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

• Published in: American Journal of Physiology: Cell Physiology Vol. 326; no. 5; pp. C1423 - C1436
• Main Authors: Zhai, Yougang, Chavez, Jose A, D'Aquino, Katharine E, Meng, Rong, Nawrocki, Andrea R, Pocai, Alessandro, Wang, Lifeng, Ma, Li-Jun
• Format: Journal Article
• Language: English
• Published: United States American Physiological Society 01.05.2024
• Subjects: Cell culture; Down-regulation; Epithelial cells; Gene expression; Kidneys; Kynurenine 3-monooxygenase; NAD; Spheroids; Tryptophan; de novo NAD+ synthesis; nicotinamide adenine dinucleotide; Kynurenine 3-monooxygenase; kynurenine pathway; renal proximal tubule epithelial cell
• ISSN: 0363-6143; 1522-1563; 1522-1563
• DOI: 10.1152/ajpcell.00445.2023

Nicotinamide adenine dinucleotide (NAD ) is a pivotal coenzyme, essential for cellular reactions, metabolism, and mitochondrial function. Depletion of kidney NAD levels and reduced NAD synthesis through the tryptophan-kynurenine pathway are linked to acute kidney injury (AKI), while augmenting NAD shows promise in reducing AKI. We investigated NAD biosynthesis using models to understand its role in AKI. 2D cultures of human primary renal proximal tubule epithelial cells (RPTECs) and HK-2 cells showed limited NAD synthesis, likely due to low pathway enzyme gene expression. Employing 3D spheroid culture model improved the expression of tubular-specific markers and enzymes involved in NAD synthesis. However, NAD synthesis remained elusive in the 3D spheroid culture, regardless of injury conditions. Further investigation revealed that 3D cultured cells couldn't metabolize tryptophan (Trp) beyond kynurenine (KYN). Intriguingly, supplementation of 3-hydroxyanthrilinic acid into RPTEC spheroids was readily incorporated into NAD . In a human precision-cut kidney slice (PCKS) ex vivo model, 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 3D spheroids successfully reinstated NAD synthesis from Trp. Additionally, in vivo study demonstrated that NAD+ synthesis is intact in the kidney of the healthy adult mice. Our findings highlight disrupted tryptophan-kynurenine NAD synthesis in cellular models and an kidney model, primarily attributed to KMO downregulation.