The Association of Serum Carotenoids, Tocopherols, and Ascorbic Acid With Rapid Kidney Function Decline: The Coronary Artery Risk Development in Young Adults (CARDIA) Study

ORIGINAL RESEARCH

The Association of Serum Carotenoids, Tocopherols, and Ascorbic Acid With Rapid Kidney Function Decline: The Coronary Artery Risk Development in Young Adults (CARDIA) Study Kristin M. Hirahatake, PhD, RD,* David R. Jacobs, PhD,† Myron D. Gross, PhD,† Kirsten B. Bibbins-Domingo, MD, PhD,‡ Michael G. Shlipak, MD, MPH,‡ Holly Mattix-Kramer, MD, MPH,§ and Andrew O. Odegaard, PhD, MPH* Objective: Nutritional intervention targeting dietary intake modification is a major component of treatment for chronic kidney disease; however, little is known about the relationship between dietary intake and kidney function decline in individuals with preserved kidney function. Design and methods: In this prospective cohort study we examined the association of biomarkers of dietary intake with kidney function decline over a 5-year interval in 2,152 men and women with cystatin-C–based estimated glomerular filtration rate . 60 mL/minute/ 1.73 m2 from the Coronary Artery Risk Development in Young Adults study. The biomarkers of interest included carotenoids, tocopherols, and ascorbic acid. Multivariable logistic regression was used to explore the relationship between serum concentrations of the sum of 4 carotenoids (a-carotene, b-carotene, b-cryptoxanthin, and lutein/zeaxanthin), lycopene, a-tocopherol, g-tocopherol, and ascorbic acid and rapid kidney function decline, defined as .15% decline in cystatin-C–based estimated glomerular filtration rate over 5 years. Results: During the 5-year follow-up, 290 participants (13.5%) experienced rapid kidney function decline. Relative to individuals in the lowest quartile of serum carotenoids, those in the highest quartile had significantly lower odds of rapid kidney function decline in the fully adjusted model (odds ratio, 0.51; 95% confidence interval [CI], 0.32-0.80; P trend, .02). No association of levels of serum tocopherols, ascorbic acid, or lycopene with kidney function decline was found. There was no evidence that results differed for individuals with hypertension or diabetes. Conclusions: These results demonstrate that higher serum carotenoid levels, reflective of a fruit- and vegetable-rich dietary pattern, inversely associate with rapid kidney function decline in early middle adulthood and provide insight into how diet might play a role in chronic kidney disease prevention. Ó 2018 by the National Kidney Foundation, Inc. All rights reserved.

Introduction HRONIC KIDNEY DISEASE (CKD) is a major economic and public health burden in the United States. According to a recent report from the Centers for Disease Control, 26 million American adults have kidney disease, and 1 in 3 adults are at high risk for developing kidney disease.1 Diabetes and hypertension are the leading

causes of CKD, with other risk factors including age, cardiovascular disease, obesity, and smoking.2 A substantial body of evidence supports a reduced risk or delayed development of CKD with optimal management of these conditions; however, little research has examined potentially modifiable factors that may influence CKD risk before the onset of major clinical risk factors.3

* Department of Epidemiology, School of Medicine, University of California, Irvine, Irvine, California. † Division of Epidemiology and Community Health, School of Public Health, University of Minnesota, Minneapolis, Minnesota. ‡ School of Medicine, University of California, San Francisco, San Francisco, California. § Public Health Sciences, Loyola University Medical Center, Maywood, Illinois. Support: The Coronary Artery Risk Development in Young Adults Study (CARDIA) is supported by contracts HHSN268201300025C, HHSN268201300026C, HHSN268201300027C, HHSN268201300028C, HHSN268201300029C,

and HHSN268200900041C from the National Heart, Lung, and Blood Institute and the Intramural Research Program of the National Institute on Aging. The Young Adult Longitudinal Trends in Antioxidants study is supported by the grant R01 HL 53560. Financial Disclosure: The authors declare that they have no relevant financial interests. Address correspondence to Andrew O. Odegaard, PhD, MPH, Department of Epidemiology, School of Medicine, University of California, Irvine, Irvine, California 92697-7550. E-mail: [email protected] Ó 2018 by the National Kidney Foundation, Inc. All rights reserved. 1051-2276/$36.00 https://doi.org/10.1053/j.jrn.2018.05.008

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Journal of Renal Nutrition, Vol -, No - (-), 2018: pp 1-9

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A limited body of evidence suggests that a diet characterized by high fruit, vegetable, whole grain, low-fat dairy, nut/seed, and legume intake and low in salt, saturated fat and cholesterol, sugar-sweetened beverages, and red and processed meats may be associated with lower risk of CKD in populations across the risk spectrum.3-7 However, existing studies of diet and CKD risk have focused on dietary patterns or specific nutrients derived from self-reported dietary intake and have not examined objectively measured dietary biomarkers in relation to kidney health. Although dietary questionnaires represent a valuable tool to estimate dietary patterns in large cohort studies, limited inference can be made from this information concerning the individual bioavailability of nutrients from the diet and their relationship to disease risk. Biomarkers from serum or other biological fluids, however, can provide a more reliable assessment of the association between individual dietary intakes and disease outcomes.8,9 Serum carotenoids and ascorbic acid are biomarkers of fruit and vegetable consumption10,11 because blood concentrations are generally responsive to dietary intake12 and supplementation13,14 in a linear fashion. Previous prospective epidemiological research has shown an inverse association between serum carotenoid concentrations and the 2 leading causes of CKD, hypertension15 and type 2 diabetes;16 however, whether an association exists with incident kidney disease, mediated by or independent of the aforementioned associations, is not known. Tocopherols are often used as markers of nut/seed and oil intake17 but are more tightly regulated by homeostatic mechanisms and thus less reflective of dietary intake than carotenoids.12 These biomarkers have also been shown to associate with healthy dietary patterns, as well as smoking and body mass index (BMI).18-20 A more comprehensive evidence base for the topic of diet and incident CKD risk will improve dietary recommendations and provide a more rigorous guide for dietary interventions for populations at high risk for developing CKD. To address this gap, we examined the association of objective biomarkers of dietary intake (serum carotenoids, tocopherols, and ascorbic acid) with kidney function decline over a 5-year interval in participants with preserved kidney function from the Coronary Artery Risk Development in Young Adults (CARDIA) study. We hypothesized that higher concentrations of serum carotenoids (sum of a-carotene, b-carotene, b-cryptoxanthin, and lutein/zeaxanthin), alpha-tocopherol, and ascorbic acid would be inversely associated with kidney function decline, whereas serum gamma-tocopherol would be positively associated with kidney function decline. In addition, we hypothesized that this association would be more pronounced for individuals in a high metabolic risk state, such as having diabetes or hypertension.

Methods The CARDIA and Young Adult Longitudinal Trends in Antioxidants (YALTA) Studies The CARDIA study is a prospective, multicenter cohort study initiated in 1984 to investigate the development and determinants of cardiovascular disease in young adults, previously described in detail elsewhere.21 Briefly, 5,115 black and white men and women, aged 18 to 30 years, were recruited between 1985 and 1986 from 4 US cities: Birmingham, Alabama; Chicago, Illinois; Minneapolis, Minnesota; and Oakland, California. The initial examination included standardized measures of known risk factors as well as psychosocial, dietary, and exercise-related characteristics. Reexamination occurred after 2, 5, 7, 10, 15, 20, 25, and 30 years. The Young Adult Longitudinal Trends in Antioxidants (YALTA) study is ancillary to CARDIA and aimed to examine the relationship between biomarkers related to antioxidants and cardiovascular disease.22 Serum concentrations of carotenoids and tocopherols were measured in most study participants at years 0, 7, and 15 and ascorbic acid at year 15. Complete data for the biomarkers of interest were available for 2,916 participants at year 15. Cystatin-C was also measured as part of an ancillary study to CARDIA on all available stored plasma samples from examinations at year 10, 15, and 20. This analysis is based on data from the 2,152 subjects (black and white men and women aged 3345 years) from the CARDIA study with preserved kidney function (defined as cystatin-C–estimated glomerular filtration rate (eGFRcys) . 60 mL/minute/1.732) and blood measures of dietary biomarkers at year 15, along with concurrent clinical risk factor data and measures of cystatin-C at years 15 and 20. The CARDIA Study was approved by the institutional review board at each clinical center, and informed consent was obtained from all participants before enrollment. Serum Biomarkers Serum obtained at CARDIA year 15 was used in the YALTA study to assay ascorbic acid; a- and g-tocopherol; and the carotenoids a-carotene, b-carotene, b-cryptoxanthin, lutein/zeaxanthin, and lycopene using a modified high-performance liquid chromatography method extensively described elsewhere (Molecular Epidemiology and Biomarker Research Laboratory, University of Minnesota, Minneapolis, MN).23,24 Quality-control procedures included routine analysis of plasma and serum control pools containing high and low concentrations of each analyte; the coefficients of variation were ,10% for all analytes and control pools. Previous work by this laboratory showed that the intraclass correlation coefficients (ratio of between-person variance to between- and within-person variance) were 0.93 for a-carotene, 0.98 for b-carotene, 0.73 for lutein plus zeaxanthin, 0.97 for b-cryptoxanthin, 0.73 for lycopene, and 0.93 for a-tocopherol.24

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Kidney Function Decline Estimated glomerular filtration rate (eGFR) was calculated using cystatin-C, which has been shown to be a more accurate measure of kidney function in high eGFR ranges25 and thus enable the detection of changes in kidney function earlier than creatinine.26 Cystatin-C was measured as part of an ancillary study on all stored frozen plasma samples from years 10, 15, and 20 simultaneously by nephelometry using the N Latex Cystatin-C kit (Dade Behring) and later calibrated to most recent cystatin-C standardization. Kidney function (eGFR in mL/minute/ 1.73 m2) was estimated by the 2012 CKD-EPI (Chronic Kidney Disease Epidemiology Collaboration) cystatin-C equation, which is for use with calibrated cystatin-C.27 We examined presence of rapid eGFRcys decline defined as .15% decline between years 15 and 20 (.3% per year) and incident CKD (eGFRcys , 60 mL/minute/ 1.73 m2 at study year 20) by quartiles of the dietary biomarkers. The definition employed for rapid decline has been used in previous studies28 and represents a magnitude of change 3 times the expected rate associated with aging.29 Other Measures The covariates age, sex, race, education, weekly alcohol consumption, and smoking were obtained from structured interview or by self-administered questionnaire at each follow-up visit. Alcohol intake (mL/day) was calculated from the self-reported frequency of weekly beer, wine, and liquor consumption. Smoking status was defined as never, past, or current use. Type 2 diabetes was defined as use of diabetes medication or a fasting plasma blood glucose of $7 mmol/L (126 mg/dL) at year 15. A physical activity score was derived from the CARDIA Physical Activity Questionnaire administered at year 15.30 At the year-15 follow-up visit, body weight with light clothing was measured to the nearest 0.09 kg, and height without shoes was measured to the nearest 0.5 cm. BMI was computed as weight divided by height squared (kg/m2). Plasma lipids were measured from blood samples collected at year 15 by the University of Washington Northwest Lipid Research Clinic Laboratory. Total triglycerides and total high-density lipoprotein (HDL) cholesterol were measured by enzymatic procedures, HDL cholesterol was measured after dextran sulfate–magnesium precipitation, and low-density lipoprotein (LDL) cholesterol was calculated using the Friedewald equation. Systolic and diastolic blood pressures (BPs) were measured at each examination after a 5-minute rest period, and the average of the second and third random zero sphygmomanometer measurements was used. At year 20, the Omron measure was used, calibrated to the zero sphygmomanometer. Albuminuria was determined from a single, untimed (spot) urine sample collected at the year-15 examination. Urine albumin concentrations were measured using a nephelometric procedure with a specific antialbumin

monoclonal antibody, and creatinine was assessed using the Jaffe method. Urine albumin-creatinine ratios were standardized to sex and race and expressed in milligrams per gram of creatinine. Albuminuria was defined as urine albumin-creatinine ratio . 30 mg/g. Serum highly sensitive C-reactive protein (CRP measured in mg/L) was measured at the year-15 examination with a BNII nephelometer (Dade Behring, Deerfield, IL).

Statistical Analysis For this analysis, serum biomarkers at year 15 were ranked into quartiles in participants with eGFRcys .60 mL/minute/1.73 m2 at baseline (study year 15) for whom complete data on all predictor and outcome variables were available (n 5 2,152). In earlier analyses of the YALTA biomarker data, findings were similar for 4 carotenoids (a-carotene, b-carotene, lutein/zeaxanthin, and b-cryptoxanthin) but epidemiologically quite different for lycopene.19,20,22,31 Thus, for this analysis, the 4 carotenoids were grouped into one variable and evaluated separately from lycopene. Multivariable logistic regression was used to estimate the odds ratio (OR) of incident rapid kidney function decline over 5 years by quartile of dietary biomarker. Model 1 included the covariates age, race, sex, CARDIA study center, education, smoking status, alcohol intake, physical activity, BMI, and lipids. We then added Y15 diabetes status, hypertension, CRP (as a marker of acute inflammation), and albuminuria individually to the model to determine if the associations between serum biomarkers and kidney function decline were mediated by these factors. The fully adjusted multivariate model included the model 1 covariates along with these 4 potential mediators added simultaneously. Previous work in CARDIA suggested that blood concentrations of fat-soluble antioxidants may be confounded by blood lipids.32 Thus, as recommended by Gross et al., both models were adjusted for serum lipids to standardize concentrations of the tocopherols and carotenoids. We also tested for effect modification by obesity (BMI $ 30 kg/m2), hypertension (systolic BP . 140 mm Hg, diastolic BP . 90 mm Hg, or currently taking antihypertensive medication), smoking status (never, former, current), and diabetes (yes/no). In addition, we carried out a priori analyses examining whether the association was dependent on baseline eGFRcys levels, race, or sex. Dietary supplement use was another covariate of interest, but data were missing for 692 (32%) of participants. We therefore performed a sensitivity analysis using a multipleimputation procedure for the missing supplement-use data. All analyses were performed using SAS 9.4 (SAS Institute Inc.), with a significance level of 0.05 for 2-sided tests.

Results At baseline, the distribution of age was consistent across quartiles of serum carotenoid concentrations. Compared

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in Supplemental Table 5 for reference. A total of 290 (13.5%) cases of rapid kidney function decline and 32 cases (1.5%) of incident CKD occurred between study years 15 and 20. The number of cases of incident CKD was inadequate for further analysis. There was a significant inverse association between higher sum of serum carotenoid concentration and rapid eGFRcys decline (P trend, .02). Individuals in the highest quartile had 47% lower odds than those in the lowest quartile in the fully adjusted model (OR, 0.51; 95% CI, 0.320.80) (Table 2). Adjustment of model 1 for incident diabetes, hypertension, CRP, and albuminuria individually did not appreciably change the association. In a post hoc sensitivity analysis with adjustment for baseline eGFRcys, the associations were slightly attenuated, but the magnitude

with the lower quartiles, the upper quartiles of carotenoid concentrations had more females, whites, former and never smokers, fewer prevalent cases of diabetes and albuminuria, were more educated and physically active, and had higher baseline eGFRcys. BMI, alcohol intake, systolic and diastolic BP, CRP, and triglycerides were lower in the groups with higher serum carotenoid concentrations (Table 1). Similar trends were also observed for most characteristics in the upper quartiles of serum a-tocopherol and ascorbic acid concentrations, but not g-tocopherol or lycopene (Supplemental Tables 1-4). Albuminuria was less frequent, and CRP was lower in the upper quartiles of ascorbic acid and lycopene. Macronutrient and food group intake details from the Y20 CARDIA Dietary History Questionnaire across quartiles of carotenoid concentrations are presented

Table 1. Baseline Characteristics of Participants According to Quartile of Baseline Sum of 4 Serum Carotenoid Concentrations, CARDIA Study Year 15, 2000-2001 Quartile of Sum of 4 Serum Carotenoid Concentrations Characteristic* N Sum serum carotenoids quartile ranges (mg/dL) Age (y) Sex (% female) Race (% white) Education (y) Smoking status (%) Never Former Current Alcohol (mL/d) BMI (kg/m2) Physical activity (EU/wk)† eGFRcys (mL/min/1.732)‡ Cholesterol (mg/dL) Total cholesterol LDL cholesterol HDL cholesterol Triglycerides (mg/dL) Hypertension§ (%) Diabetesjj (%) Albuminuria{ (%) C-reactive protein (mg/L) Serum biomarker concentration (mg/dL) Sum4Carot** A-Tocopherol G-Tocopherol Ascorbic acid Lycopene

Q1

Q2

Q3

Q4

538 5.24-40.18 39.8 (3.7) 44.4 48.9 14.2 (2.5)

538 40.19-56.86 40.0 (3.6) 48.3 56.5 14.7 (2.3)

538 56.88-81.36 40.3 (3.5) 48.3 59.1 15.4 (2.5)

538 81.41-466.4 40.6 (3.3) 55.4 70.1 16.1 (2.3)

51.0 15.0 34.0 14.9 (36.8) 30.7 (7.1) 324.4 (276.1) 105.0 (16.0)

57.4 18.8 23.8 11.9 (30.6) 29.5 (6.0) 336.2 (274.9) 108.2 (14.8)

64.1 21.2 14.7 10.5 (20.2) 27.3 (5.3) 370.4 (278.0) 111.4 (13.3)

73.2 20.9 6.0 9.6 (15.0) 25.4 (4.4) 430.5 (299.6) 112.6 (12.2)

177.2 (35.1) 107.1 (31.7) 46.3 (14.5) 120.7 (88.4) 21.4 7.06 5.02 4.16 (5.9)

183.9 (32.4) 114.3 (29.8) 48.2 (13.1) 111.1 (112.5) 16.7 3.72 3.35 3.36 (4.8)

188.0 (34.0) 116.4 (31.6) 51.7 (14.1) 97.0 (64.7) 11.5 3.35 2.42 2.50 (4.1)

193.7 (35.4) 119.8 (32.8) 54.9 (14.5) 94.1 (68.4) 7.3 0.93 1.86 1.72 (3.1)

30.5 (6.9) 1.09 (0.4) 0.28 (0.1) 6.77 (3.8) 34.5 (15.9)

48.2 (4.9) 1.19 (0.4) 0.26 (0.1) 8.34 (3.9) 39.9 (16.5)

67.7 (7.1) 1.25 (0.4) 0.24 (0.1) 8.98 (3.6) 42.5 (18.7)

118.1 (38.9) 1.48 (0.5) 0.22 (0.1) 10.3 (3.9) 44.1 (18.4)

BMI, body mass index; BP, blood pressure; CARDIA, Coronary Artery Risk Development in Young Adults; CKD, chronic kidney disease; eGFRcys, serum cystatin-C–based estimated glomerular filtration rate; SD, standard deviation. *Values are represented as unadjusted mean (SD) for all characteristics unless noted as percentage. †Exercise units, physical activity score derived from the CARDIA physical activity history. ‡GFRcys (mL/minute/1.732) estimated from the CKD-EPI (Chronic Kidney Disease Epidemiology Collaboration) equation for calibrated cystatin-C at year 15. §Hypertension is defined as systolic BP . 140 mm Hg, diastolic BP . 90 mm Hg, or currently taking antihypertensive medication. jj Diabetes is defined as fasting serum glucose . 126 mg/dL or receiving diabetes medication. {Albuminuria is defined as urinary albumin-creatinine ratio . 30 mg/g. **Sum of a-carotene, b-carotene, b-cryptoxanthin, and lutein 1 zeaxanthin.

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DIETARY BIOMARKERS AND KIDNEY FUNCTION Table 2. Odds Ratios for the Incidence of Rapid Decline in Kidney Function Over 5 Years According to Baseline Sum of 4 Serum Carotenoid Concentrations and Lycopene Concentrations Rapid eGFRcys Decline (.15% Over 5 y) Serum Biomarker (Quartile) Sum4Carot* Q1 Q2 Q3 Q4 P linear trend Lycopene Q1 Q2 Q3 Q4 P linear trend

% (n Cases/Person-Years)

Model 1 OR (95% CI)†

Model 2 OR (95% CI)‡

18.8 (101/538) 13.0 (70/538) 12.3 (66/538) 9.85 (53/538)

1.0 (ref) 0.66 (0.46-0.93) 0.67 (0.46-0.97) 0.55 (0.35-0.85) .01

1.0 (ref) 0.66 (0.46-0.95) 0.69 (0.47-1.01) 0.51 (0.32-0.80) .02

14.5 (78/537) 13.0 (70/539) 12.8 (69/538) 13.6 (73/538)

1.0 (ref) 0.86 (0.60-1.24) 0.89 (0.62-1.29) 0.89 (0.61-1.30) .16

1.0 (ref) 0.84 (0.58-1.22) 0.84 (0.58-1.22) 0.84 (0.58-1.24) .11

BMI, body mass index; CARDIA, Coronary Artery Risk Development in Young Adult; CRP, C-reactive protein; OR, odds ratio. *Sum of a-carotene, b-carotene, b-cryptoxanthin, and lutein 1 zeaxanthin. †Adjusted for potential confounding by age, race, sex, CARDIA center, education, smoking status, alcohol intake, physical activity, BMI, and lipids. ‡Additionally adjusted for potential mediators such as incident diabetes, hypertension, albuminuria, and CRP.

and direction of the estimates and significance did not materially change (data not shown). A suggestive inverse association was observed for higher ascorbic acid concentration and presence of rapid eGFR decline (P trend, .04), but the odds ratios from the quartile analysis did not reach statistical significance (Table 3). There was no association observed between serum lycopene, a- or g-tocopherol concentration, and presence of rapid eGFR decline. The

Hosmer-Lemeshow goodness of fit test indicated that the fully adjusted models fit the data well and showed no evidence of interactions in the fully adjusted model. We carried out a series of sensitivity analyses to better interpret the main results. A logistic regression analysis using year 7 sum of serum carotenoids and tocopherols with rapid kidney function decline over 10 years (study years, 10-20) was consistent with the main study results despite

Table 3. ORs for the Incidence of Rapid Decline in Kidney Function Over 5 Years According to Baseline Tocopherol and Ascorbic Acid Concentrations Rapid eGFRcys Decline (.15% Over 5 y) Serum Biomarker (Quartile) a-Tocopherol Q1 Q2 Q3 Q4 P linear trend g-Tocopherol Q1 Q2 Q3 Q4 P linear trend Ascorbic Acid Q1 Q2 Q3 Q4 P linear trend

% (n Cases/n)

Model 1 OR (95% CI)*

Model 2 OR (95% CI)†

14.0 (75/537) 13.2 (71/537) 13.3 (72/540) 13.4 (72/538)

1.0 (ref) 0.83 (0.58-1.21) 0.83 (0.56-1.23) 0.74 (0.47-1.16) .35

1.0 (ref) 0.82 (0.56-1.20) 0.83 (0.55-1.24) 0.76 (0.48-1.20) .41

12.4 (67/541) 11.7 (63/537) 14.5 (78/538) 15.3 (82/536)

1.0 (ref) 0.89 (0.61-1.30) 0.99 (0.68-1.45) 0.97 (0.64-1.45) .67

1.0 (ref) 0.90 (0.61-1.33) 1.00 (0.68-1.47) 0.96 (0.63-1.47) .56

16.6 (89/537) 14.2 (77/543) 12.7 (68/535) 10.4 (56/537)

1.0 (ref) 0.91 (0.64-1.29) 0.93 (0.65-1.34) 0.79 (0.54-1.15) .06

1.0 (ref) 0.91 (0.64-1.30) 0.94 (0.65-1.35) 0.74 (0.50-1.09) .04

BMI, body mass index; CARDIA, Coronary Artery Risk Development in Young Adult; CRP, C-reactive protein; OR, odds ratio. *Adjusted for potential confounding by age, race, sex, CARDIA center, education, smoking status, alcohol intake, physical activity, BMI, and lipids. †Additionally adjusted for potential mediators such as incident diabetes, blood pressure, albuminuria, and CRP.

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the misalignment of biomarker data (data not shown). This sensitivity finding is supported by the correlation of serum carotenoid concentrations at years 0, 7, and 15 (r 5 0.520.64; P ,.0001). Lycopene, a-tocopherol, and g-tocopherol concentrations were also found to be correlated over time (r 5 0.29-0.46, 0.42-0.54, and 0.33-0.46, respectively; P , .0001). In formal tests for effect modification, there was no evidence that the association materially differed by baseline eGFR, hypertensive status, diabetes status, smoking status, BMI (,30 vs. $30 kg/m2), sex, or race. The inclusion of supplement-use data from a multiple-imputation procedure did not change the observed associations. Furthermore, results were consistent when a cutoff of eGFRcys , 90 mL/minute/1.73 m2 at baseline was used for inclusion instead of 60 mL/minute/ 1.73 m2, and when subjects with at least 15% rapid kidney function decline between years 10 and 15 were excluded.

Discussion We observed a strong inverse association between higher concentration of serum carotenoids and rapid eGFR decline over 5 years in black and white adults with baseline eGFR . 60 mL/minute/1.73 m2, and this association was independent of comorbid conditions and metabolic risk status. An association of ascorbic acid, a-tocopherol, gtocopherol, or lycopene with rapid eGFR decline was not observed. The use of biomarkers as an objective measure of diet is a major strength of this study as it reduces biases related to the known limitations of self-reported dietary data.8 Although dietary intake data were not collected at year 15 for comparison in our study, previous findings have shown that blood carotenoid concentrations are highly sensitive to dietary intake because they are not regulated by homeostatic mechanisms.33 Therefore, they provide a reliable proxy for foods in which carotenoids are part of the food matrix, such that high concentrations may be interpreted as representing a dietary pattern with high fruit and vegetable intake.18,34,35 This may explain in part why no association was found for the other biomarkers. It is plausible that because of greater endogenous regulation of ascorbic acid, tocopherols, and lycopene, blood concentrations of these compounds do not correlate as strongly with absolute intake from the diet.36 For example, plasma concentrations of ascorbic acid plateau at the upper end of the normal range and thus are not a sensitive measure of fruit and vegetable intake for individuals using supplements containing vitamin C or consuming fortified foods.37,38 Higher serum ascorbic concentrations have also been associated with nonsmoking, a lower BMI, higher physical activity, and higher whole-grain consumption, suggesting it may serve more as a general marker of a health conscious diet and lifestyle,39,40 and with the exception of whole-grain consumption, these factors were adjusted for in our regression models. Furthermore, tocopherols are less specific bio-

markers, and serum concentrations do not correlate with absolute dietary intake as well as carotenoids.12 Our results for serum carotenoids are consistent with previous research showing a protective association between healthy dietary patterns, with high fruit and vegetable intake, and kidney function decline. Through a prior work by Chang et al.3 using a subpopulation of the CARDIA cohort, it was found that poor diet quality, based on a Dietary Approach to Stop Hypertension (DASH) diet score, was significantly associated with higher odds of albuminuria (OR, 2.0; 95% CI, 1.1-3.4). In a study by Nettleton et al.,4 a dietary pattern characterized by high consumption of whole grains, fruits, vegetables, and lowfat dairy was associated with a 20% lower albumin-tocreatinine ratio across quintiles (P for trend, .004). Results from a study by Lin et al.6 using the Nurses’ Health Study cohort showed that the highest quartile of Western diet score compared with the lowest quartile was positively associated with microalbuminuria (OR, 2.17; 95% CI, 1.183.66) and risk of rapid eGFR decline (OR, 1.77; 95% CI, 1.03-3.03), and women in the top quartile of DASH score had a reduced risk of rapid decline (OR, 0.55; 95% CI, 0.38-0.80). Finally, Rebholz et al.7 recently found that participants with baseline eGFR $ 60 mL/minute/1.73 m2 from the prospective Atherosclerosis Risk in Communities Study with a DASH diet score in the lowest tertile were 16% more likely to develop kidney disease than those with scores in the highest tertile (hazard ratio [HR], 1.16; 95% CI, 1.07-1.26; P for trend, .001). The mechanism(s) underlying how a diet rich in fruits and vegetables is associated with a reduced risk of eGFR decline is not clear. One possible explanation for our findings is that high fruit and vegetable intake is a marker of an overall healthy dietary pattern, as it has been consistently demonstrated that a dietary pattern high in fruit and vegetable intake tends to cluster with higher intakes of other foods with demonstrated healthful benefits such as whole grains, legumes, and nuts and seeds and with lower intakes of other less healthful or potentially harmful foods. Such healthful dietary patterns are known to reduce the risk of a number of chronic diseases, many of which themselves are risk factors for CKD.41 In addition, oxidative stress is known to contribute to the pathogenesis of CKD,42,43 and thus, high fruit and vegetable intake, through bioactive compounds such as carotenoids, may help reduce damage to the kidneys from reactive oxygen species and inflammation. In vitro and in vivo studies have suggested that carotenoids, the most abundant lipidsoluble phytochemicals in fruits and vegetables, have antioxidant, antiapoptotic, and anti-inflammatory properties, many of which are linked to the modulation of inflammatory and oxidative stress signaling pathways.44 Consistent with previous work in CARDIA,22 individuals in the upper quartiles of serum carotenoid concentration had lower CRP levels, an objective marker of inflammation, than

DIETARY BIOMARKERS AND KIDNEY FUNCTION

those in the lower quartiles. It is plausible that the relationship between serum carotenoids and kidney function decline is potentially mediated by other conditions such as hypertension, as a prospective study in CARDIA by Hozawa, et al. demonstrated that the sum of 4 carotenoids (excluding lycopene) was inversely associated with 20year hypertension incidence after adjustment for baseline systolic BP and other confounding factors (relative hazard per standard deviation increase of sum of 4 carotenoids: 0.91; 95% confidence interval, 0.84-0.99).15 However, our results were robust to adjustment for both diabetes and hypertension, suggesting that multiple mechanisms may explain the relationship between serum carotenoid levels and kidney function over time. Strengths of the study include the objectively measured dietary biomarkers, a well-characterized population with rigorous clinical measures, and measurement of rapid kidney function decline using eGFRcys, a highly sensitive and specific marker of early impairments of kidney function. It has been suggested that GFR decline may even precede other commonly used markers of early kidney function decline.45 The interpretation of these results also requires acknowledging several limitations and other study considerations. We note that participants in the highest quartiles of carotenoid concentration differed on many clinical and lifestyle factors from those in the lowest quartiles; however, results remained significant even after adjustment for these covariates in the regression model. Nonetheless, our findings may still be influenced by residual confounding related to clinical, lifestyle, or dietary factors. Although the observed results did not differ by smoking status or BMI in this population, higher BMI and being a current smoker associate with lower levels of serum carotenoids in this population. Several epidemiological studies have demonstrated an inverse relationship between cigarette smoking and serum concentrations of antioxidant compounds, including carotenoids, tocopherols, and ascorbic acid,46-49 and BMI has been shown to negatively associate with serum carotenoids.19,46,50 For both smoking and BMI, it is not clear whether lower concentrations are due to the impact of smoking and greater adiposity on metabolism of carotenoids or if people who smoke or have a higher BMI simply eat fewer fruits and vegetables. In addition, unmeasured dietary components may have added residual confounding to the analyses. Although there are multiple components and biomarkers of dietary intake for which we did have measures of, objective protein intake is an area that may have been most informative as a confounder. High protein intake is associated with progression of kidney function decline in individuals with preexisting kidney disease;51 however, it remains unclear whether it is also related to kidney function decline in individuals with normal renal function.52 Experimental evidence from animal studies suggests that long-term high dietary protein

7

intake may cause glomerular hyperfiltration and proinflammatory gene expression, known risk factors for kidney disease, but the overall effect on kidney health in individuals without kidney disease is inconclusive.52 Because the cutoff for inclusion in our study was baseline eGFR . 60 mL/minute/1.73 m2, it is plausible that some individuals in the lower ranges may have been susceptible to the effect of dietary protein intake on kidney function decline during the follow-up period. Individuals in the lower quartiles of serum carotenoid concentration did have lower mean eGFR levels at baseline; however, the quartile means were all above 100 mL/minute/1.73 m2. Our results did not change when a sensitivity analysis was performed using a baseline eGFR cutoff of 90 mL/minute/1.73 m2. Still, it is possible that dietary protein intake, in addition to overall high diet quality, as measured by serum carotenoid concentrations, influenced our findings to an unknown degree. There are inherent limitations to accurately quantifying nutrient intake from food frequency questionnaire (FFQ) data,53 and calculated dietary protein intake was not available in this dataset; thus, we were unable to account for dietary protein intake in our analytical models. Furthermore, because there were few cases of CKD during the follow-up interval, we were unable to measure the association of the dietary biomarkers with actual CKD incidence. The follow-up time period was short and may not have been long enough to fully determine whether the rapid decline in eGFR translated to persistent CKD in these young adults, although the results using misaligned biomarker data (year 7) with rapid eGFR decline from 10 to 20 were consistent with our main results. Serum carotenoid concentrations were correlated throughout the follow-up period. Although levels are directly affected by dietary intake and high day-to-day as well as individual variations are common, the observed correlation suggests that they are a good marker of long-term consumption as diet quality is generally a stable characteristic over time.54 We note the potential for inflated type I errors, as correction for multiple comparisons was not done based on the strength of our a priori hypotheses. Finally, the incomplete data on dietary supplement use within the study sample limit the extent of the inference that may be garnered for statistical modeling with this covariate, although efforts were made to address this using a multiple-imputation procedure. In conclusion, our results suggest that higher serum carotenoid levels are inversely associated with rapid loss of renal function in early adulthood and provide insight for dietary recommendations and approaches related to CKD prevention. This is an understudied topic, so there are multiple feasible avenues where future research can contribute to knowledge on this topic. Studies that include additional dietary biomarkers, or that align with more specific dietary assessment methods, may further elucidate how diet relates to kidney function. Our findings of an independent association between dietary biomarkers and kidney decline

8

HIRAHATAKE ET AL

suggest that diet quality and composition are important for kidney function preservation not only in at-risk populations or individuals with CKD but also for young and middle-aged adults with normal kidney function.

Practical Application A dietary pattern rich in fruits and vegetables may be associated with a reduced risk of kidney function decline in young adults. Nutrition education to increase fruit and vegetable intake and establish healthy eating patterns represents a practical intervention for kidney disease prevention in both high-risk individuals and the general population.

Acknowledgments Authors’ contributions: K.M.H. and A.O.O. designed research, analyzed data, and wrote the article. D.R.J. and M.D.G. were involved with primary data collection. K.B.B.-D. was involved with cystatin-C data collection. D.R.J., M.G.S., H.M.-K., and M.D.G. provided a critical review of the statistical analyses and manuscript. A.O.O. had the primary responsibility for the final content. All authors read and approved the final manuscript.

Supplementary data

Supplementary data associated with this article can be found in the online version at https://doi.org/10.1053/j. jrn.2018.05.008.

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