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

Abstract

Inhibitors of poly (ADP-ribose) synthetase protect rat proxiIschemia followed by reperfusion of the kidney inmal tubular cells against oxidant stress. volves a complex and interrelated sequence of events Background. The generation of reactive oxygen species (ROS) that result in the injury of renal cells and eventual cell has been implicated in the pathogenesis of renal ischemiadeath due to apoptosis and necrosis [1, 2]. Although reperfusion injury. ROS produce DNA strand breaks that lead reperfusion is essential for the survival of ischemic tissue, to the activation of the DNA-repair enzyme poly (ADP-ribose) synthetase (PARS). Excessive PARS activation results in the there is good evidence that reperfusion itself causes addidepletion of its substrate, nicotinamide adenine dinucleotide tional cell injury (reperfusion-injury) [3], which has been (NAD) and subsequently of adenosine 59-triphosphate (ATP), attributed to calcium overload, neutrophil infiltration leading to cellular dysfunction and eventual cell death. The and generation of reactive oxygen species (ROS) [1–3]. aim of this study was to investigate the effect of various PARS inhibitors on the cellular injury and death of rat renal proximal ROS–mediated cell injury and death has been implicated tubular (PT) cells exposed to hydrogen peroxide (H2O2). in the pathogenesis of renal ischemia-reperfusion injury Methods. Rat PT cell cultures were incubated with H2O2 and associated renal failure [1, 2]. Within the kidney, (1 mm) either in the presence or absence of the PARS inhibitors the situation is particularly complex, as ischemia itself 3-aminobenzamide (3-AB, 3 mm), 1,5-dihydroxyisoquinoline can cause early irreversible damage, which appears to (0.3 mm) or nicotinamide (Nic, 3 mm), or increasing concentrations of desferrioxamine (0.03 to 3 mm) or catalase (0.03 to 3 be mediated by ROS [2]. Furthermore, the proximal U/ml). Cellular injury and death were determined using the tubule (PT) appears to be particularly susceptible to MTT and lactate dehydrogenase (LDH) assays, respectively. reperfusion-injury [1, 2]. H2O2-mediated PARS activation in rat PT cells and the effects ROS produce cellular injury and necrosis via several of PARS inhibitors on PARS activity were determined by mechanisms including peroxidation of membrane lipids, measurement of the incorporation of [H]NAD into nuclear proteins. protein denaturation and DNA damage [3]. Evidence obResults. Incubation of rat PT cells with H2O2 significantly tained using cultured cells demonstrate that ROS produce inhibited mitochondrial respiration and increased LDH restrand breaks in DNA, which triggers energy-consuming lease, respectively. Both desferrioxamine and catalase reduced DNA repair mechanisms and activates the nuclear enH2O2-mediated cellular injury and death. All three PARS inhibitors significantly attenuated the H2O2-mediated decrease zyme poly (ADP-ribose) synthetase (PARS) [EC 2.4.2.30, in mitochondrial respiration and the increase in LDH release. also referred to as poly (ADP-ribose) polymerase Incubation with H2O2 produced a significant increase in PARS (PARP) or poly (ADP-ribose) transferase (pADPRT)] activity that was significantly reduced by all PARS inhibitors. [4]. PARS is an abundant, chromatin-bound enzyme con3-Aminobenzoic acid (3 mm) and nicotinic acid (3 mm), strucstitutively expressed in numerous cell types [5], which tural analogs of 3-AB and Nic, respectively, which did not inhibit PARS activity, did not reduce the H2O2-mediated injury when activated catalyzes the transfer of ADP-ribose and necrosis in cultures of rat PT cells. moieties from NAD to nuclear proteins including hisConclusion. We propose that PARS activation contributes tones and onto PARS itself (automodification) with the to ROS-mediated injury of rat PT cells and, therefore, to the concomitant formation of nicotinamide [6]. However, cellular injury and cell death associated with conditions of oxidant stress in the kidney. there is now good evidence that exposure of cells to oxidant stress in the form of superoxide anions, hydrogen peroxide (H2O2) and hydroxyl radicals causes strand