Iron chelation or anti-oxidants prevent renal cell damage in the rewarming phase after normoxic, but not hypoxic cold incubation

It has now been firmly established that, not only ischemia/reperfusion, but also cold itself causes damage during kidney transplantation. Iron chelators or anti-oxidants applied during the cold plus rewarming phase are able to prevent this damage. At present, it is unknown if these measures act only during the cold, or whether application during the rewarming phase also prevents damage. We aimed to study this after cold normoxic and hypoxic conditions. LLC-PK1 cells were incubated at 4 °C in Krebs–Henseleit buffer for 6 or 24 h, followed by 18 or 6 h rewarming, respectively. Cold preservation was performed under both normoxic (95% air/5% CO2) and hypoxic (95% N2/5% CO2) conditions. The iron chelator 2,2′-DPD (100 μM), anti-oxidants BHT (20 μM) or sibilinin (200 μM), and xanthine oxidase inhibitor allopurinol (100 μM) were added during either cold preservation plus rewarming, or rewarming alone. Cell damage was assessed by LDH release (n = 3–9). Addition of 2,2′-DPD and BHT during cold hypoxia plus rewarming did, but during rewarming alone did not prevent cell damage. When added during rewarming after 6 h cold normoxic incubation, BHT and 2,2′-DPD inhibited rewarming injury compared to control (p < 0.05). Allopurinol did not prevent cell damage in any experimental set-up. Our data show that application of iron chelators or anti-oxidants during the rewarming phase protects cells after normoxic but not hypoxic incubation. Allopurinol had no effect. Since kidneys are hypoxic during transplantation, measures aimed at preventing cold-induced and rewarming injury should be taken during the cold.