ABSTRACT The development of acute kidney (AKI) injury in hospitalized patients may be catastrophic. In general, the in- hospital mortality rate for patients with AKI has been estimated between 20% to 25%. AKI prolongs hospitalization, increases costs, and requires the utilization of additional resources. Drugs are implicated in roughly one-quarter of hospitalized patients who develop AKI, and roughly half of the cases are related to antibiotics. While broad spectrum antibiotics are essential to treat infections, they carry risk. Clinicians can alter modifiable risk factors, but many patients are inherently at an increased risk of AKI. The exact molecular mechanisms of antibiotic induced AKI are unclear, but emerging data supports oxidative stress and mitochondrial dysfunction. Melatonin, a hormonal dietary supplement, has antioxidant properties via the nuclear factor erythroid 2–related factor 2 (NRF2) pathway and can also restore mitochondrial bioenergetics. As such, melatonin is an attractive option for kidney protection. This proposal will test the hypothesis that melatonin reduces the risk of antibiotic-associated AKI through activation of the NRF2 transcriptional pathway and balancing of mitochondrial function and bioenergetics. To test this hypothesis, we propose a translational strategy to investigate mechanism and efficacy in parallel. First, in our in vitro studies, we will expose human renal proximal tubule (RPT) cells to nephrotoxic antibiotics in the presence /absence of melatonin. RPT cells will include control cells as well as those with disrupted NRF2 and KEAP1 function. Analyses will include 1) targeted biomarkers of mitochondrial/cellular health and 2) transcriptomics to identify complementary and alternative reno-protective pathways. Next, we will enroll 300 hospitalized patients prescribed vancomycin and piperacillin-tazobactam and randomize them 1:1 to melatonin 5 mg or matched placebo. Whole blood will be analyzed for gene expression (NRF2/KEAP1) and plasma to evaluate antibiotic and melatonin biodistribution. Further, traditional and novel biomarkers of kidney injury and mitochondrial stress will be assessed. The studies described in the two aims will provide evidence to support the use of melatonin and provide evidence of the mechanism.