Identification and Expression of a Putative Facilitative Urea Transporter in Three Species of True Frogs (Ranidae): Implications for Terrestrial Adaptation

Andrew J Rosendale,Richard E Lee,Andor J Kiss,Jon P Costanzo

doi:10.1155/2014/148276

Abstract

Urea transporters (UTs) help mediate the transmembrane movement of urea and therefore are likely important in amphibian osmoregulation. Although UTs contribute to urea reabsorption in anuran excretory organs, little is known about the protein’s distribution and functions in other tissues, and their importance in the evolutionary adaptation of amphibians to their environment remains unclear. To address these questions, we obtained a partial sequence of a putative UT and examined relative abundance of this protein in tissues of the wood frog (Rana sylvatica), leopard frog (R. pipiens), and mink frog (R. septentrionalis), closely related species that are adapted to different habitats. Using immunoblotting techniques, we found the protein to be abundant in the osmoregulatory organs but also present in visceral organs, suggesting that UTs play both osmoregulatory and nonosmoregulatory roles in amphibians. UT abundance seems to relate to the species’ habitat preference, as levels of the protein were higher in the terrestrialR. sylvatica, intermediate in the semiaquaticR. pipiens, and quite low in the aquaticR. septentrionalis. These findings suggest that, in amphibians, UTs are involved in various physiological processes, including solute and water dynamics, and that they have played a role in adaptation to the osmotic challenges of terrestrial environments.

Highlights

During periods of osmotic stress, amphibians accumulate various balancing osmolytes that help them maintain water balance
To elucidate the potential physiological roles of urea transporters (UTs) in anurans, we examined the tissue distribution and abundance of a putative UT isolated from the urinary bladder of three species of true frogs, Rana sylvatica, R. pipiens, and R. septentrionalis, which differ markedly in their hydric requirements and, susceptibility to osmotic stress
In a validation experiment [21], the optimal concentration of each primer set was determined over a 10x dilution series of cDNA, and we found that the amplification efficiencies for UT (92.3%) and GAPDH (92.9%) were similar among primers sets. Quantitative real-time PCR (qPCR) reactions were analyzed using an iCycler thermal cycler (BioRad)

Summary

Introduction

During periods of osmotic stress, amphibians accumulate various balancing osmolytes that help them maintain water balance. High levels of urea in the body fluids reduce water loss and improve ability to survive osmotic stresses [1, 2] while at the same time replacing ionic solutes that are more detrimental to the animal [3]. Urea accumulation occurs by increasing the synthesis of urea by altering urea-cycle activity and/or increasing the retention of urea [7]. The latter can be accomplished by decreasing urine production through a reduction in glomerular filtration rate and by reabsorbing urea from the filtrate in the kidneys and urinary bladder [8, 9]. Growing evidence suggests the involvement of transport proteins, facilitative urea transporters (UTs), in the reabsorption process [10,11,12], but the specific roles of these proteins require additional investigation

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Discussion

Conclusion