25-hydroxyvitamin D3 (25[OH]D3), vitamin D2binding globulin mass, and vitamin D2binding globulin isoform using liquid chromatography2tandem mass spectroscopy on an Xevo TQ tandem mass spectrometer (Waters).11,15 Interassay coefficients of variation for these vitamin D metabolite assays across a range of measured concentrations are 3.5% to 10.4% (Table S1, available as online supplementary material). We calculated total 1,25(OH)2D as the sum of 1,25(OH)2D2 and 1,25(OH)2D3 and calculated total 25(OH)D as the sum of 25(OH) D2 and 25(OH)D3. We estimated bioavailable 25(OH)D based on measured serum 25(OH)D, vitamin D2binding globulin mass, vitamin D2binding globulin isoform, and serum albumin level according to published equations.16 For participants who had heterozygous vitamin D2binding globulin isoforms (2 different alleles), we averaged the binding affinity of the 2 alleles to estimate bioavailable vitamin D.17

Vascular Function Tests Vascular function measurements included flow-mediated and nitroglycerin-mediated brachial artery dilation to assess endothelium-dependent and endothelium-independent functions and carotid-femoral and carotid-radial PWVs to assess arterial stiffness of the aorta and peripheral arteries. The HFM Study vascular function personnel were trained by the HFM Study Vascular Function Core laboratory at Boston University before the

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Vitamin D and Vascular Function start of the study, and tests were performed using a standardized protocol. Study personnel performed vascular function testing in the arm in which the fistula was to be created, unless a functioning access was already present in that arm. Patients who were receiving maintenance hemodialysis underwent vascular function testing on a nondialysis day. When possible, all vascular function tests were performed on a single day prior to AVF creation surgery, with the arterial PWVs assessed first, followed by flowmediated dilation and then nitroglycerin-mediated dilation. Three supine blood pressure and pulse measurements were obtained 1 minute apart with the SunTech 247 diagnostic station (SunTech Medical). Radial and femoral pulses were located and a tape measure was used to record the distances between each of these pulses to the nearest centimeter. The pulse was monitored until 10 consecutive waves were obtained with similar amplitudes before capturing the measurements. Next, radial and carotid artery pulse wave measurements were obtained using the SphygmoCor CPV system (AtCor Medical). The procedure was repeated for femoral artery measurements. Carotid-radial and carotid-femoral PWVs are expressed in meters per second. The procedure was repeated if the relative standard deviation, that is, the coefficient of

variation of the captured repeated measurements, was .10%, and the test was rejected on technical grounds if this could not be achieved. The brachial artery was examined using a high-resolution ultrasound system. After participants rested for 10 minutes in a supine position, imaging was optimized by visualizing an artery segment over at least 1 cm to obtain baseline 2-dimensional (2D) and Doppler recordings over 15 seconds and 10 cardiac cycles. A blood pressure cuff was inflated to the higher of 200 mm Hg or 50 mm Hg above the systolic blood pressure for 5 minutes, followed by immediate cuff deflation. Doppler recording over 10 cardiac cycles and 2D images over a 1-minute 15-second interval were obtained. If any portion of the procedure was unsuccessful, the participant was asked to wait for 2 hours before the procedure was repeated. The nitroglycerin portion of the test was omitted if the participant had any of the following conditions: (1) baseline systolic blood pressure , 100 mm Hg; (2) history of migraine headaches; (3) history of nitrate intolerance; (4) use of sildenafil, vardenafil, or tadalafil in the previous 7 days; or (5) pregnancy. Otherwise, after completion of the flow-mediated dilation test, the participant again

Table 1. Baseline Characteristics by Categories of Bioavailable Vitamin D Bioavailable Vitamin D

Age, y Male sex Race/ethnicity White Black Other Maintenance dialysis Diabetes mellitus Education #High school Some college $Completed college Smoking Never Former Current BMI, kg/m2 Systolic blood pressure, mm Hg Albumin, g/dL Hemoglobin, g/dL C-Reactive protein, g/dL B-Type natriuretic peptide, pg/mL Oral medication use Ergocalciferol Cholecalciferol Paricalcitol Calcitriol ACE inhibitor Angiotensin receptor blocker Calcium channel blocker Statin Diuretic

,1.67 ng/mL (n 5 186)

1.67-2.89 ng/mL (n 5 186)

$2.9 ng/mL (n 5 186)

51.4 6 12.3 118 (63)

55.7 6 12.8 132 (71)

58.0 6 14.5 144 (77)

52 (28) 116 (62) 18 (10)

89 (48) 79 (42) 18 (10)

123 (66) 46 (25) 17 (9)

124 (67) 124 (67)

118 (63) 107 (58)

117 (63) 105 (56)

57 (31) 113 (61) 9 (5)

51 (27) 102 (55) 28 (15)

43 (23) 106 (57) 34 (18)

79 (42) 65 (35) 40 (22)

86 (46) 61 (33) 38 (20)

93 (50) 72 (39) 20 (11)

31.4 6 8.0 156.6 6 23.5 3.2 6 0.6 10.1 6 1.7 6.5 [2.8-22.0] 179.0 [79.0-531.0]

30.6 6 7.4 150.1 6 23.0 3.6 6 0.5 10.6 6 1.6 4.9 [2.3-16.8] 191.0 [72.0-481.8]

29.0 6 7.2 148.0 6 24.0 3.8 6 0.5 10.8 6 1.5 4.2 [1.6-11.8] 161.0 [67.0-377.0]

25 (13) 13 (7) 17 (9) 35 (19) 74 (40) 18 (10) 127 (68) 111 (60) 88 (47)

23 (12) 34 (18) 14 (8) 47 (25) 73 (39) 23 (12) 124 (67) 95 (51) 100 (54)

28 (15) 52 (28) 14 (8) 50 (27) 58 (31) 23 (12) 112 (60) 105 (56) 87 (47)

Note: N 5 558. Values for categorical variables are given as number (percentage); values for continuous variables, as mean 6 standard deviation or median [interquartile range] if skewed. Abbreviations: ACE, angiotensin-converting enzyme; BMI, body mass index. Am J Kidney Dis. 2017;69(6):805-814

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van Ballegooijen et al rested in the supine position for 10 minutes. Baseline 2D images over 15 seconds were obtained in the same segment of the brachial artery. The participant was then given 0.4 mg of sublingual nitroglycerin. After 2 minutes 45 seconds and confirming with the participant that the nitroglycerin had dissolved, additional 2D images over a 1-minute interval were obtained. All brachial artery ultrasound images were read at a central core laboratory at Boston University using customized software with automated edge detection. Flow-mediated dilation is primarily expressed as percent change in diameter (millimeters) at 1 minute after cuff deflation relative to baseline, and nitroglycerin-mediated dilation similarly, as percent change in prenitroglycerin baseline diameter after nitroglycerin administration. For both flow-mediated dilation and nitroglycerin-mediated dilation, absolute pre-to-post changes in diameter were also examined as secondary measures.

Other Study Variables Personnel of the HFM Study collected information from participants about demographic characteristics, presence of comorbid conditions, and use of oral medications, including vitamin D supplements and vitamin D receptor agonists, at the time of study entry. Intravenous medications given during hemodialysis were not assessed in HFM. Study participants completed questionnaires to report race, smoking status, and the highest level of education attained. Study staff measured height, weight, and 3 blood pressures at rest in the nonaccess arm. We categorized race as black or

nonblack; level of education as some high school or less, some college/technical school certificate, or completed college or more; and smoking status as never, former, or current. We combined hemodialysis and peritoneal dialysis into a single dialysis category because there were only 4 participants who were receiving peritoneal dialysis at baseline. We measured serum concentrations of creatinine and albumin on a Beckman-Coulter DXC automated chemistry analyzer and calculated estimated glomerular filtration rate for participants not receiving dialysis using the 2009 CKDEPI (CKD Epidemiology Collaboration) creatinine equation.18

Statistical Analyses We summarized continuous values by mean 6 standard deviation (SD) or median and interquartile range (IQR) if notably skewed. We described univariate associations among measured vitamin D metabolites using Pearson correlation coefficients. We used linear regression to estimate cross-sectional associations of baseline vascular function measurements with vitamin D metabolites after adjustment for characteristics that could plausibly confound this association. Our first model examined associations adjusted for age, sex, race, and study site. Our second model added adjustments for dialysis status, diabetes status, body mass index, current smoking, education, use of vitamin D medications (ergocalciferol, or cholecalciferol, calcitriol, and paricalcitol), angiotensin-converting enzyme inhibitors, angiotensin receptor blockers, and calcium channel blockers. We also show mean

Table 2. Associations of Vitamin D Metabolites With Brachial Artery Flow-Mediated Dilation Model 1

Model 2

Bioavailable vitamin D Adjusted mean FMD, by tertile ,1.67 ng/mL (n 5 186) 1.67-2.89 ng/mL (n 5 186) $2.9 ng/mL (n 5 186) Difference in FMD, per 1 SD (1.82 ng/mL) higher P

3.97% (3.29% to 4.66%) 5.02% (4.26% to 5.79%) 5.19% (4.47% to 5.90%) 0.36% (20.05% to 0.77%) 0.09

4.23% (3.53% to 4.93%) 4.95% (4.18% to 5.72%) 4.99% (4.29% to 5.69%) 0.13% (20.29% to 0.55%) 0.5

Total 25(OH)D Adjusted mean FMD, by category ,20 ng/mL (n 5 175) 20-30 ng/mL (n 5 174) .30 ng/mL (n 5 209) Difference in FMD, per 1 SD (13.9 ng/mL) higher P

4.12% (3.41% to 4.82%) 5.27% (4.46% to 6.08%) 4.79% (4.15% to 5.42%) 0.38% (20.02% to 0.79%) 0.07

4.37 (3.63% to 5.12%) 5.34% (4.54% to 6.13%) 4.50% (3.85% to 5.15%) 0.14% (20.32% to 0.60%) 0.5

Total 1,25(OH)2D Adjusted mean FMD, by tertile ,8.8 pg/mL (n 5 186) 8.8-20.4 pg/mL (n 5 186) $20.5 pg/mL (n 5 186) Difference in FMD, per 1 SD (17.2 pg/mL) higher P

4.09% (3.43% to 4.75%) 4.73% (4.01% to 5.44%) 5.37% (4.56% to 6.17%) 0.33% (20.12% to 0.77%) 0.2

4.03% (3.36% to 4.70%) 4.77% (4.07% to 5.46%) 5.36% (4.58% to 6.13%) 0.31% (20.14% to 0.76%) 0.2

24,25(OH)2D3 Adjusted mean FMD, by tertile ,0.35 ng/mL (n 5 181) 0.35-0.86 ng/mL (n 5 190) .0.86 (n 5 187) Difference in FMD, per 1 SD (0.88 ng/mL) higher P

4.19% (3.50% to 4.88%) 4.72% (4.01% to 5.42%) 5.25% (4.44% to 6.07%) 0.31% (20.10% to 0.72%) 0.2

4.36% (3.66% to 5.06%) 4.69% (3.99% to 5.38%) 5.10% (4.33% to 5.88%) 0.17% (20.23% to 0.57%) 0.4

Note: Values in parentheses are 95% CIs. Model 1 adjusted for age, sex, race, and study site. Model 2 adds adjustment for dialysis status, diabetes, body mass index, current smoking, education, use of vitamin D medications, use of angiotensin-converting enzyme inhibitors, use of angiotensin receptor blockers, and use of calcium channel blockers. Abbreviations: 1,25(OH)2D, 1,25-dihydroxyvitamin D; 24,25(OH)2D3, 24,25-dihydroxyvitamin D3; 25(OH)D, 25-hydroxvitamin D; CI, confidence interval; FMD, flow-mediated dilatation; SD, standard deviation. 808

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Vitamin D and Vascular Function values for categories of 25(OH)D according to Endocrine Society guidelines19 and for tertiles of bioavailable vitamin D, 1,25(OH)2D, and 24,25(OH)2D3 adjusted to mean values of model covariates to more fully portray their associations with arterial functions. For all analyses, we multiply imputed vascular function measurements for HFM Study participants who had missing values for flow-mediated dilation (n 5 48), nitroglycerin-mediated dilation (n 5 135), and PWV (n 5 136), and rarely, other covariates.20 Multiple imputations were generated by chained equations using a comprehensive set of HFM covariates and results combined across imputations using Rubin’s rules to account for variability in the imputation procedure.21 Minimum detectable effect sizes with 80% and 90% power per standard deviation difference in vitamin D metabolites were 0.124 and 0.143 SDs of flow-mediated dilation and 0.136 and 0.157 SDs of nitroglycerinmediated dilation and both PWVs. We conducted analyses with R statistical software, version 3.3.0 (R Foundation for Statistical Computing). We considered 2-sided P , 0.05 to be statistically significant.

RESULTS Description of HFM Study Population Mean age of the 558 HFM Study participants in this ancillary study was 55 6 13 years, 394 (71%) were men, and 336 (60%) had a history of diabetes. There were 363 (65%) participants who were receiving maintenance dialysis at the time of AVF creation surgery, with a median dialysis vintage of 0.4 (IQR, 0.21.2) years. Among the 195 participants who were not receiving dialysis, median estimated glomerular filtration rate was 12 (IQR, 10-16) mL/min/1.73 m2.

Participants who had higher serum bioavailable vitamin D concentrations were older, more likely to be white, and more likely to be taking oral cholecalciferol or calcitriol (Table 1). Correlations Among Vitamin D Metabolites Bioavailable vitamin D and total 25(OH)D levels were strongly correlated with each other (r 5 0.78), and each was moderately correlated with 24,25(OH)2D3 levels (r 5 0.38 and r 5 0.41, respectively). Total serum 25(OH)D level was more modestly correlated with total 1,25(OH)2D level (r 5 0.21; Table S2). Associations of Vitamin D Metabolites With FlowMediated Vasodilation Mean brachial artery diameters at rest and postdeflation were 4.56 6 0.81 mm and 4.76 6 0.77 mm, respectively, corresponding to a mean flow-mediated dilation of 4.76%64.93%. Initial and final brachial artery diameters were highly correlated (r 5 0.97). Mean flow-mediated dilation values tended to be greater for higher categories of bioavailable vitamin D, 1,25(OH)2D, and 24,25(OH)2D3 (Table 2). However, in continuous analyses adjusted for age, race, sex, and study site, none of the vitamin D metabolites was significantly associated with flow-mediated dilation. Associations remained statistically nonsignificant after further adjustment for dialysis status,

Figure 1. Associations of continuous serum mineral metabolism concentrations with vascular functions stratified by dialysis status. Analyses adjusted for age, sex, race, study site, diabetes, body mass index, current smoking, education, use of vitamin D medications, use of angiotensin-converting enzyme inhibitors, use of angiotensin receptor blockers, and use of calcium channel blockers. The xaxes depict adjusted difference in each vascular function measurement per standard deviation increment in each vitamin D metabolism marker. Solid circles represent estimated adjusted differences, and solid horizontal bars represent 95% confidence intervals. The dashed vertical line indicates the null association (adjusted difference of 0). Abbreviations: BAVD, bioavailable vitamin D; FMD, flow-mediated vasodilation; NMD, nitroglycerin-mediated vasodilation; PWV CF, carotid femoral pulse wave velocity; PWV CR, carotid radial pulse wave velocity. Am J Kidney Dis. 2017;69(6):805-814

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diabetes, body mass index, smoking, education, and medications. Analyses of absolute rather than relative change in vessel diameter yielded substantively similar results. Associations of vitamin D metabolites with flow-mediated dilation were statistically similar (overlapping confidence intervals) among participants who were receiving maintenance dialysis at the time of surgery and those who were not receiving dialysis (Fig 1). Associations of Vitamin D Metabolites With Nitroglycerin-Mediated Vasodilation Mean nitroglycerin-mediated dilation was 7.11%6 6.15%. There was moderate correlation between flow-mediated dilation and nitroglycerin-mediated dilation measurements (r 5 0.51). Similar to findings for flow-mediated dilation, higher categories of vitamin D metabolites tended to track with higher values of nitroglycerin-mediated dilation (Table 3). After basic adjustment for demographics and study site, each 1-SD higher bioavailable vitamin D and 1,25(OH)2D concentration was associated with an estimated

0.62% and 0.58% greater nitroglycerin-mediated dilation, respectively. However, full adjustment for model covariates markedly attenuated the association for bioavailable vitamin D and modestly attenuated the association for 1,25(OH)2D. After full adjustment, none of the vitamin D metabolites remained significantly associated with nitroglycerin-mediated dilation. Analyses of absolute rather than relative change in vessel diameter yielded similar findings. The size of associations of vitamin D metabolites with flow-mediated and nitroglycerin-mediated dilation did not differ significantly by race or maintenance dialysis status. Associations of Vitamin D Metabolites With PWVs Mean values for carotid-femoral and carotid-radial PWVs were 10.7 6 3.2 and 8.8 6 1.7 m/s, respectively. In continuous analyses adjusted for basic demographic variables, only bioavailable vitamin D concentrations were associated with carotid-femoral PWV (Tables 4 and 5). None of the vitamin D metabolites was associated with carotid-femoral or carotid-radial PWVs in fully adjusted analyses.

Table 3. Associations of Vitamin D Metabolites With Brachial Artery Nitroglycerin-Mediated Dilation Model 1

Bioavailable vitamin D Adjusted mean NMD, by tertile ,1.67 ng/mL (n 5 186) 1.67-2.89 ng/mL (n 5 186) $2.9 ng/mL (n 5 186) Difference in NMD, per 1 SD (1.82 ng/mL) higher P Total 25(OH)D Adjusted mean NMD, by category ,20 ng/mL (n 5 175) 20-30 ng/mL (n 5 174) .30 ng/mL (n 5 209) Difference in NMD, per 1 SD (13.9 ng/mL) higher P Total 1,25(OH)2D Adjusted mean NMD, by tertile ,8.8 pg/mL (n 5 186) 8.8-20.4 pg/mL (n 5 186) $20.5 pg/mL (n 5 186) Difference in NMD, per 1 SD (17.2 pg/mL) higher P 24,25(OH)2D3 Adjusted mean NMD, by tertile ,0.35 ng/mL (n 5 181) 0.35-0.86 ng/mL (n 5 190) .0.86 ng/mL (n 5 187) Difference in NMD, per 1 SD (0.88 ng/mL) higher P

6.61% 7.31% 8.17% 0.62%

(5.66% to (6.41% to (7.26% to (0.00% to 0.05

Model 2

7.57%) 8.21%) 9.08%) 1.24%)

6.67% (5.73% to 7.61%) 7.78% (6.75% to 8.81%) 7.60% (6.72% to 8.48%) 0.40% (20.16% to 0.96%) 0.2

6.57% 7.17% 8.35% 0.58%

(5.65% to (6.22% to (7.42% to (0.04% to 0.04

7.49%) 8.12%) 9.27%) 1.11%)

6.57% (5.72% to 7.42%) 7.38% (6.46% to 8.30%) 8.11% (6.98% to 9.25%) 0.35% (20.34% to 1.05%) 0.3

6.97% (5.99% to 7.95%) 7.27% (6.37% to 8.17%) 7.87% (6.95% to 8.78%) 0.33% (20.29% to 0.96%) 0.3

7.01% (6.05% to 7.96%) 7.87% (6.89% to 8.86%) 7.25% (6.32% to 8.19%) 0.08% (20.57% to 0.72%) 0.8

6.61% (5.67% to 7.54%) 7.22% (6.31% to 8.13%) 8.27% (7.35% to 9.18%) 0.53% (20.02% to 1.08%) 0.06

6.94% (6.06% to 7.81%) 7.40% (6.50% to 8.31%) 7.75% (6.61% to 8.88%) 0.07% (20.61% to 0.74%) 0.9

Note: Values in parentheses are 95% confidence intervals. Model 1 adjusted for age, sex, race, and study site. Model 2 adds adjustment for dialysis status, diabetes, body mass index, current smoking, education, use of vitamin D medications, use of angiotensin-converting enzyme inhibitors, use of angiotensin receptor blockers, and use of calcium channel blockers. Abbreviations: 1,25(OH)2D, 1,25-dihydroxyvitamin D; 24,25(OH)2D3, 24,25-dihydroxyvitamin D3; 25(OH)D, 25-hydroxvitamin D; NMD, nitroglycerin-mediated dilatation; SD, standard deviation. 810

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Vitamin D and Vascular Function Table 4. Associations of Vitamin D Metabolites With Carotid-Femoral PWV Model 1

Model 2

Bioavailable vitamin D Adjusted mean carotid-femoral PWV, in m/s, by tertile ,1.67 ng/mL (n 5 186) 1.67-2.89 ng/mL (n 5 186) $2.9 ng/mL (n 5 186) Difference in carotid-femoral PWV, in m/s, per 1 SD (1.82 ng/mL) higher P

11.17 (10.66 to 11.68) 10.85 (10.41 to 11.30) 10.27 (9.81 to 10.73) 20.31 (20.61 to 0.00) 0.05

11.10 (10.62 to 11.57) 10.95 (10.53 to 11.38) 10.27 (9.83 to 10.71) 20.26 (20.57 to 0.05) 0.1

Total 25(OH)D Adjusted mean carotid-femoral PWV, in m/s, category ,20 ng/mL (n 5 175) 20-30 ng/mL (n 5 174) .30 ng/mL (n 5 209) Difference in carotid-femoral PWV, in m/s, per 1 SD (13.9 ng/mL) higher P

10.97 (10.44 to 11.50) 10.91 (10.48 to 11.34) 10.47 (10.00 to 10.95) 20.2 (20.5 to 0.1) 0.2

10.90 (10.39 to 11.41) 10.97 (10.56 to 11.38) 10.50 (10.04 to 10.96) 20.17 (20.48 to 0.15) 0.3

Total 1,25(OH)2D Adjusted mean carotid-femoral PWV, in m/s, by tertile ,8.8 pg/mL (n 5 186) 8.8-20.4 pg/mL (n 5 186) $20.5 pg/mL (n 5 186) Difference in carotid-femoral PWV, in m/s, per 1 SD (17.2 pg/mL) higher P

11.05 (10.57 to 11.53) 10.58 (10.09 to 11.08) 10.66 (10.2 to 11.13) 20.2 (20.4 to 0.0) 0.09

11.1 (10.65 to 11.55) 10.52 (10.05 to 11.00) 10.69 (10.25 to 11.13) 20.14 (20.37 to 0.08) 0.2

24,25(OH)2D3 Adjusted mean carotid-femoral PWV, in m/s, by tertile ,0.35 ng/mL (n 5 181) 0.35-0.86 ng/mL (n 5 190) .0.86 ng/mL (n 5 187) Difference in carotid-femoral PWV, in m/s, per 1 SD (0.88 ng/mL) higher P

10.77 (10.29 to 11.25) 10.80 (10.34 to 11.26) 10.72 (10.22 to 11.22) 20.03 (20.32 to 0.26) 0.9

10.67 (10.20 to 11.15) 10.85 (10.42 to 11.28) 10.79 (10.30 to 11.28) 0.03 (20.27 to 0.32) 0.9

Note: Values in parentheses are 95% confidence intervals. Model 1 adjusted for age, sex, race, and study site. Model 2 adds adjustment for dialysis status, diabetes, body mass index, current smoking, education, use of vitamin D medications, use of angiotensin-converting enzyme inhibitors, use of angiotensin receptor blockers, and use of calcium channel blockers. Abbreviations: 1,25(OH)2D, 1,25-dihydroxyvitamin D; 24,25(OH)2D3, 24,25-dihydroxyvitamin D3; 25(OH)D, 25-hydroxvitamin D; PWV, pulse wave velocity; SD, standard deviation.

Further, none of the associations of vitamin D metabolites with vascular functions changed significantly after adjustment for the natural log of C-reactive protein concentrations (Table S3).

DISCUSSION In summary, serum concentrations of bioavailable vitamin D, 25(OH)D, 1,25(OH)2D, and 24,25(OH)2D3 were not associated with vasodilator functions or central or peripheral arterial stiffness among 558 patients with CKD awaiting AVF creation surgery. To our knowledge, this is the largest study of vitamin D metabolites and vascular function in such a population that includes comprehensive vascular testing procedures and detailed covariate assessment to address potential confounding. Null associations were observed in the context of vitamin D measurements that were quantified using precise liquid chromatography2tandem mass spectrometry assays and vascular function tests that were performed by trained study personnel under standardized conditions. Although associations still may exist in the late-stage CKD population, the upper limits of the Am J Kidney Dis. 2017;69(6):805-814

95% confidence intervals suggest that potential associations are likely to be quantitatively small. Our investigation of vitamin D metabolites and vascular functions was motivated by mechanistic and clinical evidence suggesting that disturbances in vitamin D metabolism may contribute to the high prevalence of vascular disease in CKD. Serum concentrations of 1,25(OH)2D3 (calcitriol), the activated form of vitamin D, progressively decline during the course of CKD due to loss of functional renal mass, and inhibitory effects of fibroblast growth factor 23.10,22,23 Calcitriol modulates a diverse set of target genes that play important roles in vascular function, including renin, proinflammatory cytokines, and tissue factor.3-7 Previous small cross-sectional studies have suggested correlations of 1,25(OH)2D3 concentrations with flowmediated dilation in dialysis patients.12 Moreover, clinical trials of vitamin D receptor analogues in patients with CKD have demonstrated improvements in flow-mediated dilation and reductions in albuminuria, a marker of endothelial function.24 811

van Ballegooijen et al Table 5. Associations of Vitamin D Metabolites With Carotid-Radial PWV Model 1

Bioavailable vitamin D Adjusted mean carotid-radial PWV, in m/s, by tertile ,1.67 ng/mL (n 5 186) 1.67-2.89 ng/mL (n 5 186) $2.9 ng/mL (n 5 186) Difference in carotid-radial PWV, in m/s, per 1 SD (1.82 ng/mL) higher P Total 25(OH)D Adjusted mean carotid-radial PWV, in m/s, by category ,20 ng/mL (n 5 175) 20-30 ng/mL (n 5 174) .30 ng/mL (n 5 209) Difference in carotid-femoral PWV, in m/s, per 1 SD (13.9 ng/mL) higher P Total 1,25(OH)2D Adjusted mean carotid-radial PWV, in m/s, by tertile ,8.8 pg/mL (n 5 186) 8.8-20.4 pg/mL (n 5 186) $20.5 pg/mL (n 5 186) Difference in carotid-femoral PWV, in m/s, per 1 SD (17.2 pg/mL) higher P 24,25(OH)2D3 Adjusted mean carotid-radial PWV, in m/s, by tertile ,0.35 ng/mL (n 5 181) 0.35-0.86 ng/mL (n 5 190) .0.86 ng/mL (n 5 187) Difference in carotid-femoral PWV, in m/s, per 1 SD (0.88 ng/mL) higher P

Model 2

9.09 8.65 8.79 20.08

(8.81 to 9.37) (8.38 to 8.93) (8.51 to 9.06) (20.25 to 0.08) 0.3

9.08 8.67 8.77 20.06

(8.80 to 9.36) (8.40 to 8.94) (8.50 to 9.05) (20.23 to 0.10) 0.5

8.96 8.85 8.74 20.05

(8.66 to 9.26) (8.56 to 9.13) (8.49 to 8.99) (20.22 to 0.13) 0.6

8.94 8.86 8.74 20.03

(8.64 to 9.24) (8.58 to 9.14) (8.49 to 8.99) (20.21 to 0.16) 0.8

9.01 8.73 8.79 20.05

(8.74 to 9.28) (8.48 to 8.98) (8.54 to 9.04) (20.21 to 0.12) 0.6

8.80 (8.53 to 9.07) 8.88 (8.62 to 9.15) 8.84 (8.55 to 9.13) 0.10 (20.08 to 0.27) 0.3

8.94 (8.66 to 9.22) 8.72 (8.48 to 8.96) 8.86 (8.60 to 9.12) 0.01 (20.16 to 0.17) 0.9

8.78 (8.53 to 9.04) 8.87 (8.61 to 9.13) 8.87 (8.58 to 9.16) 0.11 (20.05 to 0.27) 0.2

Note: Values in parentheses are 95% confidence intervals. Model 1 adjusted for age, sex, race, and study site. Model 2 adds adjustment for dialysis status, diabetes, body mass index, current smoking, education, use of vitamin D medications, use of angiotensin-converting enzyme inhibitors, use of angiotensin receptor blockers, and use of calcium channel blockers. Abbreviations: 1,25(OH)2D, 1,25-dihydroxyvitamin D; 24,25(OH)2D3, 24,25-dihydroxyvitamin D3; 25(OH)D, 25-hydroxvitamin D; PWV, pulse wave velocity; SD, standard deviation.

Nonetheless, we did not detect associations of circulating markers of vitamin D metabolism with vasodilator functions or measures of vascular stiffness in a contemporary cohort of patients with CKD awaiting AVF surgery. It is possible that serum concentrations of the selected vitamin D metabolites do not adequately inform vitamin D activity within cells, particularly in the setting of ESRD. The value of serum 25(OH)D concentrations may be diminished in patients with ESRD due to markedly suppressed 1,25(OH)2D synthesis and catabolism.9,22 Although bioavailable vitamin D may provide a more precise estimate of available vitamin D stores by incorporating protein binding, this measurement is subject to similar limitations of other vitamin D substrate measurements in patients with kidney disease. Serum 1,25(OH)2D measurements theoretically provide a more direct readout of vitamin D activity. However, circulating 1,25(OH)2D concentrations have a short biological half-life, are tightly regulated by other metabolic processes, and may not reflect intracellular vitamin D activity. 812

It is also possible that potential vasodilatory, antithrombotic, and immunomodulatory properties of vitamin D are blunted by long-standing hypertension and vascular disease that are highly prevalent in flowmediated dilation ESRD. Moreover, inherent imprecision of the noninvasive vascular function tests, even when carefully performed under standardized conditions, may have also contributed to the null findings. Although vitamin D metabolism markers were measured with high laboratory precision, biological variation in these markers within individuals may have also weakened potential associations with vascular function outcomes. Our null findings for serologic markers of vitamin D do not exclude a mechanistic contribution of vitamin D metabolism to vascular disease or a potential therapeutic role of vitamin D treatments on vascular function. However, our results question the value of laboratory measurements of vitamin D metabolites as indicators of vascular function in ESRD. Strengths of our study include use of dedicated liquid chromatography2tandem mass spectrometry Am J Kidney Dis. 2017;69(6):805-814

Vitamin D and Vascular Function

assays to simultaneously measure multiple interrelated vitamin D metabolites with high precision, including measurement of vitamin D2binding globulin mass and isoform to estimate bioavailable vitamin D. The internal validity of our findings is further enhanced by standardized study procedures used to measure flowmediated dilation, nitroglycerin-mediated dilation, and PWV and by collection of important comorbid condition data to address potential confounding. The HFM Study population was recruited from multiple centers across the United States, increasing the generalizability of our results. Limitations of our study include the observational design, cross-sectional ascertainment of vitamin D metabolites and vascular functions at a single time point late during the course of kidney disease, and potential misclassification of study measurements. In summary, serum concentrations of bioavailable vitamin D, 25(OH)D, 1,25(OH)2D, and 24,25(OH)2D3 were not meaningfully associated with vasodilator functions or measures of arterial stiffness in a cohort of 558 patients with CKD awaiting AVF surgery. Clinical trials of vitamin D treatments offer the most direct approach for determining whether vitamin D has biological or clinical impact on vascular functions in populations with ESRD or earlier stages of CKD.

ACKNOWLEDGEMENTS Support: The HFM Study is funded by the following grants from the National Institute of Diabetes and Digestive and Kidney Diseases: U01DK066597, U01DK082179, U01DK082189, U01DK082218, U01DK082222, U01DK082236, and U01DK082240. This ancillary study was funded by grant R01 DK094891 and by an unrestricted fund from the Northwest Kidney Centers and by the Niels Stensen Fellowship 2014. None of the funders of this study had any role in the collection, analysis, and interpretation of data for this study. Financial Disclosure: Dr Kestenbaum reports receiving consulting fees from Keryx Biopharmaceuticals, Medscape Inc, and Sanofi Inc. Dr de Boer reports receiving fees from Amgen, Boehringer-Ingelheim, Ironwood, and Janssen. Dr Cheung reports receiving fees from TVA Medical and Hemocytes. Dr Hamburg reports receiving consulting fees from Acceleron Pharma. The other authors declare that they have no other relevant financial interests. Contributions: Research idea and study design: ANH, NMH, PR-C, AKC, Y-TES, JH, PBI, JWK, BK; data acquisition: NMH, JH, JWK, BK; data analysis/interpretation: AJvB, LZ, CR-C, IHdB, GB, PBI, BK; statistical analysis: AJvB, LZ; supervision or mentorship: IHdB, BK. Each author contributed important intellectual content during manuscript drafting or revision and accepts accountability for the overall work by ensuring that questions pertaining to the accuracy or integrity of any portion of the work are appropriately investigated and resolved. Peer Review: Evaluated by 2 external peer reviewers, a Statistics/Methods Editor, and an Acting Editor-in-Chief.

SUPPLEMENTARY MATERIAL Table S1: Interassay coefficients of variation for vitamin D metabolite assays. Am J Kidney Dis. 2017;69(6):805-814

Table S2: Pearson correlations among the vitamin D metabolites. Table S3: Association of vitamin D metabolites with vascular functions before and after adjustment for CRP. Note: The supplementary material accompanying this article (http://dx.doi.org/10.1053/j.ajkd.2017.01.049) is available at www.ajkd.org

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