Aristolochic acid nephropathy is characterized by a progressive interstitial fibrosis located in the superficial cortex, along with a tubular atrophy and a loss of proximal tubules due to the ingestion of aristolochic acids. In different study models of nephropathy, the effects of sirtuins 1 and 3 have already been investigated as they have been shown to have protective roles against oxidative stress or mitochondrial impairment. However, the roles of these enzymes have never been investigated in a model of aristolochic acid nephropathy. In the first part of this study, an aristolochic acid I (AAI)-induced cytotoxicity in vitro model is used on human kidney-2 cells to determine these putative roles. To allow sirtuins 1 and 3 gene expression analysis in cells exposed to an AAI, independently of cell death, we have characterized the effects of low concentrations (0, 0.1, 0.25, 0.5, 0.75 or 1 µM) of AAI on cell proliferation and cell toxicity by MTT and LDH assays for 12, 24, 48 or 72 h. After setting up the cell model, the second part concerns the analysis of the ATP content of cells incubated with low concentrations of AAI by a bioluminescent method. Indeed, mitochondrial fragmentation observed in cells exposed to a toxic AAI concentration might be accompanied by ATP production alterations. These low concentrations don’t affect the ATP contents. The third part of this work covers the investigation of the putative effects of low concentrations of AAI on the abundance of sirtuins 1 and 3 at the transcriptomic and protein levels. No significant difference is observed on the expression of sirtuin 1 and sirtuin 3. The last part relates to the impact of the activation or the inhibition of sirtuins 1 and 3, by the use of four molecules. In order to evaluate a putative sensitisation by sirtuins inhibitors (3-TYP and EX527) or a potential protection by sirtuins activators (Coumarin and SRT2104) against the AAI-induced cytotoxicity, western blot analysis, MTT and LDH assays are performed. A slight activation of sirtuin 3 was noted with the use of 10 µM of Coumarin. No change is observed in the cytotoxicity by the use of 10 µM of 3-TYP, whereas a decrease of the cell proliferation is observed. As regards sirtuin 1, a reduction of the cell proliferation and an increase in the cytotoxicity are observed with the use of 25 µM of SRT2104. Moreover, a decrease of the cell proliferation is observed 72 hours after the incubation with 10 µM of EX527. This study opens the scope for additional experiments such as the analysis of the morphology of the mitochondrial population in human kidney-2 cells exposed to low concentrations of aristolochic acid I or an increase of the concentrations of activators or inhibitors of sirtuins used in this work, in order to obtain a more marked effect of these four molecules.
|Date of Award||Sept 2019|
|Supervisor||Nathalie Caron (Supervisor) & Thierry Arnould (Co-Supervisor)|
Study of the putative roles of sirtuins 1 and 3 in the aristolochic acid I-induced cytotoxicity in human kidney-2 cells
Harvent, M. (Author). Sept 2019
Student thesis: Master types › Master in biochemistry and molecular and cell biology Research focus