Inhibition of mitochondrial respiration and oxygen uptake in isolated rat renal tubular fragments by atractyloside

Abstract

Atractyloside (ATR) is widely used as a specific inhibitor of mitochondrial adenine nucleotide translocase and it is also a potent nephrotoxin that selectively injures the proximal tubule in vivo. This regioselectivity has been attributed to the prominence of mitochondria in the proximal tubule cells, but there have been no investigations to confirm this. In order to better understand the molecular basis of ATR-induced renal injury, oxidative phosphorylation was studied in freshly isolated rat proximal tubular and glomeruli fragments, and in isolated rat renal cortical mitochondria. In isolated renal mitochondrial, ATR significantly inhibited state 3 respiration in a dose-dependent manner, with the maximum inhibition achieved at the highest ATR concentration. Low doses of ATR (53 μM) inhibited respiration by 50%, an effect which was reversed by 2.5 μmol ADP. 2,4-Dinitrophenol (5 mM), which stimulated respiration in control mitochondria, failed to do this in the presence of ATR. Basal oxygen consumption was significantly inhibited by ATR (> 50 μM) in proximal tubule previously incubated for 1 h at 37 °C. The concentration-dependent inhibition of oxygen uptake by the proximal tubule was maintained in the presence of 1 mM ouabain or 0.25 mg/ml nystatin. Glomeruli have active mitochondrial respiration (about half that of the proximal tubules), but were not affected by ATR at concentrations up to 500 μM. These data demonstrates that both purified renal mitochondria and freshly isolated fragments of the proximal tubule exposed to ATR in vitro exhibit similar alteration in respiratory parameters that demonstrate inhibition of state 3 mitochondrial respiration, but there was no significant effect on glomeruli cells. Thus, the inhibition of oxidative phosphorylation may be an early event in ATR-induced nephrotoxicity, where the prominence of mitochondria in the proximal tubule explain, in part, the localised injury. The resistance of the glomeruli suggest that preferential transport of ATR may also contribute to the sensitivity of the proximal tubule.