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

Abstract

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), while 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. 2D 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. Employing 3D 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 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, 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 3D spheroids successfully reinstated de novo NAD+ synthesis from Trp. Additionally, 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.

Unraveling the nexus of NAD+ metabolism and diabetic kidney disease: insights from murine models and human data

“…Our data supplemented their study with evidence that decreased tubular KMO might be responsible for disruption of glomerular renal NAD+ homeostasis in early phase of DKD. This was further supported by the work of Yougang Zhai et al., wherein the overexpression of KMO in primary cultured human proximal tubular cells exhibited otherwise undetectable downstream intermediate metabolites such as 3-HK, QA and consequently restored the NAD+ levels originating form de novo pathway ( 19 ).…”

“…The discrepancy in NAD+ levels might be due to different diabetic animal models and distinct mouse strains. In the kidney, the salvage pathway dominates NAD+ biosynthesis ( 18 , 19 ). Overexpression of NAMPT, an enzyme of NAD+ salvage pathway, was reported to protect diabetic kidney injury via regulating renal NAD+ level and NAD+-dependent deacetylase SIRT6 ( 12 ).…”

“…De novo pathway, also known as the kynurenine pathway, consists of eight steps, and generate NAD+ from dietary tryptophan (TRP) ( Figure 1A ). Although salvage pathway is responsible for most NAD+ production ( 18 , 19 ), supplementation with TRP has long been recognized as a treatment for vitamin B3 deficiency, suggesting an important role for the de novo pathway in NAD+ homeostasis ( 20 ). Besides, manipulation of the de novo pathway has been shown to change NAD+ levels and resistance to acute kidney injury (AKI) ( 13 , 21 – 24 ).…”