Exploring the role of OAT1/3 on mitochondrial function in human renal cells

Tijmen B.T. van den Berge1*, Jelle Vriend1, Frans G. Russel1, Martijn J. Wilmer1

*Presenting author (Email: [email protected])

1 Department of Pharmacology and Toxicology, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, Nijmegen, The Netherlands.

 

Introduction: Transcellular transport in renal proximal tubular cells is key in active drug secretion. Mitochondria are the main source for cellular energy and therefore have a pivotal role in ATP-dependent transport mechanisms. Hence, drug excretion could be hampered due to drug-induced mitochondrial toxicity. Organic anion transporter 1 and 3 (OAT1; OAT3) function as organic anion/dicarboxylate exchangers, thereby possibly interfering with the cell’s metabolism and play a role in mitochondrial toxicity. Here, we study the relation between OAT1/3 and mitochondrial activity in a human renal cell line, ciPTEC.

Methods: Transporter function upon full inhibition of mitochondrial oxygen consumption was analyzed in ciPTEC parent and ciPTEC overexpressing OAT1 or OAT3 (ciPTEC-OAT1; ciPTEC-OAT3), upon exposure to mitochondrial complex-III inhibitor antimycin A(AA; 0,05-15 nM; 24h). O2-consumption linked to oxidative phosphorylation (OXPHOS) was measured with an Oroboros-oxygraph. Viability was measured using live/dead stains based on Hoechst, Yo-Pro and PI. OAT1/3 activity was determined by the uptake of the OAT-substrate fluorescein. Next to ATP production, lactate levels were measured using the enzyme lactate-dehydrogenase (LDH). Renal drug-transporter activity of apical Multidrug-Resistance-associated-Protein (MRP) was studied using CMFDA.

Results: Cell viability was intact upon OXPHOS inhibition, while a dose-dependent decrease in fluorescein uptake by OAT1/3 was observed upon AA exposure, together with a decreased O2-consumption. Increased intracellular lactate levels suggested a shift from OXPHOS towards glycolysis, and resulted in intact ATP levels. Activity of MRP2/4 was not affected by OXPHOS inhibition.

Conclusion: Decreased renal organic anion transport associated with OXPHOS inhibition could impair drug elimination during hypoxia.

 Keywords: Renal proximal tubular epithelial cells, OAT1, OAT3, Oxidative phosphorylation, Hypoxia