Inhibitors of poly (ADP-ribose) synthetase protect rat proximal tubular cells against oxidant stress

Inhibitors of poly (ADP-ribose) synthetase protect rat proximal tubular cells against oxidant stress.

Background

The generation of reactive oxygen species (ROS) has been implicated in the pathogenesis of renal ischemia-reperfusion injury. ROS produce DNA strand breaks that lead to the activation of the DNA-repair enzyme poly (ADP-ribose) synthetase (PARS). Excessive PARS activation results in the depletion of its substrate, nicotinamide adenine dinucleotide (NAD) and subsequently of adenosine 5′-triphosphate (ATP), leading to cellular dysfunction and eventual cell death. The aim of this study was to investigate the effect of various PARS inhibitors on the cellular injury and death of rat renal proximal tubular (PT) cells exposed to hydrogen peroxide (H₂O₂).

Methods

Rat PT cell cultures were incubated with H₂O₂ (1 mM) either in the presence or absence of the PARS inhibitors 3-aminobenzamide (3-AB, 3 mM), 1,5-dihydroxyisoquinoline (0.3 mM) or nicotinamide (Nic, 3 mM), or increasing concentrations of desferrioxamine (0.03 to 3 mM) or catalase (0.03 to 3 U/ml). Cellular injury and death were determined using the MTT and lactate dehydrogenase (LDH) assays, respectively. H₂O₂-mediated PARS activation in rat PT cells and the effects of PARS inhibitors on PARS activity were determined by measurement of the incorporation of [³H]NAD into nuclear proteins.

Results

Incubation of rat PT cells with H₂O₂ significantly inhibited mitochondrial respiration and increased LDH release, respectively. Both desferrioxamine and catalase reduced H₂O₂-mediated cellular injury and death. All three PARS inhibitors significantly attenuated the H₂O₂-mediated decrease in mitochondrial respiration and the increase in LDH release. Incubation with H₂O₂ produced a significant increase in PARS activity that was significantly reduced by all PARS inhibitors. 3-Aminobenzoic acid (3 mM) and nicotinic acid (3 mM), structural analogs of 3-AB and Nic, respectively, which did not inhibit PARS activity, did not reduce the H₂O₂-mediated injury and necrosis in cultures of rat PT cells.

Conclusion

We propose that PARS activation contributes to ROS-mediated injury of rat PT cells and, therefore, to the cellular injury and cell death associated with conditions of oxidant stress in the kidney.