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