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Abstract
Ischaemia/reperfusion (IR) is the commonest cause of acute kidney injury which is a major cause of morbidity and an important contributing factor to mortality in patients with a wide range of medical and surgical conditions. IR often occurs in an unpredictable manner but in certain situations, most notably organ transplantation its occurrence can be pre-empted thus allowing consideration of preventative therapies.
IR injury is an inflammatory process involving complex interactions between resident and infiltrating cells of both innate and cognate arms of the immune system. The immediate sequelae of renal IR are necrosis of tubular epithelial cells associated with an acute decline in renal function. Chronic kidney injury may follow and is preceded by endothelial cell injury and loss of peritubular capillaries. The mechanisms linking acute and chronic injury, or more specifically acute inflammatory tubular damage and long-term endothelial cell injury, are not well understood.
The tissue factor (TF) initiated coagulation pathway has been shown to contribute to renal IR injury. Signalling by TF and the coagulation protease thrombin leads to chemokine upregulation, neutrophil infiltration and kidney injury after IR. These effects are primarily dependent on activation of the thrombin receptor protease activated receptor (PAR)-1. TF and thrombin may promote survival and growth of endothelial cells; therefore it is possible that inhibition of TF or PAR-1 signalling could have deleterious effects on endothelial cells after IR.
The primary aims of the studies described in this thesis were two-fold:-
1. To characterise the expression of factors contributing to endothelial cell survival or apoptosis after renal IR and determine whether the TF-thrombin-PAR-1 system is involved in their regulation.
2. To determine whether signalling by the cytoplasmic domain of TF, which does not promote thrombin production but may signal directly, is involved in renal IR injury.
Results
A murine model of 25 min of warm renal ischaemia was used to explore the regulation of angiogenic factors. Differential regulation of the three predominant isoforms of vascular endothelial growth factor (VEGF) was detected in kidneys during the first 24 h after ischaemia. The expression of VEGF164 and 188 was suppressed after IR. Despite this down-regulation of the main isoforms, the level of VEGF protein in kidneys was not altered by IR. Circulating levels of VEGF were increased suggesting that VEGF was generated at extra-renal sites after IR. Angiopoietin-1 (Ang-1) was also down-regulated in kidneys after IR. The expression of VEGF receptors, angiopoietin-2 and angiopoietin receptor were not significantly altered after IR. Thus, angiogenic factors were down-regulated in kidneys after IR, potentially predisposing endothelial cells to apoptosis. Thrombin inhibition did not significantly alter this pattern of expression. However, thrombin inhibition or PAR-1 deletion prevented up-regulation of the anti-angiogenic factor thrombospondin-1 and the pro-apoptotic caspase-3, suggesting that PAR-1 signalling could potentially contribute to endothelial cell injury after IR.
Mice with deletion of the cytoplasmic domain of TF generated on a mixed genetic background (TFCT/CT) developed more severe renal impairment and tubular injury after IR than wild type strain control mice. The expression of the chemokine KC was greater in TFCT/CT mice than wild-type and this was associated with a higher number of infiltrating neutrophils in kidneys. PAR-1 was up-regulated to a greater degree in TFCT/CT mice and pharmacologic inhibition of PAR-1 abrogated their more severe injury, suggesting that the cytoplasmic domain of TF may inhibit PAR-1 signalling. PAR-2 deletion did not alter renal IR injury. The effect of the TF cytoplasmic domain on renal injury appeared to be strain dependent, because the renal impairment of fully backcrossed mice with TF cytoplasmic domain deletion was not different to wild-type mice after IR.