Absence of endonuclease activation during acute cell death in renal proximal tubules.

Abstract

The role of endonuclease and poly(ADP-ribose) polymerase activation in various types of cell injuries and death to rabbit renal proximal tubule suspensions was examined. Proximal tubules were exposed to the mitochondrial inhibitor antimycin A (0.1 microM), the protonophore carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP, 1 microM), the calcium ionophore ionomycin (5 microM), or the oxidant t-butyl hydroperoxide (TBHP, 0.5 mM) in the absence or presence of the endonuclease inhibitor aurintricarboxylic acid or the poly(ADP-ribose) polymerase inhibitor 3-aminobenzamide. Lactate dehydrogenase (LDH) release was used as a marker of cell death and analysis of genomic DNA for internucleosomal cleavage was used as a marker of endonuclease activation. Aurintricarboxylic acid and 3-aminobenzamide had no effect on the proximal tubule LDH release produced by 1 h exposure to antimycin A, FCCP, or ionomycin, or 2 h exposure to TBHP. Furthermore, there was no evidence of DNA fragmentation with any compound prior to or after cell death began. As a positive control, proximal tubules exposed to digitonin in the absence of metabolic substrates resulted in the chelator-inhibitable fragmentation of DNA, indicating that the endonuclease is present in proximal tubules. These results show that endonuclease activation did not occur prior to or after cell death began. Furthermore, these results suggest that endonuclease and poly(ADP-ribose) polymerase activation do not play a role in this model of acute renal proximal tubule cell injury and death induced by agents that cause oxidative stress, mitochondrial dysfunction, or increases in cytosolic free calcium.