Seasonal variation and response to osmotic challenge in urea transporter expression in the dehydration- and freeze-tolerant wood frog, Rana sylvatica.

Urea accumulation is a universal response to osmotic challenge in anuran amphibians, and facilitative urea transporters (UTs) seem to play an important role in this process by acting in the osmoregulatory organs to mediate urea retention. Although UTs have been implicated in urea reabsorption in anurans, little is known about the physiological regulation of UT protein abundance. We examined seasonal variation in and effects of osmotic challenge on UT protein and mRNA levels in kidney and urinary bladder of the wood frog (Rana sylvatica), a terrestrial species that tolerates both dehydration and tissue freezing. Using immunoblotting techniques to measure relative UT abundance, we found that UT numbers varied seasonally, with a low abundance prevailing in the fall and winter, and higher levels occurring in the spring. Experimental dehydration of frogs increased UT protein abundance in the urinary bladder, whereas experimental urea loading decreased the abundance of UTs in kidney and bladder. Experimental freezing, whether or not followed by thawing, had no effect on UT numbers. UT mRNA levels, assessed using quantitative real-time polymerase chain reaction, did not change seasonally nor in response to any of our experimental treatments. These findings suggest that regulation of UTs depends on the nature and severity of the osmotic stress and apparently occurs posttranscriptionally in response to multiple physiological factors. Additionally, UTs seem to be regulated to meet the physiological need to accumulate urea, with UT numbers increasing to facilitate urea reabsorption and decreasing to prevent retention of excess urea.