Circulating factors as a mechanism of improvements in vascular endothelial function with inorganic nitrate in patients with chronic kidney disease

PROJECT SUMMARY Cardiovascular disease (CVD) is the leading cause of death in patients with chronic kidney disease (CKD). Vascular endothelial dysfunction is a key antecedent to CVD in patients with CKD. A primary mechanism of endothelial dysfunction in CKD is a decline in the vasoprotective molecule nitric oxide (NO). Reactive oxygen species (ROS)-related oxidative stress, a key source of which is mitochondria, mediates reductions in NO bioavailability in CKD. Establishing new therapies to enhance NO bioavailability and lower ROS-related oxidative stress to improve endothelial function in patients with CKD is a biomedical research priority. Targeting the nitrate-nitrite-NO pathway represents a promising approach for enhancing NO bioavailability and improving endothelial function in CKD. I have shown in older adults without CKD that targeting the nitrate- nitrite-NO pathway with inorganic nitrite (sodium nitrite) improves NO-mediated endothelial function. The improvements are associated with changes in circulating factors in plasma that reduce endothelial cell total and mitochondria-specific ROS bioactivity. Sodium nitrite also alters the plasma metabolome, and changes in select metabolites with treatment are associated with lower plasma-induced endothelial cell ROS bioactivity. To translate these findings to CKD, I am completing a randomized clinical trial testing the effects of 3 months of treatment with nitrate-rich beetroot juice vs. placebo (nitrate-depleted beetroot juice) for improving endothelial function in individuals with CKD (K01DK115524). My preliminary findings suggest that nitrate-rich beetroot juice improves NO-mediated endothelial function. My preliminary results also suggest nitrate-rich beetroot juice may change circulating factors in plasma to improve endothelial cell function, as shown by an increased acetylcholine (ACh)-stimulated NO production and reduced ROS bioactivity in human aortic endothelial cells (HAECs) exposed to plasma from subjects taken before/after active treatment vs. placebo. The purpose of this R03 application is to leverage samples from my K01-supported clinical trial to show changes in ‘circulating factors’ as a novel mechanism contributing to improvements in endothelial function with nitrate-rich beetroot juice supplementation in patients with CKD and to identify the specific circulating molecular transducers of the benefits of nitrate-rich beetroot juice on endothelial function in CKD. Aim 1: To assess before/after 3 months of nitrate-rich beetroot juice or placebo (double-blind, randomized) in men and women ≥50 years of age with stage II-IV CKD: i) ACh-stimulated NO production; and ii) total and mitochondria-specific ROS bioactivity in HAECs exposed to plasma from subjects before/after the intervention. Aim 2: a) To determine the effects of nitrate-rich beetroot juice on metabolites in plasma via targeted metabolomics analysis of plasma samples taken before and after nitrate-rich beetroot juice treatment vs. placebo; and b) determine NO production as well as total and mitochondrial-specific ROS bioactivity in HAECs cultured with subject plasma with vs. without normalization metabolites changed with treatment.