20-HETE-mediated cytotoxicity and apoptosis in ischemic kidney epithelial cells.
20-HETE, a metabolite of arachidonic acid, has been implicated as a mediator of free radical formation and tissue death following ischemia-reperfusion (IR) injury in the brain and heart. The present study examined the role of this pathway in a simulated IR renal injury model in vitro. Modified self-inactivating lentiviral vectors were generated to stably overexpress murine Cyp4a12 following transduction into LLC-PK(1) cells (LLC-Cyp4a12). We compared the survival of control and transduced LLC-PK(1) cells following 4 h of ATP depletion and 2 h of recovery in serum-free medium. ATP depletion-recovery of LLC-Cyp4a12 cells resulted in a significantly higher LDH release (P < 0.05) compared with LLC-enhanced green fluorescent protein (EGFP) cells. Treatment with the SOD mimetic MnTMPyP (100 microM) resulted in decreased cytotoxicity in LLC-Cyp4a12 cells. The selective 20-HETE inhibitor HET-0016 (10 microM) also inhibited cytotoxicity significantly (P < 0.05) in LLC-Cyp4a12 cells. Dihydroethidium fluorescence showed that superoxide levels were increased to the same degree in LLC-EGFP and LLC-Cyp4a12 cells after ATP depletion-recovery compared with control cells and that this increase was inhibited by MnTMPyP. There was a significant increase (P < 0.05) of caspase-3 cleavage, an effector protease of the apoptotic pathway, in the LLC-Cyp4a12 vs. LLC-EGFP cells (P < 0.05). This was abolished in the presence of HET-0016 (P < 0.05) or MnTMPyP (P < 0.01). These results demonstrate that 20-HETE overexpression can significantly exacerbate the cellular damage that is associated with renal IR injury and that the programmed cell death is mediated by activation of caspase-3 and is partially dependent on enhanced CYP4A generation of free radicals.
Nilakantan
Vani
V
Department of Transplant Surgery, Medical College of Wisconsin, Milwaukee, WI 53226, USA. vnilakan@mcw.edu
Maenpaa
Cheryl
C
Jia
Guangfu
G
Roman
Richard J
RJ
Park
Frank
F
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D053148:Caspase 3 / Q000378:metabolism; D002460:Cell Line; D042926:Cytochrome P-450 CYP4A / Q000235:genetics / Q000378:metabolism*; D004847:Epithelial Cells / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors; D006893:Hydroxyeicosatetraenoic Acids / Q000378:metabolism*; D007527:Isoenzymes / Q000378:metabolism; D007687:Kidney Tubules, Proximal / Q000378:metabolism / Q000502:physiology*; D016086:Lentivirus; D051379:Mice; D015427:Reperfusion Injury / Q000378:metabolism*; D013481:Superoxides / Q000378:metabolism; D013552:Swine; D014161:Transduction, Genetic; D018116:Vesiculovirus
D006893:Hydroxyeicosatetraenoic Acids; D007527:Isoenzymes; D013481:Superoxides; C055987:20-hydroxy-5,8,11,14-eicosatetraenoic acid; D000255:Adenosine Triphosphate; D042926:Cytochrome P-450 CYP4A; D053148:Caspase 3