Metabolic impact of genetic and chemical ADP/ATP carrier inhibition in renal proximal tubule epithelial cells

Mitochondrial dysfunction is pivotal in drug-induced acute kidney injury (AKI), but the underlying mechanisms remain largely unknown. Transport proteins embedded in the mitochondrial inner membrane form a significant class of potential drug off-targets. So far, most transporter-drug interactions have been reported for the mitochondrial ADP/ATP carrier (AAC). Since it remains unknown to what extent AAC contributes to drug-induced mitochondrial dysfunction in AKI, we here aimed to better understand the functional role of AAC in the energy metabolism of human renal proximal tubular cells. To this end, CRISPR/Cas9 technology was applied to generate AAC3^-/- human conditionally immortalized renal proximal tubule epithelial cells. This AAC3^-/- cell model was characterized with respect to mitochondrial function and morphology. To explore whether this model could provide first insights into (mitochondrial) adverse drug effects with suspicion towards AAC-mediated mechanisms, wild-type and knockout cells were exposed to established AAC inhibitors, after which cellular metabolic activity and mitochondrial respiratory capacity were measured. Two AAC3^-/- clones showed a significant reduction in ADP import and ATP export rates and mitochondrial mass, without influencing overall morphology. AAC3^-/- clones exhibited reduced ATP production, oxygen consumption rates and metabolic spare capacity was particularly affected, mainly in conditions with galactose as carbon source. Chemical AAC inhibition was stronger compared to genetic inhibition in AAC3^-/-, suggesting functional compensation by remaining AAC isoforms in our knockout model. In conclusion, our results indicate that ciPTEC-OAT1 cells have a predominantly oxidative phenotype that was not additionally activated by switching energy source. Genetic inhibition of AAC3 particularly impacted mitochondrial spare capacity, without affecting mitochondrial morphology, suggesting an important role for AAC in maintaining the metabolic spare respiration.

ADP/ATP carrier; CRISPR/Cas9; Drug-induced mitochondrial dysfunction; Nephrotoxicity; Off-target; Oxidative metabolism.