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 treatme...