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Absence of endonuclease activation during acute cell death in renal proximal tubules
R G Schnellmann1, A R Swagler, M M Compton
1Department of Physiology and Pharmacology, College of Veterinary Medicine, University of Georgia, Athens 30602-7389.
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.
Insights
Endonuclease and poly(ADP-ribose) polymerase activation do not contribute to acute renal proximal tubule cell death. These enzymes were not involved in cell injury models involving mitochondrial dysfunction or oxidative stress.
Area of Science:
- Nephrology
- Cell Biology
- Biochemistry
Background:
- Acute kidney injury involves complex cellular mechanisms.
- Endonuclease and poly(ADP-ribose) polymerase (PARP) activation are implicated in cell death pathways.
- Their specific roles in renal proximal tubule injury require elucidation.
Purpose of the Study:
- To investigate the involvement of endonuclease and PARP activation in rabbit renal proximal tubule cell injury and death.
- To determine if these enzymes contribute to cell death induced by mitochondrial inhibitors, ionophores, or oxidants.
Main Methods:
- Rabbit renal proximal tubule suspensions were exposed to antimycin A, FCCP, ionomycin, or t-butyl hydroperoxide (TBHP).
- Cell death was assessed by lactate dehydrogenase (LDH) release.
- Endonuclease activation was evaluated by genomic DNA fragmentation.
- Inhibitors of endonuclease (aurintricarboxylic acid) and PARP (3-aminobenzamide) were used.
Main Results:
- Neither aurintricarboxylic acid nor 3-aminobenzamide affected LDH release induced by the tested agents.
- No DNA fragmentation was observed prior to or after cell death.
- Positive control with digitonin confirmed the presence of functional endonuclease in proximal tubules.
Conclusions:
- Endonuclease activation does not precede or follow cell death in this model.
- Endonuclease and PARP activation do not play a significant role in acute renal proximal tubule cell injury and death induced by oxidative stress, mitochondrial dysfunction, or increased cytosolic calcium.