Effect of osmotic stress on expression of a putative facilitative urea transporter in the kidney and urinary bladder of the marine toad, Bufo marinus

Anuran amphibians accumulate a large amount of urea in their extracellular fluids to avoid a severe dehydration under dry and hyper-saline environments. To clarify the mechanisms of urea retention, we examined structure and distribution of the urea transporter (UT) in the kidney of the marine toad (Bufo marinus), and its expression in the kidney and urinary bladder following exposure to dry and hyper-saline conditions by means of cDNA cloning, semiquantitative RT-PCR, immunoblot analysis and immunohistochemistry. The Bufo UT cDNA cloned from the kidney encodes a 390-amino-acid residue protein, which is 80% identical to Rana esculenta UT with the functional characteristics of a urea transporter. The Bufo UT mRNA was abundantly expressed in the kidney and urinary bladder, but not in the skin. In immunoblot analysis using a specific antibody raised against the Bufo UT, a 52 kDa protein similar to the glycosylated forms of mammalian UT-A2 (∼55 kDa) was detected in extracts from plasma membrane fractions of the kidney and urinary bladder. When toads were acclimated to dry and hyper-saline environments for 7 days, UT mRNA expression was upregulated in the kidney and urinary bladder and there was an elevated plasma urea concentration and osmolality. Immunohistochemistry showed that the UT was specifically localized on the apical membrane of the early distal tubule, known to be the diluting segment, in the kidney and the epithelial cells of urinary bladder. Immunoreactive cells were not detected along the late distal tubule, the connecting tubule or the collecting duct in the kidney. The present findings suggest that the Bufo UT probably contributes to urea transport in the kidney and urinary bladder in response to hyperosmotic stresses such as body fluid hypertonicity and dehydration.