MO006CHANGES IN THE KYNURENINE PATHWAY LEAD TO ALTERATIONS IN NAD BALANCE AND BIOENERGETICS PARAMETERS IN GLOMERULAR CELLS IN VITRO AND CONTRIBUTE TO PROTEINURIA IN A ZEBRAFISH MODEL*

Abstract Background and Aims Tryptophan catabolism is carried out by the enzymes of kynurenine pathway leading to de novo synthesis NAD production a series bioactive metabolites. Kynurenine 3-Monooxigenase (KMO) key component this it one responsible for degradation kynurenine. The metabolites participate in various cellular processes, so systemic dysregulation tryptophan metabolism, marked increased circulation, has been linked onset severity wide range pathologies, such as chronic kidney disease associated co-morbidities. Since are expressed cleared urine, we aim describe effects changes on glomerular cells, both vitro vivo. Method Modulation KMO expression or enzymatic function was performed transgenic zebrafish line that allows monitoring fluorescently labelled protein circulation an indicator proteinuria. Morpholinos targeting three were injected into fish embryos, knockdown Afmid, Kmo Kynu. Additionally, dechorionated larvae treated with inhibitor administered via embryo rearing media, starting at 48hpf. In all cases 96hpf, circulating fluorescent levels determined, larval phenotype scored based edema, samples collected metabolite analysis fixed prepared imaging. results NAD, enzyme located outer mitochondrial membrane, cultured murine parietal epithelial cells well immortalized human mouse podocytes incubated inhibitor. Changes NAD+ NADH, alterations membrane polarization assessed. oxygen consumption rate measured order determine if inhibition leads bioenergetics parameters vitro. Results modification Kynu morpholino mediated lead accumulation upstream larvae, confirmed mass spectrometry analysis. Following our previous results, alteration led development yolk sac pericardial effusion loss from accompanied enlargement Bowman’s space nephrin glomerulus larvae. Under cell culture conditions, reduction size focal adhesion proteins (podocalyxin). NAD+/NADH ratio polarity also altered. spare respiratory capacity, coupling efficiency proton leak suggest might impair cell’s ability adapt its bioenergetic profile response stress. Conclusion Taken together these modulation through may contribute maintaining structural integrity cytoskeleton flexible energy metabolism podocytes. Moreover, vivo model imbalances ultimately impact filtration barrier.