Donor kidney cold (4°C) ischemia (CI) of >30 hrs is associated with increased apoptosis of renal tubular epithelial cells (RTEC) and is an important cause of DGF. The 13-lined ground squirrel (GS) is a mammal that undergoes winter hibernation, when its core body temperature falls to 4°C for up to 18 days. We have previously shown that GS kidneys are protected from apoptosis and caspase-3 activation after several days of CI. We sought to determine the mechanism of protection from apoptosis. Methods: Kidneys of C57BL6 mice and hibernating GSs were exposed to CI in UW solution for 72 hrs. Apoptotic RTECs were scored by a pathologist. Immunoblots were performed for caspase-3, XIAP (an inhibitor of caspase-3 & apoptosis) & phosphoAKT (pAKT) which converts BAD to pro-survival factor phosphoBAD (pBAD). Results: RTEC apoptosis was significantly increased in mouse vs. GS whole kidneys subjected to ex vivo CI. Anti-apoptotic XIAP, pAKT and pBAD were significantly increased in hibernating GS kidneys, but were undetectable in mouse kidneys (table 1).

To determine the mechanism of resistance to apoptosis, RTECs were isolated from GS and mouse kidneys, and treated with cisplatin (10 & 50ΜM), an agent known to cause severe apoptosis. GS RTEC subjected to cisplatin had significantly less apoptosis (Fig 1A), no active caspase-3 (Fig 1B), and a 50 % increase in XIAP, pAkt and pBAD protein vs. mouse (M1) RTEC.

To demonstrate that XIAP and pAKT are required for protection against apoptosis, GS RTEC were treated with shRNA to reduce XIAP and pAKT expression. Treated cells had significantly increased apoptosis and caspase-3(Figs. 1C & D). Conclusion: We have shown for the first time that protection of GS RTEC from apoptotic stimuli such as prolonged CI of several days duration and cisplatin exposure is mediated by upregulation of XIAP and pBAD. Application of these findings to human donor preservation may help prevent RTEC apoptosis and DGF.

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