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 (NFκB), Activator Protein 1 (AP1) and the Heat Shock Factor I (HSF1) as well as the mitogen activated protein kinases (MAPK), p38, JNK and ERK 1 /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 37°C after 30 minutes or 12 hours of hypothermia. NFκB was activated within minutes of a hypothermic insult, correlating with the phosphorylation of the p38 and ERK 1 /2 MAPKs (p<0.01). Inhibition of p38 had no effect on NFκB translocation, but inhibition of ERK 1 /2 prevented subsequent NFκB activation (p<0.01). In contrast AP1 was not significantly up-regulated until 12 hours of hypothermia and HSF1 was down regulated during hypothermia. The downstream effects of NFκB activation were investigated by measuring the production of the inflammatory cytokines IL-6, IL-8 and TNFα. All three cytokines were up-regulated during hypothermia and reperfusion and the inhibition of NFκB 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 hypothermia. HUVECS did not release von willebrand factor (VWF), a marker of endothelial dysfunction during hypothermia, however, cells did produce lactate after prolonged storage indicating a degree of hypoxia developed during hypothermic stress. To extrapolate the in vitro work to a clinical setting 17 preservation solutions were collected from cadaveric donor kidneys. Levels of lactate, VWF, IL-6, IL-8, TNFα and the anti-inflammatory IL-4 were measured. A large range in values was found for all markers in the 17 preservation solutions, however, no correlation was found between increasing cold or warm ischaemic times, early graft function or rejection with the markers chosen. IL-6 was significantly increased in donor kidneys that had experienced greater than 30 minutes warm ischaemia (p<0.05). The response of a donor kidney may be regulated by polymorphisms which the graft cells contain. Microsatellite polymorphisms in the NFκB and TNFα genes and restriction enzyme sites in the IL-6 and NAD(P)H genes were investigated in 50 donor DNA samples. No correlation was found with rejection episodes with any of these polymorphisms. However, in the 17 preservation solutions, donors with the IL-6 allele which is linked to high protein production had higher IL-6 levels than donors with the low production allele. The role of IL-6 in transplantation remains unclear. However, this study has demonstrated that IL-6 may be a useful marker of stress. The ability to block cytokines by inhibiting transcription factors such as NFκB may have a therapeutic potential in ischaemic injury.