Reduced nicotinamide mononucleotide (NMNH) as a potent NAD+ enhancer Rubén Zapata-Pérez1, Alessandra Tammaro2, Bauke V. Schomakers1,3, Angelique M. L. Scantlebery1, Simone Denis1, Hyung L. Elfrink1,3, Judith Giroud-Gerbetant4, Carles Cantó4, Carmen López-Leonardo5, Rebecca L. McIntyre1, Michel van Weeghel1,3, Álvaro Sánchez-Ferrer6 & Riekelt H. Houtkooper1 1Laboratory Genetic Metabolic Diseases, Amsterdam Gastroenterology, Endocrinology, and Metabolism, Amsterdam Cardiovascular Sciences, Amsterdam UMC, University of Amsterdam, Meibergdreef 9, Amsterdam, The Netherlands 2Pathology department, Amsterdam UMC, University of Amsterdam, Meibergdreef 9, The Netherlands 3Core Facility Metabolomics, Amsterdam UMC, University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, the Netherlands 4Nestlé Institute of Health Sciences, Nestlé Research, EPFL Innovation Park, 1015, Lausanne, Switzerland 5Department of Organic Chemistry, University of Murcia, Murcia, Spain. 6Department of Biochemistry and Molecular Biology-A, University of Murcia, Murcia, Spain. Nicotinamide adenine dinucleotide (NAD+) plays a crucial role in energy metabolism. The fact that NAD+ is the limiting substrate of sirtuins has renewed the interest in strategies to increase NAD+ bioavailability to activate these enzymes and combat disease. NAD+ repletion by means of supplementation with the NAD+ precursors nicotinamide mononucleotide (NMN) or nicotinamide riboside (NR) emerged as a promising strategy to prevent or ameliorate diseases such as obesity, diabetes or fatty acid-induced liver disease in mice. However, no beneficial effects have been achieved with the current set of NAD+ precursors in the clinical trials carried out so far. This lack of efficacy could be due to the inefficiency of NMN and NR to boost NAD+ in humans. Therefore, we are now in need for more potent NAD+ boosting molecules. In this work we developed an enzymatic synthesis route of a reduced form of nicotinamide mononucleotide, termed NMNH, and identified it as a new and potent NAD+ precursor. We show that NMNH has a greater NAD+-enhancing potential than NMN. In contrast to NMN, the conversion of NMNH to NAD+ is dependent on adenosine kinase (AK), instead of NR kinase. NMNH administration in mice leads to a rapid surge of NAD+ in blood, which is maintained for much longer than in NMN-treated animals. This NAD+ increase was also observed in other tissues, including liver, kidney, muscle and brain. Finally, by using an in vitro model of acute kidney injury (AKI), we demonstrate that NMNH administration massively increases NAD+ levels under hypoxia, leading to a potential protection against renal damage, as highlighted by a sharp decrease in the major kidney damage marker Kim-1 (kidney injury molecule 1). Together, our data highlight NMNH as a new NAD+ precursor with therapeutic potential for acute kidney injury, confirm the existence of a novel pathway for the recycling of reduced NAD+ precursors and establish NMNH as a member of the new family of reduced NAD+ precursors.