Abstract: TH-PO1073
Inhibition of Kynurenine 3-Monooxigenase (KMO) Alters NAD Balance and Mitochondrial Function in Murine and Human Glomerular Cells and Contributes to Proteinuria in Zebrafish
Session Information
- Glomerular Diseases: Podocyte Biology - I
November 07, 2019 | Location: Exhibit Hall, Walter E. Washington Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Glomerular Diseases
- 1204 Podocyte Biology
Authors
- Bolanos-Palmieri, Patricia, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany
- Schenk, Heiko Joachim, Hannover Medical School, Hannover, Germany
- Schroder, Patricia Ann, Mount Desert Island Biological Laboratory, Salisbury Cove, Maine, United States
- Schiffer, Mario, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany
Background
KMO is an enzyme of the kynurenine pathway (KP) and as such it is key for tryptophan (trp) catabolism. KMO uses kynurenine (kyn) as a substrate and participates in the production of important bioactive metabolites. Systemic KP dysregulation has been linked to a variety of pathologies, including CKD. Since the enzymes of the KP are expressed in the kidney, we aim to describe the effects of their dysregulation on glomerular cells.
Methods
KMO inhibition was performed in a transgenic zebrafish line that facilitates the detection of proteinuria. Embryos were treated with a KMO inhibitor or kyn starting at 48hpf. At 96hpf the phenotype was recorded and samples were collected for metabolite analysis by mass spectrometry. Since KMO is located in the mitochondria and participates in the production of NAD, mouse parietal epithelial cells (PEC), as well as human and murine podocytes (POD) were incubated with a KMO inhibitor; the levels of NAD+, NADH and mitochondrial membrane polarization were determined. Alterations in the bioenergetics parameters were also assessed by measuring the oxygen consumption rate after KMO inhibition.
Results
Our results show that inhibition of KMO leads to the accumulation of upstream kynurenine metabolites in the treated larvae. Additionally, proteinuria and edema were observed after KMO inhibition, in line with our previous results from morpholino mediated knockdowns. Treating the larvae with exogenous kyn also leads to mild proteinuria. In vitro data show that KMO inhibition reduces the NAD+/NADH ratio in POD, and leads to mitochondrial depolarization in POD and PEC. A reduction in spare respiration, coupling efficiency and an increase in proton leak accompanied the mitochondrial phenotype in POD. We also show that glomerular cells are capable of taking up kyn from the media, suggesting that kyn is not only relevant as a biomarker, but may directly contribute to disease etiology.
Conclusion
Taken together our results highlight the importance of trp catabolism via the KP within the context of renal cell biology, where a reduction in KMO activity alters the energy metabolism of POD and leads to kyn metabolite imbalances that ultimately may impact the glomerular filtration barrier.
Funding
- Government Support - Non-U.S.