Effect of Resveratrol on Endothelial Function in Patients with CKD and Diabetes

Visual Abstract

Abstract

Background

Patients with CKD and diabetes are at higher risk of developing cardiovascular disease, in part, because of impaired endothelial function. Cardioprotective compounds such as resveratrol could improve endothelial function and attenuate the cardiovascular burden in patients with CKD and diabetes. We hypothesized that resveratrol supplementation would improve endothelial function in patients with CKD and diabetes.

Methods

Twenty-eight adults aged 68±7 years (84% men) with stage 3 CKD and diabetes were enrolled in a randomized, double-blind, placebo-controlled, crossover study to investigate the effects of 6-week resveratrol supplementation (400 mg/d) on endothelial function. Endothelial function was determined through brachial artery flow-mediated dilation.

Results

The mean values for eGFR and hemoglobin A_1c were 40±9 ml/min per 1.73 m² and 7.36%±0.72%, respectively. Compared with placebo, resveratrol supplementation increased flow-mediated dilation (ratio of geometric mean changes and 95% confidence interval for between-group comparisons, 1.43 (1.15 to 1.77); P value = 0.001). eGFR, hemoglobin A_1c, BP, and nitroglycerin-mediated dilation were unchanged with resveratrol or placebo (P = 0.15), suggesting the observed change in flow-mediated dilation was likely independent of changes in traditional cardiovascular risk factors.

Conclusions

Resveratrol supplementation improved endothelial function in patients with CKD and diabetes.

Clinical Trial registry name and registration number

Resveratrol and Vascular Function in CKD, NCT03597568.

Podcast

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Introduction

CKD is a major public health concern affecting approximately 10% of the global population.¹ In addition, CKD is an independent risk factor of cardiovascular disease,^2,3 and patients with CKD are far more likely to die from cardiovascular disease than to progress to kidney failure.⁴ Diabetes is the most common cause of CKD, comprising nearly half of all cases,⁵ and contributes a significant portion of the cardiovascular risk associated with CKD.⁶ Indeed, patients with CKD and diabetes have a greater risk of cardiovascular disease compared with those with CKD alone.⁷ Therefore, interventions aimed at lowering cardiovascular risk in patients with CKD and diabetes are needed.

Endothelium-dependent dilation determined by brachial artery flow-mediated dilation is a well-established end point of endothelial dysfunction, linked with numerous cardiovascular risk factors.⁸ In addition, lower flow-mediated dilation is strongly associated with impaired coronary artery vasodilation,⁹ suggesting flow-mediated dilation provides clinically meaningful insight into coronary artery endothelial dysfunction and atherosclerotic cardiovasular disease. Along these lines, reduced flow-mediated dilation is associated with cardiovascular disease in patients with CKD,¹⁰ and patients with CKD and diabetes have diminished endothelial function compared with healthy, age-matched controls.^11–13 Importantly, a 1% increase in flow-mediated dilation is associated with a 13% lower relative risk of cardiovascular events in adults at risk of cardiovascular disease.¹⁴ Thus, it is plausible that interventions to improve flow-mediated dilation could attenuate cardiovascular disease risk in patients with CKD and diabetes.

Polyphenols are naturally occurring compounds found in various fruits and plants that attenuate atherosclerotic vascular disease,¹⁵ likely because of their ability to limit oxidative stress and inflammation in endothelial cells.¹⁶ Resveratrol is a widely studied polyphenol that increases endothelial function in various populations¹⁷; however, whether resveratrol improves endothelial function in patients with CKD and diabetes is unknown. The purpose of this pilot study was to determine the effects of 6-week resveratrol supplementation on flow-mediated dilation in patients with CKD and diabetes. We hypothesized that 6-week resveratrol supplementation would result in clinically meaningful improvements in flow-mediated dilation in patients with CKD and diabetes.

Methods

Patient Population

Twenty-eight participants were recruited from the Nephrology Clinics at the University of Iowa Hospital and Clinics and the Iowa City Veterans Affairs Health Care System to complete a study investigating the effects of 6-week resveratrol supplementation on endothelial function. Individuals were considered eligible for participation if they were between the ages 45 and 80 years, able to provide informed consent, and had stage 3 CKD on the basis of an eGFR of 30–60 ml/min per 1.73 m², diabetes defined as current treatment with oral hypoglycemic agents or insulin, or fasting glucose ≥126 mg/dl, and controlled hypertension defined as having received an angiotensin-converting enzyme inhibitor or angiotensin II receptor blocker for >3 months before the study. Exclusion criteria included consuming more than two glasses of red wine per day, current use or use of resveratrol or a vitamin C supplement within the past 12 months, life expectancy of less than 1 year, body mass index (BMI) >40 kg/m², pregnant, breastfeeding, unwilling to use adequate birth control, uncontrolled hypertension, uncontrolled diabetes mellitus with hemoglobin A_1c (HbA_1c) >8.5%, currently taking anticoagulants (coumadin, dalteparin, enoxaparin, heparin, and clopidogrel), severe liver disease, severe systolic heart failure, hospitalization within the past 3 months, acute infection or antibiotic therapy, immunosuppressive therapy within the past year, or current participation in another research study. All procedures were approved by the University of Iowa and Iowa City Veterans Association Institutional Review Boards and conducted in accordance with the Declaration of Helsinki. All participants provided written informed consent before study participation. The study was registered with ClinicalTrials.gov; NCT03597568 first posted on July 24, 2018. We had planned to obtain a 6-minute walk test, but once the coronavirus disease 2019 pandemic ensued, hospital procedures and mask mandates led us to remove this outcome.

Study Procedures

This study followed a randomized, double-blind, placebo-controlled, crossover design. The randomization sequence was generated through randomization.com by the Investigative Drug Service. Participants were randomized in blocks of two to either placebo first then resveratrol or resveratrol first then placebo. On randomization, and after baseline measurements, patients were provided with a nonlabeled container consisting of 100-mg tablets of high-purity (99%) trans-resveratrol (ResVida, Endurance Products Company) or placebo. The patients were instructed to take two 100-mg tablets of resveratrol or placebo twice per day for 6 weeks. After 6 weeks of supplementation, the patients completed a 2-week washout and were then crossed over to the opposing arm of the intervention for the remaining 6 weeks. To minimize residual carryover effects introduced by the brief washout period, follow-up visits in each intervention arm were compared with the original baseline visit in preference to baseline measurements taken at the beginning of each period (Figure 1). All procedures were performed in the Institute for Clinical and Translational Science at the University of Iowa Hospitals and Clinics.

Figure 1.

Study flow chart. COVID-19, coronavirus disease 2019.

Adherence was measured at the end of the trial by pill count. The number of pills ingested was calculated as follows: number of pills provided−number of pills returned. Percentage adherence was then calculated as follows: number of pills ingested/number of pills provided ×100.

Clinical Characteristics

Race, ethnicity, smoking status, and preexisting cardiovascular disease were evaluated by using questionnaires. Smoking status was defined as a history of smoking (current or former) or no history of smoking. Preexisting cardiovascular disease was defined as a history of myocardial infarction, stroke, arrhythmia, or heart failure. BP was obtained in the seated position in triplicate after 10 minutes of quiet rest using an automated oscillometric cuff (GE Dinamap). BMI was computed after measurement of weight and height in kg/m². Clinical laboratory test results were measured at the University of Iowa Diagnostics laboratories and included fasting lipid panel, HbA_1c, creatinine, cystatin C, and urinary albumin-creatinine ratio (UACR). eGFR was calculated from cystatin C using the Chronic Kidney Disease Epidemiology Collaboration formula as previously described.¹⁸

Primary Outcome

The primary outcome was the 6-week change in brachial artery flow-mediated dilation. Flow-mediated dilation was measured using high-resolution ultrasonography (GE Logiq E9) as previously described by our laboratory.¹⁹ After baseline diameter measurements, a cuff placed on the upper forearm was inflated to suprasystolic pressure (250 mm Hg) for 5 minutes. After 5 minutes of occlusion, the cuff was rapidly deflated, and measurements continued for an additional 2 minutes. Baseline and deflation images were analyzed using offline software (Vascular Analysis Tools 5.5, Medical Imaging Applications, LLC, Coralville, IA) with automatic edge detection to determine peak change in the brachial artery diameter in response to reactive hyperemia. Flow-mediated dilation was calculated using the following equation:

$$\%\hspace{0.17em}dilation=\frac{∆diameter}{baseline\hspace{0.17em}diameter}\times 100.$$

Secondary and Other Outcomes

Peak nitroglycerin-mediated dilation, an index of vascular smooth muscle function, was determined by imaging brachial artery vasodilation for 10 minutes after administration of 0.4-mg sublingual nitroglycerin (GE Logiq E9). Peak nitroglycerin dilation was calculated using the same equation used to assess flow-mediated dilation.

In a subset of patients (n=7), liquid chromatography–mass spectrometry (Waters Xevo TQ-S) was used to quantify the following resveratrol metabolites from plasma samples: trans-resveratrol 3-sulfate (sc-213070), trans-resveratrol 3-O-β-D-glucuronide (sc-213069), and trans-resveratrol 4′-O-β-D-glucuronide (sc-213071) purchased from Santa Cruz Biotechnology (Santa Cruz, CA).²⁰ Samples were extracted with 1-ml acetonitrile and 10 μl of internal standard consisting of resveratrol-¹³C₆ and resveratrol-3-O-β-D-glucuronide-¹³C₆ both at 5 μg/ml. Each sample was vortexed for 2 minutes and then put in −20°C freezer for 40 minutes. Samples were then centrifuged for 10 minutes at 4°C. Afterward, the samples were moved to a new tube and combined with 4 μl of 4-mg/ml ascorbic acid and vortexed. Samples were then reconstituted with 100 μl of 1-mM ammonium acetate, vortexed, and centrifuged. The flow rate was 0.2 ml/min, and injection volume was 15 μl. Multiple reaction monitoring was used to detect resveratrol metabolites in prepared samples.

Statistical Analysis

Normally distributed data are presented as mean±SD, non-normally distributed data as median (interquartile range), and categorical data as count (percentage of patients). To determine the effects of resveratrol supplementation on our primary and secondary outcomes, a generalized linear mixed-effects model was used to examine the significance of condition, time, and their interaction for normally distributed variables, and a gamma mixed-effects model was used for non-normally distributed variables. In each model, a random intercept was built for each patient to account for inherent between-patient variability. A complete case analysis was used when analyzing primary and secondary outcomes. A Wilcoxon rank-sum test was used to test differences in resveratrol metabolites between the resveratrol and placebo arms. Significance was set at an α level of 0.05. All analyses were performed in R, version 4.2.3 (R Foundation for Statistical Computing, Vienna, Austria).

Results

Clinical Characteristics

A total of 28 patients with stage 3 CKD and diabetes were screened and consented; of those, three patients withdrew before randomization (Figure 1). Of the 25 patients randomized, two (8%) withdrew from this study (Figure 1). Baseline clinical characteristics are presented in Table 1. Patients' mean age was 68±7 years, and 84% were men. On average, patients had moderate to severely decreased eGFR and normal to mildly increased albuminuria per Kidney Disease Improving Global Outcomes prognosis. Most of the patients were considered overweight or obese according to BMI and had suboptimal glycemic control with a mean HbA_1c of 7.36%±0.72%. Most of the patients were nonsmoking (92%) and had no history of cardiovascular disease (80%), whereas the remaining patients had a history of myocardial infarction (12%), stroke (4%), and arrhythmia (8%).

Table 1.

Clinical characteristics (n=25)

Variable	Value
Age, yr	68±7
Sex, no. (%)
Female	4 (16)
Male	21 (84)
Race, no. (%)
White	25 (100)
Ethnicity, no. (%)
Non-Hispanic or Latino	25 (100)
History of smoking, no. (%)
No	23 (92)
Yes	2 (8)
History of cardiovascular disease, no. (%)
Arrhythmia	2 (8)
Myocardial infarction	3 (12)
Stroke	1 (4)
Diabetes mellitus, no. (%)
Type 1	0 (0)
Type 2	25 (100)
Antihypertensive medication, no. (%)
ACE inhibitor/ARB	25 (100)
Calcium channel blockers	7 (28)
β blockers	14 (56)
Diuretics	14 (56)
Diabetic medication, no. (%)
SGLT2 inhibitor	5 (20)
BMI, kg/m2	32.7±4.0
Systolic BP, mm Hg	136±15
Diastolic BP, mm Hg	74±5
HbA1c, %	7.4±0.7
Total cholesterol, mg/dl	131±25
Triglycerides, mg/dl	155 (117–209)
LDL, mg/dl	62±22
HDL, mg/dl	35 (30–38)
Serum creatinine, mg/dl	1.7±0.4
Cystatin C, mg/dl	1.6 (1.4–1.8)
eGFR, ml/min per 1.73 m2	40±9
Albumin-creatinine ratio, mg/g	28 (6–240)
Brachial artery flow-mediated dilation, %	2.8 (2.1–3.7)
Nitroglycerin dilation, %	14.0±5.4

Variables are presented as mean±SD, median (interquartile range), or number (%). ACE, angiotensin-converting enzyme; ARB, angiotensin receptor blockers; BMI, body mass index; HbA_1c, hemoglobin A_1c; SGLT2, sodium-glucose cotransporter-2.

Primary Outcome

Of the 23 participants who completed this study, two had images of poor quality for flow-mediated dilation. Thus, 21 participants had complete data for the primary outcome. As presented in Figure 2 and Table 2, resveratrol supplementation at 400 mg per day for 6 weeks versus placebo increased flow-mediated dilation significantly, with the mean ratio for in between-group comparison=1.43 (95% confidence interval, 1.15 to 1.77; P = 0.001). We then evaluated the interaction term for the order of the treatment to examine whether the treatment that a participant received first had a substantial relation with the effect that resveratrol had on their flow-mediated dilation. Of note, the interaction term was not significant (mean ratio, 0.87; 95% confidence interval, 0.54 to 1.40; P value = 0.57).

Figure 2.

Six weeks of resveratrol supplementation improved brachial artery flow-mediated dilation in patients with CKD and diabetes. (A) Placebo. (B) Resveratrol. Data were analyzed using a gamma mixed model and are presented as median and interquartile range. *P = 0.01.

Table 2.

Primary and secondary outcomes

Variable	Placebo versus Baseline	Resveratrol versus Baseline	Between-Group Comparisons
	Mean Differencea/Ratiob (95% CI)	Mean Differencea/Ratiob (95% CI)	Mean Difference in Changea/Ratio of Changeb (95% CI)
Brachial artery flow-mediated dilation, %	0.9 (0.7 to 1.1)b	1.3 (1.1 to 1.6)b,c	1.4 (1.2 to 1.8)b,d
Nitroglycerin dilation, %	−1.6 (−4.4 to 1.2)a	0.5 (−2.4 to 3.3)a	2.1 (−0.8 to 4.9)a
Systolic BP, mm Hg	0.1 (−4.7 to 4.9)a	0.08 (−4.7 to 4.8)a	−0.02 (−4.8 to 4.8)a
Diastolic BP, mm Hg	2.4 (−0.6 to 5.4)a	1.2 (−1.8 to 4.1)a	−1.2 (−4.3 to 1.8)a
HbA1c, %	0.01 (−0.2 to 0.2)a	−0.08 (−0.3 to 0.1)a	−0.10 (−0.3 to 0.1)a
eGFR, ml/min per 1.73 m2	0.06 (−2.4 to 2.5)a	−1.7 (−4.1 to 0.7)a	−1.8 (−4.2 to 0.7)a
UACR, mg/g	0.9 (0.7 to 1.1)b	0.91 (0.71 to 1.2)b	1.0 (0.8 to 1.3)b

CI, confidence interval; HbA_1c, hemoglobin A_1c; UACR, urinary albumin-creatinine ratio.

^aFor variables that are normally distributed, we show the mean difference.

^bFor variables that are right skewed, we show the mean ratio.

^cP = 0.01.

^dP = 0.001.

Secondary Outcomes

Vascular smooth muscle function determined from nitroglycerin-mediated dilation was not significantly changed with either resveratrol or placebo (P = 0.15, Table 2). In addition, eGFR, HbA_1c, and systolic and diastolic BP were not significantly changed after resveratrol or placebo (Table 2), suggesting the improvement in flow-mediated dilation was likely independent of changes in traditional cardiovascular risk factors. Finally, the metabolite analyses indicated that byproducts of resveratrol metabolism reach the circulation with resveratrol supplementation, but not placebo (Table 3).

Table 3.

Resveratrol metabolite analysis

Metabolite	Resveratrol, ng/ml	Placebo, ng/ml	P Value
Trans-resveratrol 3-sulfate	631.4±711.8	0±0	0.001
Trans-resveratrol 3-O-β-D-glucuronide	228.7±396.9	−2.3±3.8	0.001
Trans-resveratrol 4′-O-β-D-glucuronide	186.0±256.9	−0.4±2.5	<0.001

Data are presented as mean±SD. A Wilcoxon rank-sum test was used in a subset of patients (n=7) to compare circulating resveratrol metabolites in the resveratrol and placebo arms.

Adverse Events and Adherence

No evidence of differences was found in any category of adverse events between resveratrol and placebo (P = 0.50). Overall, 21 patients (88%) reported no side effects from resveratrol, and 19 patients (83%) reported no side effects from placebo. In the resveratrol arm, there were two reports of dizziness (8%) and one report of diarrhea (4%), but no reports of nausea, vomiting, or abdominal pain. Median study supplement adherence was 96% (89–100) with resveratrol and 98% (93–100) with placebo (P = 0.48).

Discussion

In this randomized, double-blind, placebo-controlled, crossover study involving patients with stage 3 CKD and diabetes, 6 weeks of resveratrol supplementation improved endothelial function, as assessed through flow-mediated dilation, and this was independent of changes in eGFR, UACR, HbA_1c, BP, and vascular smooth muscle function. These data identify resveratrol as a promising nutraceutical compound that may lower subclinical cardiovascular risk in patients with CKD and diabetes and suggests that the resveratrol-mediated benefits on endothelial function are because of mechanisms other than improved BP or glycemic control.

To the best of our knowledge, no studies have tested the effectiveness of resveratrol supplementation on flow-mediated dilation in patients with CKD and diabetes; however, our findings agree with previous studies demonstrating the ability of resveratrol to improve endothelial function in different populations.^21–24 Wong et al. found that 6-week supplementation with 75 mg/d of resveratrol (resVida; DSM Nutritional Products Ltd.) improved flow-mediated dilation in 28 obese middle-aged and older adults.²¹ In addition, a study performed in 34 older adults with metabolic syndrome revealed that 100 mg/d of resveratrol (Longevinex; Resveratrol Partners LLC) for 3 months increased flow-mediated dilation independent of changes in waist circumference, BP, HbA_1c, and lipid profile.²² In 40 older patients with stable coronary artery disease and prior myocardial infarction, 10 mg/d of resveratrol (Admarc Med Diagnostics and Nutraceuticals) for 3 months enhanced flow-mediated dilation; however, this was not significant when compared with placebo, possibly because of the low dosage used in this study.²³ In older adults, 6-week supplementation with 1000–1500 mg/d of resveratrol (RevGenetics Corporation) improved endothelial function and skeletal muscle mitochondrial function.²⁴ Collectively, our study extends previous findings in clinical and nonclincal populations and identifies patients with stage 3 CKD and diabetes as a novel population in which resveratrol supplementation improves endothelial function.

Brachial artery flow-mediated dilation is a measure of endothelium-dependent dilation in response to an acute increase in shear stress stimulus after prolonged occlusion²⁵ and is largely dependent on the production of nitric oxide by endothelial nitric oxide synthase (eNOS).²⁶ However, eNOS activity and/or nitric oxide bioavailability are impaired when the endothelium is exposed to elevated levels of oxidative stress and inflammation, subsequently leading to impaired endothelium-dependent dilation.²⁷ Resveratrol is an activator of sirtuin 1 (SIRT1), a NAD-dependent histone deacetylase that regulates a number of downstream signaling pathways, including those involved in oxidative stress and inflammation.²⁸ Notably, SIRT1 lowers oxidative stress and increases eNOS expression in human umbilical vein endothelial cells (HUVECs) and enhances endothelium-dependent dilation in mice.²⁹ Therefore, it is plausible that resveratrol increases endothelium-dependent dilation through SIRT1-dependent mechanisms in CKD.³⁰ In this regard, HUVECs treated with resveratrol had higher eNOS expression and nitric oxide production compared with control cells.³¹ Moreover, ex vivo vascular reactivity experiments performed on superior thyroid arteries obtained from patients with hypertension and dyslipidemia demonstrated that resveratrol increased nuclear erythroid 2-related factor 2 (Nrf2)–dependent expression of mitochondrial antioxidant proteins, reduced endothelial cell oxidative stress, and augmented eNOS activity.³² Resveratrol also lowers inflammation, which could further explain the increase in flow-mediated dilation seen in our study. A recent meta-analysis of 17 randomized clinical trials revealed that resveratrol supplementation lowered the levels of inflammatory markers TNF-α and C-reactive protein.³³ Similarly, Pan et al. treated cultured HUVECs with TNF-α to induce cellular injury through elevated oxidative stress and inflammation.³⁴ After TNF-α–induced injury, HUVECs treated with resveratrol resulted in increased SIRT1 expression, reduced expression of the inflammatory transcription factor nuclear factor kappa B (NFκB), and attenuated reactive oxygen species.³⁴ Given these findings, it is plausible that resveratrol activated SIRT1 in the endothelial cells of our participants, thereby repressing oxidative stress and inflammation and increasing eNOS expression and flow-mediated dilation. In CKD, we found one study that evaluated biomarkers of oxidative stress and inflammation. In this study, 500 mg/d of resveratrol (Officilab, Rio de Janeiro, Brazil) was administered to 20 nondialyzed patients with CKD for 4 weeks. No change was observed in mRNA levels of Nrf2 and NFκB measured in isolated PBMCs.³⁵ Of note, mRNA expression measured in isolated PBMCs may not reflect nuclear translocation and gene transcription by Nrf2 and NFκB in endothelial cells, thus limiting the findings of the previous study.³⁵ Considering our limited ability to identify the specific cellular mechanisms by which resveratrol increases endothelial function in patients with CKD and diabetes, further research is needed.

Albuminuria is independently associated with cardiovascular and kidney progression events in patients with diabetes³⁶; therefore, interventions that reduce or slow the progression of UACR could prevent cardiovascular disease and further decline in kidney function in patients with CKD and diabetes. In our study, resveratrol did not alter UACR significantly. Our results differ from those of Sattarinezhad et al., who found 500 mg/d of resveratrol (Merck KGaA, Darmstadt, Germany) for 90 days, in combination with 12.5 mg/d of losartan, significantly reduced UACR in patients with diabetic nephropathy.³⁷ However, it should be noted that most of our participants had little or no albuminuria (UACR of <30 mg/g), whereas those in the previous study had a UACR of >120 mg/g.³⁷ Therefore, it is likely that differences in our patient populations could explain the divergent results regarding the effect of resveratrol on UACR.

There were several limitations to our study. First, this is a small study evaluating one measure of endothelial dysfunction over a relatively short duration of time. Our findings need to be confirmed in larger studies and to evaluate other indicators of endothelial dysfunction. Second, our study consisted of only White individuals who were predominantly male, and as a result, we cannot generalize our findings regarding the effect of resveratrol on flow-mediated dilation to individuals of a different race, ethnicity, or sex. Given the existing disparities in CKD and diabetes,^38–40 it is important that future studies evaluate whether resveratrol improves endothelial function in more diverse populations. Third, our participants had relatively low levels of albuminuria, and additional studies are needed to extend our findings to patients with clinically significant albuminuria. However, although recent trials have focused on patients with CKD and significant albuminuria,⁴¹ a large portion of individuals with CKD have minimal or no albuminuria.⁴² Seeing that reduced GFR is an independent risk factor of cardiovascular disease,² it is critical to evaluate potential therapies in patients with moderate to severe CKD and minimal or no albuminuria.

In conclusion, our pilot study suggests that resveratrol improves endothelial function in patients with CKD and diabetes and informs the design of larger clinical trials to confirm our findings. In addition, further investigation is required to identify the mechanisms by which resveratrol improves endothelial function in patients with CKD and diabetes.

Disclosures

C.J. Gimblet reports research funding from the American Heart Association. D.I. Jalal reports research funding from AstraZeneca and Corvidia; honoraria from K-INBRE, Reata, PER (Physicians Education Resource), and Sullivan conference-University of Kansas; and an advisory or leadership role for Reata. G.L. Pierce reports research funding from Microvascular Health Solutions (free supplement for clinical study) and an advisory or leadership role for North American Artery, treasurer (unpaid). All remaining authors have nothing to disclose.

Funding

D.I. Jalal: National Heart, Lung, and Blood Institute (R01HL134738). M. Sun and N.T. Kruse: National Institute of Diabetes and Digestive and Kidney Diseases (DK007690). C.J. Gimblet: American Heart Association (23PRE1012593). P.T. Eyck and L.H. Wendt: National Institutes of Health (UM1TR004403). G.L. Pierce: Russell B. Day and Florence D. Day Endowed Chair in Liberal Arts and Sciences Fund.

Author Contributions

Conceptualization: Diana I. Jalal.

Data curation: Katharine Geasland, Jeni Michelson, and Mingyao Sun.

Formal analysis: Patrick Ten Eyck and Linder H. Wendt.

Funding acquisition: Diana I. Jalal.

Investigation: Colin J. Gimblet, Diana I. Jalal, Nicholas T. Kruse, and Mingyao Sun.

Methodology: Nicholas T. Kruse, Safur Rehman Mandukhail, Jeni Michelson, Gary L. Pierce, and Mingyao Sun.

Project administration: Katharine Geasland, Colin J. Gimblet, Diana I. Jalal, and Nicholas T. Kruse.

Supervision: Diana I. Jalal.

Writing – original draft: Colin J. Gimblet.

Writing – review & editing: Patrick Ten Eyck, Katharine Geasland, Diana I. Jalal, Nicholas T. Kruse, Gary L. Pierce, Mingyao Sun, and Linder H. Wendt.

Data Sharing Statement

Original data created for the study are available in Figshare: https://figshare.com/articles/dataset/Table_of_raw_de-identified_data/23959293.