Study of mitochondrial dysfunction in in vivo and in vitro models of aristolochic acids nephropathy

Abstract

Aristolochic acids (AA) are toxic compounds naturally present in Aristolochiaceae plants which are particularly dangerous for kidneys. Indeed, upon exposure to AA, patients can develop a nephropathy termed aristolochic acid-induced nephropathy (AAN), characterized by oxidative stress, inflammation, interstitial fibrosis, and more recently by mitochondrial dysfunction. Mitochondrial dysfunction has been reported in various types of kidney diseases. It is particularly deleterious to renal proximal tubular epithelial cells. These cells are enriched in mitochondria to ensure their energy supply, used for absorption and secretion functions. Mitochondrial dysfunction may result from excessive reactive oxygen species (ROS) generation, alteration of mitochondrial structure, or alteration of their energy production capacities. Despite the recent research, the exact molecular mechanisms of mitochondrial dysfunction are not fully characterized yet. Moreover, supplementation with nicotinamide riboside (NR) may be a valuable approach to circumvent oxidative stress during AAN. Aim. This study aimed to investigate mitochondrial dysfunction in both in vivo and in vitro models of AAN. In addition, NR was used in the in vitro model to alleviate AA-intoxication effects.Methods. Previously, male C57BL/6J (n=8) were submitted to intraperitoneal injection of AA-I (3.5 mg/kg of bodyweight) or of saline solution for 4 days, and then sacrificed after 12, 24, 48, 72, 96 or 216 hours of treatment. Kidney tissue, urine and blood were collected to evaluate different toxicity parameters. Parallelly, HK-2 cells were treated with 0 or 10 µM of AA, and/or 0.5 mM of NR for 24, 48 or 72 hours. Then, cell viability and cytotoxicity tests were performed along with confocal microscopy analyses. In both models, mRNA and protein expression of actors regulating mitochondrial function and anti-oxidative stress response were assessed.Results. Gene and protein expression of actors related to mitochondrial function were nearly all downregulated in AA-treated mice, in a time-dependent manner. By contrast, results performed on HK-2 cells were partly different. NR supplementation in HK-2 cells treated with AA seemed ameliorate their viability evaluated by enzymatic assays. In addition, ROS generation was lower in cells treated with both AA and NR. Conclusion. Data indicate that mitochondrial dysfunction is present in AA-treated mice. The expression level of the actors involved in mitochondrial biogenesis and its dynamics are decreased by AA in both models. In HK-2 cells, treatment with NR seems to be beneficial, particularly against the AA cytotoxicity and the ROS production. More experiments should be performed in HK-2 cells to better characterize the AA-related mitochondrial dysfunction and the NR relative protection.

Date of Award	17 Jan 2023
Original language	English
Awarding Institution	University of Namur
Supervisor	Charles Nicaise (Supervisor)