Plasma metabolite profiles, cellular cholesterol efflux, and non-traditional cardiovascular risk in patients with CKD

https://doi.org/10.1016/j.yjmcc.2017.05.001Get rights and content

Highlights

  • Cholesterol efflux in humans varies in terms of cellular components.

  • We report a cellular defect in ABCA1-mediated cholesterol efflux in CKD patients.

  • Plasma metabolites predicted cardiovascular events in CKD patients.

Abstract

Background

Patients with chronic kidney disease (CKD) experience high rates of atherosclerotic cardiovascular disease and death that are not fully explained by traditional risk factors. In animal studies, defective cellular cholesterol efflux pathways which are mediated by the ATP binding cassette transporters ABCA1 and ABCG1 are associated with accelerated atherosclerosis. We hypothesized that cholesterol efflux in humans would vary in terms of cellular components, with potential implications for cardiovascular disease.

Methods

We recruited 120 CKD patients (eGFR < 30 mL/min/1.73 m2) and 120 control subjects (eGFR ≥ 60 mL/min/1.73 m2) in order to measure cholesterol efflux using either patients' HDL and THP-1 macrophages or patients' monocytes and a flow cytometry based cholesterol efflux assay. We also measured cell-surface levels of the common β subunit of the IL-3/GM-CSF receptor (IL-3Rβ) which has been linked to defective cholesterol homeostasis and may promote monocytosis. In addition, we measured plasma inflammatory cytokines and plasma metabolite profiles.

Results

There was a strong positive correlation between cell-surface IL-3Rβ levels and monocyte counts in CKD (P < 0.001). ABCA1 mRNA was reduced in CKD vs. control monocytes (P < 0.05), across various etiologies of CKD. Cholesterol efflux to apolipoprotein A1 was impaired in monocytes from CKD patients with diabetic nephropathy (P < 0.05), but we found no evidence for a circulating HDL-mediated defect in cholesterol efflux in CKD. Profiling of plasma metabolites showed that medium-chain acylcarnitines were both independently associated with lower levels of cholesterol transporter mRNA in CKD monocytes at baseline (P < 0.05), and with cardiovascular events in CKD patients after median 2.6 years of follow-up.

Conclusions

Cholesterol efflux in humans varies in terms of cellular components. We report a cellular defect in ABCA1-mediated cholesterol efflux in monocytes from CKD patients with diabetic nephropathy. Unlike several traditional risk factors for atherosclerotic cardiovascular disease, plasma metabolites inversely associated with endogenous cholesterol transporters predicted cardiovascular events in CKD patients. (Funded by the National Institute of Diabetes and Digestive and Kidney Diseases K23DK097288 and others.)

Introduction

Plasma high density lipoprotein cholesterol (HDL-C) levels have an independent inverse relationship with atherosclerotic cardiovascular disease; [1] however, several recent therapeutic interventions aimed at raising HDL-C levels have failed in clinical trials [2], [3], highlighting an incomplete understanding of the relationship between HDL and atherosclerosis. Recent studies have indicated that the ability of HDL to mediate cholesterol efflux from macrophages is a better predictor of atherosclerotic plaque burden [4] and future atherosclerotic cardiovascular disease events [5] than HDL-C levels [4], [5], and defects in HDL function have been described in a variety of settings [6], [7]. Although the assessment of cholesterol efflux capacity used in these earlier studies [4], [5] reflects the ability to mobilize free cholesterol from macrophages, it does not capture variation in cholesterol efflux in terms of cellular components such as endogenous cholesterol transporters [4]. Given their relationship to atherosclerosis in animal models [8], an investigation of cellular defects in cholesterol efflux warrants study in humans.

Two ATP-binding cassette transporters, ABCA1 and ABCG1, play key roles in promoting efflux of cellular cholesterol to apolipoprotein A1 (apoA1) [9] and HDL [10] and are highly expressed in cholesterol loaded macrophages and monocytes [11], [12]. ABCA1 is mutated in patients with Tangier Disease, who accumulate macrophage foam cells in tissues [13]. In animal studies, defective cellular cholesterol efflux pathways are associated with accelerated atherosclerosis [8]. Increased cell-surface levels of the common β subunit of the IL-3/GM-CSF receptor (IL-3Rβ or CD131) which can be detected by flow cytometry on the surface of monocytes appears to be a signature of defective cholesterol homeostasis and myeloid proliferation in animals lacking ABCA1 and ABCG1 [8].

Patients with chronic kidney disease (CKD) experience high rates of atherosclerotic cardiovascular disease and death [14], [15] that are not fully explained by traditional risk factors [15]. We recently discovered that mild levels of renal dysfunction are accompanied by reduced levels of HDL-C and higher levels of circulating monocytes [16]. Moreover, studies in animal models [8] have shown that cellular defects in cholesterol efflux can promote atherosclerosis independent of HDL changes.

This study was designed to investigate new mechanisms of accelerated atherosclerosis in patients with CKD. We hypothesized that monocyte counts would be positively correlated with cell-surface IL-3Rβ in CKD patients, due either to a defect in HDL or in monocytes due to decreased levels of ABCA1 or ABCG1. A secondary hypothesis was that monocytes from CKD patients would have a cellular defect in cholesterol efflux compared to controls using a flow cytometry based cholesterol efflux assay. In exploratory analyses we also measured plasma inflammatory cytokines and carried out metabolite profiling.

Section snippets

Study population

The research protocol was approved by the Institutional Review Board (IRB) of Columbia University Medical Center (CUMC) in New York. All study participants provided written informed consent. CKD patients were recruited from outpatient Nephrology clinics and Control subjects from the Associates in Internal Medicine clinic at CUMC. We used the abbreviated Modification of Diet in Renal Disease (MDRD) equation [17] to calculate the estimated GFR (eGFR). CKD patient inclusion criteria were age  18 

Results

Baseline characteristics of the study participants are shown in Table 1. 240 total subjects (120 CKD patients and 120 controls) comprised the Entire Cohort. The first 120 subjects (60 CKD patients and 60 controls) comprised the Metabolite Cohort, in which plasma was separated for cross-sectional comparison of 89 circulating metabolites, and the first 30 CKD patients and 30 controls comprised the Monocyte Cohort, in which the main goal was to measure monocyte counts and cell-surface levels of

Discussion

A cholesterol efflux assay which measures efflux of cholesterol from cultured cells to patients' HDL has recently been associated with future atherosclerotic cardiovascular disease events [5] emphasizing the potential value of this [5] and other in vitro assays [4] in guiding the development of new HDL-targeted therapies [4] which may particularly benefit patients with dysfunctional HDL [6], [7]. In this study, we report evidence for a cellular defect in ABCA1-mediated cholesterol efflux in

Sources of funding

Supported by awards to Dr. Ganda from the NIDDK of the NIH (K23DK097288), The Louis V. Gerstner, Jr. Scholars Program at Columbia College of Physicians & Surgeons, the Irving Institute for Clinical and Translational Research through the National Center for Advancing Translational Sciences (UL1 TR000040), and Merck and Pfizer Investigator-Initiated research grants to Dr. Ganda; also supported by awards from the NHLBI to Dr. Tall (1R01HL107653) and to Dr. Gerszten (1R01HL096738), an AHA

Disclosures

AT reports being a consultant to CSL, Lilly, Merck, MedImmune and Amgen.

Acknowledgments

Preliminary results were presented by Dr. Ganda on Saturday, November 7, 2015 at the American Society of Nephrology annual meeting in San Diego, CA during a Clinical Practice Session entitled ‘Non-Framingham Risk Factors for Cardiovascular Disease in the CKD Population: Inflammation and Cardiovascular Disease: New Insight from Monocytes and Macrophages’. We wish to thank the patients who participated in this study.

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