Rana sylvatica, known as the wood frog, can survive extremely cold temperatures during winter by undergoing full-body freezing, where it tolerates freezing of 65-70% of its total body water. During freezing, cellular dehydration decreases damage to the cell by preventing ice crystallization. Challenged with many stresses, these animals are forced to develop physiological adaptations to osmoregulation and osmoprotection that are necessary to ensure their survival. The purpose of this study was to elucidate a potential mechanism by which the transcription factor, NFAT5, regulates the expression of three osmoregulatory proteins (aldose reductase, SMIT, and BGT-1). These three proteins control cellular concentrations of the organic osmolytes: betaine (BGT-1), myo-inositol (SMIT), and sorbitol (aldose reductase). We studied this mechanism during the freeze-thaw stress in R. sylvatica liver, kidney, and skeletal muscle. Protein expression of BGT-1, SMIT, aldose reductase, and NFAT5 were examined using immunoblotting. We identified that the NFAT5 pathway facilitated osmoregulation in a tissue-specific manner during freezing. In skeletal muscle, we demonstrated that NFAT5 upregulation in thawing led to increases in the protein levels of BGT-1. In liver, NFAT5 was upregulated during freezing, along with aldose reductase. Furthermore, neither of these patterns of expression were observed in kidney as none of these four proteins showed differential expression during freezing or thawing. Therefore, the NFAT5 osmoregulatory pathway appears to be tissue-specific. Our novel findings on a mechanism of osmoregulation in R. sylvatica highlight the importance of studying naturally stress-tolerant animals to identify novel pro-survival pathways.
Keywords: Cryoprotection; Freezing; Nuclear factor of activated T cells; Osmotic stress; Rana sylvatica; Thaw.
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