Osmolyte regulation by TonEBP/NFAT5 during anoxia-recovery and dehydration-rehydration stresses in the freeze-tolerant wood frog (

backgroundThe wood frog,

methodsWood frogs were subjected to 24 h anoxia-4 h recovery and 40% dehydration-full rehydration experiments. Protein levels of NFAT5, BGT-1, SMIT, and aldose reductase were studied using immunoblotting in muscle, liver, and kidney tissues.

resultsImmunoblotting results demonstrated downregulations in NFAT5 protein levels in both liver and kidney tissues during anoxia (decreases by 41% and 44% relative to control for liver and kidney, respectively). Aldose reductase protein levels also decreased in both muscle and kidney tissues during anoxia (by 37% and 30% for muscle and kidney, respectively). On the other hand, BGT-1 levels increased during anoxia in muscle (0.9-fold compared to control) and kidney (1.1-fold). Under 40% dehydration, NFAT5 levels decreased in liver by 53%. Aldose reductase levels also decreased by 42% in dehydrated muscle, and by 35% in dehydrated liver. In contrast, BGT-1 levels increased by 1.4-fold in dehydrated liver. SMIT levels also increased in both dehydrated muscle and liver (both by 0.8-fold). DISCUSSION: Overall, we observed that osmoregulation through an NFAT5-mediated pathway is both tissue- and stress-specific. In both anoxia and dehydration, there appears to be a general reduction in NFAT5 levels resulting in decreased aldose reductase levels, however BGT-1 and SMIT levels still increase in certain tissues. Therefore, the regulation of osmoregulatory genes during dehydration and anoxia occurs beyond the transcriptional level, and it possibly involves RNA processing as well. These novel findings on the osmoregulatory mechanisms utilized by the wood frog advances our knowledge of osmoregulation during anoxia and dehydration. In addition, these findings highlight the importance of using this model to study molecular adaptations during stress.