The Role of Cellular Stress during Cold Ischaemic and Reperfusion Injury

The elemental physiology of the highly complex and regulated cellular response to stress remains poorly understood. Hypothermia and reperfusion are necessary and unavoidable stresses associated with the procurement, storage and transplantation of organs such as the kidney. Signal transduction pathways and transcription factors are evolutionarily conserved mediators of stress responses. This project has investigated the activation of the transcription factors Nuclear Factor kappa B (NFKB), Activator Protein I (API) and the Heat Shock Factor I (HSFI) as well as the mitogen activated protein kinases (MAPK), p38, JNK and ERK I /2, during hypothermic and reperfusion stress in cultured endothelial cells (HUVECS) as a model of kidney graft endothelial cells. HUVECS were subjected to 72 hours of hypothermia at 4°C in a renal preservation solution. For reperfusion experiments cells were returned to 3 flc after 30 minutes or 12 hours of hypothermia. NFKB was activated within minutes of a hypothermic insult, correlating with the phosphorylation ofthe p38 and ERK 1/2 MAPKs (p<O.Ol).lnhibition ofp38 had no effect on NFKB translocation. but inhibition ofERK I /2 prevented subsequent NFKB activation (p<O.Ol). In contrast API was not significantly up-regulated until 12 hours of hypothermia and HSF I was down regulated during hypothermia. The downstream effects of NFKB activation were investigated by measuring the production of the inflammatory cytokines IL-6, IL-8 and TNFa. All three cytokines were up-regulated during hypothermia and reperfusion and the inhibition of NFKB with a decoy oligonucleotide reduced the expression of these cytokines. HUVECS were not killed by hypothermia with greater than 95% cell viability for 48 hours. Similarly DNA fragmentation. an event that occurs during apoptosis was not seen during hypothermic or reperfusion stress in HUVECS. There was a consistent expression of the mitochondrial anti-apoptosis protein BCL-2 during