PROJECT SUMMARY Nicotinamide adenine dinucleotide (NAD+) and its reduced form NADH, play crucial roles in maintaining tissue homeostasis. We now know that NAD+ and NADH concentrations differ in different parts of the cell by orders of magnitude. Nevertheless, how these distinct cellular pools of NAD+/NADH enable compartment specific metabolic and regulatory functions, ensuring coordination of various cellular processes remains elusive. NAD+/NADH compartmentalization is particularly important for the kidneys, in which tubule cells perform the high-energy demanding task of reclaiming solutes filtered from the bloodstream and eliminating waste products through urine. In acute kidney injury (AKI), a common condition in elderly patients, substantial decreases in the levels of NAD+ impair energy generation in tubular epithelial cells leading to critical loss of cellular and organismal homeostasis. Patients who survive AKI frequently develop chronic kidney disease (CKD), a condition characterized by kidney fibrosis and inflammation leading to deleterious systemic effects. We and others have shown that NAD+ boosting by cytoprotective approaches protects the kidney tubules against ischemia and other insults. Furthermore, several experimental studies have demonstrated protective role for NAD+ precursor supplementation against aging and AKI but the underlying mechanisms remain elusive. In particular, while the cellular NAD+/NADH ratio is critical for energy metabolism, the extent to which it reflects versus drives metabolic physiology in vivo is poorly understood. To successfully translate NAD-based therapeutics in humans, it is essential to understand the molecular mechanisms by which this metabolite regulates kidney health. To this end, recent technological advances that allow genetic manipulation of NAD+ in different cellular compartments, have uncovered an exciting potential for revealing the multi-faceted effects of this fascinating molecule. The goal of this proposal is to identify novel mechanisms by which directly increasing the NAD+/NADH promotes kidney health in the context of injury and aging. To this end, we propose in vivo expression of Lactobacillus brevis (Lb)NOX, a bacterial water forming NADH oxidase, in the cytosol or mitochondrial of kidney tubular cells. Two specific aims are proposed. Aim 1 will determine how boosting mitochondrial NAD+/NADH ratio in tubular epithelial cells regulates kidney health following ischemic kidney injury. Aim 2 will define the role of boosting cytosolic NAD+/NADH in tubular epithelial cells as a regulator of kidney health following ischemic kidney injury. Completion of this project will generate exciting new insights in kidney biology and enable the development of urgently needed novel therapeutics that protect kidney health.