Kynurenine 3-monooxygenase limits de novo NAD + synthesis through dietary tryptophan in renal proximal tubule epithelial cell models.

Nicotinamide adenine dinucleotide (NAD ⁺ ) is a pivotal coenzyme, essential for cellular reactions, metabolism, and mitochondrial function. Depletion of kidney NAD ⁺ levels and reduced de novo NAD ⁺ synthesis through the tryptophan-kynurenine pathway are linked to acute kidney injury (AKI), whereas augmenting NAD ⁺ shows promise in reducing AKI. We investigated de novo NAD ⁺ biosynthesis using in vitro, ex vivo, and in vivo models to understand its role in AKI. Two-dimensional (2-D) cultures of human primary renal proximal tubule epithelial cells (RPTECs) and HK-2 cells showed limited de novo NAD ⁺ synthesis, likely due to low pathway enzyme gene expression. Using three-dimensional (3-D) spheroid culture model improved the expression of tubular-specific markers and enzymes involved in de novo NAD ⁺ synthesis. However, de novo NAD ⁺ synthesis remained elusive in the 3-D spheroid culture, regardless of injury conditions. Further investigation revealed that 3-D cultured cells could not metabolize tryptophan (Trp) beyond kynurenine (KYN). Intriguingly, supplementation of 3-hydroxyanthranilic acid into RPTEC spheroids was readily incorporated into NAD ⁺ . In a human precision-cut kidney slice (PCKS) ex vivo model, de novo NAD ⁺ synthesis was limited due to substantially downregulated kynurenine 3-monooxygenase (KMO), which is responsible for KYN to 3-hydroxykynurenine conversion. KMO overexpression in RPTEC 3-D spheroids successfully reinstated de novo NAD ⁺ synthesis from Trp. In addition, in vivo study demonstrated that de novo NAD ⁺ synthesis is intact in the kidney of the healthy adult mice. Our findings highlight disrupted tryptophan-kynurenine NAD ⁺ synthesis in in vitro cellular models and an ex vivo kidney model, primarily attributed to KMO downregulation. NEW & NOTEWORTHY Nicotinamide adenine dinucleotide (NAD ⁺ ) is essential in regulating mitochondrial function. Reduced NAD ⁺ synthesis through the de novo pathway is associated with acute kidney injury (AKI). Our study reveals a disruption in de novo NAD ⁺ synthesis in proximal tubular models, but not in vivo, attributed to downregulation of enzyme kynurenine 3-monooxygenase (KMO). These findings highlight a crucial role of KMO in governing de novo NAD ⁺ biosynthesis within the kidney, shedding light on potential AKI interventions.