Abstract: TH-PO941
Kynurenine 3-Monooxygenase Limits De Novo NAD+ Synthesis Through Dietary Tryptophan in Cultured Renal Proximal Tubule Epithelial Cells
Session Information
- Health Maintenance, Nutrition, Metabolism - I
November 02, 2023 | Location: Exhibit Hall, Pennsylvania Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Health Maintenance, Nutrition, and Metabolism
- 1500 Health Maintenance, Nutrition, and Metabolism
Authors
- Zhai, Yougang, Janssen Research and Development LLC, Raritan, New Jersey, United States
- Meng, Rong, Janssen Research and Development LLC, Raritan, New Jersey, United States
- Chavez, Jose A., Janssen Research and Development LLC, Raritan, New Jersey, United States
- Nawrocki, Andrea R., Janssen Research and Development LLC, Raritan, New Jersey, United States
- Pocai, Alessandro, Janssen Research and Development LLC, Raritan, New Jersey, United States
- Wang, Lifeng, Janssen Research and Development LLC, Raritan, New Jersey, United States
- Ma, Li-Jun, Janssen Research and Development LLC, Raritan, New Jersey, United States
Group or Team Name
- CVMR-PH Discovery, Janssen Research & Development LLC.
Background
Nicotinamide adenine dinucleotide (NAD+) is an essential coenzyme involved in regulation of mitochondrial function. Depletion of kidney NAD+ levels has been linked to acute kidney injury (AKI). The de novo NAD+ can be synthesized through the tryptophan-kynurenine pathway. Renal proximal tubular epithelial cells (RPTECs) are susceptible to diverse injuries in AKI and maybe important for de novo NAD+ generation. However, despite feeding cultured RPTECs with isotope labeled tryptophan, we were unable to detect the synthesis of NAD+. To address this, we aim to investigate whether the use of 3D culture of primary human RPTECs can enhance de novo NAD+ synthesis.
Methods
Primary hRPTECs were cultured in ULA plates for 4 days to form spheroids. Gene expression levels were assessed by qPCR. Human Kynurenine 3-monooxygenase (hKMO) was overexpressed in hRPTEC spheroids through transduction of Adv-hKMO vector. Cells were treated with isotope-labeled Tryptophan (*Trp) or isotope-labeled 3-hydroxyanthranilic acid intermediate (*3HAA). The relative concentrations of intracellular *Trp, *NAD+ and metabolite intermediates in the hRPTEC spheroids were analyzed by LC-MS/MS.
Results
3D culture of primary human RPTECs led to a significant increase in the expression of tubular marker genes and enzyme genes involved in de novo NAD+ synthesis. However, de novo NAD+ synthesis was not detected. To investigate why 3D tubular cells were unable to metabolize Trp beyond kynurenine (KYN) metabolite, supplement of *3HAA to hRPTEC 3D spheroids was applied and resulted in its effective incorporation into *NAD+. Furthermore, overexpressing KMO, the enzyme responsible for converting KYN to 3-hydroxykynurenine (3HK), achieved successful rescue of de novo NAD+ synthesis through Trp in the hRPTEC 3D spheroids.
Conclusion
Our data demonstrate that 1) in cultured primary RPTECs, the tryptophan-kynurenine pathway loses its function for de novo NAD+ synthesis, 2) the downregulation of a key enzyme gene KMO disrupts the conversion of KYN to 3HK in the pathway.
Funding
- Commercial Support – JNJ