New Roles for Urea in Frog Hibernation



Osmoconformers adapt to osmotic stress by accumulating one or more “compatible solutes,” members of a few classes of organic osmolytes that are benign to cellular functions, even in high concentration. Urea, despite its destabilizing effects on macromolecular structure and function, is a major balancing osmolyte in species as diverse as snails, elasmobranchs, lungfishes, and desert frogs. Urea’s role as an osmolyte has been studied with respect to saline adaptation and estivation, but its importance in other ecological contexts is virtually unknown.

One economical means to acquiring organic osmolytes is to exploit metabolic end-products. Urea, an important end-product of nitrogen metabolism in many species, has diverse physiological functions such as maintaining acid-base balance, contributing to buoyancy (elasmobranchs), nitrogen transport (ruminant mammals), and urine concentration (mammalian kidney). In this project, we sought to discover additional roles for this “waste product” in terrestrially-overwintering amphibians. Much of this work was funded by the National Science Foundation (IOB-0416750).

Urea level rises in wood frogs during hibernation

Accumulation of urea in defense of body water is a universal amphibian response to osmotic challenge, such as occurs during salt acclimation, aestivation, and transient exposure to arid environments.  We tested the idea that amphibians also accumulate urea in terrestrial hibernation.  Our study of seasonal dynamics in hydro-osmotic balance of R. sylvatica showed that plasma urea was ~50 mM in autumn and early winter, when soil moisture was scarce, but only ~2 mM in late winter and spring, when moisture availability increased (Costanzo and Lee 2005). In the laboratory, hibernating R. sylvatica accumulated ~90 mM urea under relatively dry, warm conditions.

We investigated regulation of maximal capacity for urea production, as indicated by activity and expression of carbamoyl phosphate synthetase I (CPS I), the key regulatory enzyme of the ornithine-urea cycle (Schiller et al. 2008).  Hepatic CPS I activity was maintained in autumn and winter at levels found in active summer frogs, although it decreased in spring. In no experiment did variation in enzyme activity reflect corresponding changes in CPS I quantity, suggesting that regulation is effected primarily by post-translational modifications or feedback inhibition rather than transcription and translation.




Urea is a novel cryoprotectant

Several investigations provided unequivocal evidence of urea’s efficacy as a cryoprotectant in R. sylvatica.  Urea (40 or 80 mM) markedly reduced in vitro cryoinjury to erythrocytes and organs (Costanzo and Lee 2005).  In fact, urea provided as much protection as glycerol and in some experiments was superior to glucose, theretofore the only known cryoprotectant in R. sylvatica.  In another study, augmenting urea levels in isolated gastrocnemius muscles before freezing significantly improved post-thaw isometric contractile performance relative to untreated controls (Costanzo et al. 2008).  Notably, urea conferred cryoprotection to muscles of R. sylvatica, but not to muscles of R. pipiens, an aquatic hibernator that neither accumulates urea in winter nor tolerates freezing.  Finally, we tested urea’s cryoprotective efficacy at the whole-organism level by comparing freezing survival and post-thaw recovery rates of hyperuremic and control frogs (Costanzo and Lee 2008).  All urea-loaded frogs survived freezing at –4°C and quickly recovered neurobehavioral faculties after thawing.  Significantly fewer control frogs recovered, and those that did survive incurred more extensive damage, as evidenced by protracted recovery times and leakage of the intracellular proteins, LDH, CK, and hemoglobin. Although freezing-induced mobilization of low-molecular-mass carbohydrates (glucose in R. sylvatica) has long been touted the hallmark physiological adaptation in amphibian freeze tolerance, our research demonstrated that urea accumulated preparatory to hibernation also contributes to freezing survival, establishing this compound as a novel class of natural cryoprotectant.

Urea is a metabolic depressant

Another line of research examined the putative role of urea as a metabolic depressant in hibernating R. sylvatica, as some authors had earlier proposed that, by perturbing protein structure/function, urea might contribute to hypometabolism in animals that have few or no “counteracting solutes.”  Amphibians apparently do not co-accumulate methylamines with urea.  In winter-acclimatized R. sylvatica, experimental dehydration caused plasma osmolality to rise (chiefly due to urea accumulation) and resting to fall; metabolic rate was strongly inversely correlated with plasma urea levels over the range of 7-41 mM (Muir et al. 2007).  In a separate experiment, resting metabolism in hydrated frogs receiving injections of physiological saline, or saline containing urea, did not differ initially; however, during a course of experimental dehydration, metabolism decreased sooner in the urea-loaded frogs.

Probing deeper, we compared in vitro respiration rates of R. sylvatica organs tested in the presence or absence of urea (Muir et al. 2008).  Urea’s depressive effect on was substantial in some organs (~15% in liver; ~50% in skeletal muscle) but absent in others, suggesting that metabolic sensitivity to this agent varies among tissues.  In addition, the hypometabolic effect was modulated by temperature and seasonal acclimatization. 

Subsequent study showed that urea contributes to metabolic depression in taxonomically-diverse species that accumulate urea in hibernation or estivation (Muir et al., 2010). We measured rates of oxygen consumption of excised organ samples from dormant animals in the presence or absence of physiological concentrations of urea. Three of four urea-accumulating species representing the clades Amphibia (Spea bombifrons, Ambystoma tigrinum), Reptilia (Malaclemys terrapin), and Gastropoda (Anguispira alternata), had at least one organ whose metabolism was significantly decreased by urea treatment. However, metabolism in organs from the leopard frog (R. pipiens), a species that does not accumulate urea during dormancy, was unaffected by urea treatment. Our results support the hypothesis that urea accumulation can reduce metabolic rate of dormant animals and provide a base for further investigation into the evolution of urea-induced hypometabolism.