engleski

Caspase-3 activation in a porcine kidney model of donation after cardiac death (DCD).

The organ-donor shortage has led to greater use of DCD kidneys. These organs have increased delayed graft function (DGF). We developed a porcine model of DCD to identify potentially therapeutic targets in DGF. Methods: 30 kg Male Yorkshire pigs were subjected to right-ventricular overload or myocardial infarction. The supra-renal aorta (SRA) was clamped and the infra-renal IVC vented. The pigs were exsanguinated and 1L of UW solution at 4C was infused via the SRA. The perfused kidneys were removed and stored in cold UW at 4C for 0-72 hrs. A kidney biopsy was obtained at 0, 24, 48 and 72 hrs post-retrieval. Apoptotic tubular epithelial cells and brush border injury were quantitated by a renal pathologist. Caspase-3/7 activity was measured using flourescent substrate DEVD-AMC which detects both caspases. To determine the exact caspase involved, samples were immunoblotted with an antibody that detects active caspase-3 (17 kDa), caspase-7 (20kDa), spectrin breakdown products (145/150 kDa) and Bax (23 kDa). Results: Apoptosis increased significantly with increasing cold storage. Apoptotic cells/hpf (n=4) were 0.40.2 at 0 hrs, 2.50.5 at 24 hrs, 18.93.0 at 48 hrs (p < 0.0001 vs. 0 and 24 hrs) and 28.4 at 72 hrs. Brush border injury score was 4.50.3 at 0 hrs indicating 75-80% of tubules were injured. Caspase-3/7 activity increased significantly at 72 hrs (336.5 vs 98.2 nmol/min/mg, p < 0.05). At 24 hrs active caspase-3 expression was 25 % > 0 hrs and 50% > 0 hrs at 48-72 hrs (n = 8). Active caspase-7 expression was not seen at any time point but was detected in etoposide treated Jurkat cells. Caspase-3 mediated spectrin breakdown products and pro-apoptotic bax increased 50% at 24 hrs vs 0 hrs (n=3). Conclusion: We have established a porcine model of DCD that is characterised by extensive brush border injury, increasing apoptosis, and increased expression of caspase-3, spectrin breakdown products and bax (but not caspase-7) during static cold storage. Further study of the effect of caspase inhibiton or pulsatile perfusion on the injury and apoptosis is warranted.

Caspase-3; organ donation; cardiac death

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