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Role of Increased Autophagic Flux in Renal Tubular Epithelial Cells Undergoing Warm Reperfusion (WR) after Cold Ischemia (CI), The

A. Jani, S. Jain, T. Nydam, C. Edelstein

UC Denver, Aurora, CO

Meeting: 2013 American Transplant Congress

Abstract number: D1668

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Delayed graft function (DGF) is primarily caused by cold ischemia (CI) and warm reperfusion (WR). The mechanism by which CI/WR causes renal tubular epithelial cell (RTEC) death is not known. Autophagy is a cell-survival strategy used by cells during stresses such as ischemia and reperfusion. The relationship between apoptosis and autophagy during CI/WR of donor kidneys is unknown. We hypothesized that increased autophagic flux and apoptosis occur during CI/WR of RTE cells. Methods: RTECs (LLC-PK1) were subjected to CI in UW at 4°C for 24h. To simulate rewarming, UW was replaced with DMEM containing bovine serum at 37°C for 24h. LLCPK cells incubated at 37°C served as controls. Immunoblot and densitometry were used to assess active caspase-3 (17kDa), caspase-1 (45kDa) and LC3–II (14kDa), a marker of autopahgic flux. Autophagic flux is measured by comparing LC3-II protein expression with and without Bafilomycin, a lysosomal inhibitor, and wortmannin, a PI3 kinase inhibitor. Cells were incubated with Bafilomycin (150nM) and wortmanin (250nM) 1 hour before CI. Results: During CI, caspase-1 was not increased whereas apoptosis and active caspase 3 were significantly increased vs. controls (Table 1).

Treatment with Bafilomycin and wortmannin did not change LC-3 II protein expression during CI indicating that autophagic flux was not increased. In contrast, during WR, apoptosis and caspase-3 did not increase whereas caspase-1 and necrosis increased significantly. Treatment with Bafilomycin during WR resulted in significantly more LC-3II expression (indicating increased autophagic flux)and apoptosis but had no effect on necrosis or caspase-1. Conclusions: During CI, caspase-3 and apoptosis are increased but autophagic flux is not. Bafilomycin and wortmannin had no effect on apoptosis or necrosis during CI. In contrast, autophagic flux, caspase-1 and necrosis are increased during WR. Bafilomycin and wortmannin increased caspase-3 and apoptosis during WR, but had no effect on caspase-1 and necrosis. These results suggest autophagic flux protects from apoptosis during WR but does not prevent caspase-1 activation nor necrotic cell death. Autophagy inhibitors should be used with caution during donor kidney reperfusion.

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To cite this abstract in AMA style:

Jani A, Jain S, Nydam T, Edelstein C. Role of Increased Autophagic Flux in Renal Tubular Epithelial Cells Undergoing Warm Reperfusion (WR) after Cold Ischemia (CI), The [abstract]. Am J Transplant. 2013; 13 (suppl 5). https://atcmeetingabstracts.com/abstract/role-of-increased-autophagic-flux-in-renal-tubular-epithelial-cells-undergoing-warm-reperfusion-wr-after-cold-ischemia-ci-the/. Accessed April 18, 2021.

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