Abstract
The aim of this study was to elucidate mechanisms of Pi handling in toads (Bufo bufo). We introduced toads to experimental solutions of various [Pi] and high Pi diets and measured urine and lymph [Pi]. Both lymph and urine [Pi] increased with increasing Pi loads, indicating Pi absorption across skin and intestine. An initial fragment of a NaPi-II type transporter was amplified from kidney, and the full-length sequence was obtained. The protein showed the molecular hallmarks of NaPi-IIb transporters. When expressed in Xenopus oocytes the clone showed unusual pH dependence, but apparent affinity constants for Pi and Na+ were in the range of other NaPi-II transporters. Expression profiling showed that the transporter was present in skin, intestine and kidney. Reverse transcription–polymerase chain reaction assays on dissected renal tubules indicated expression in the collecting duct system. Collecting tubules and ducts were isolated, perfused and microelectrode recordings showed electrogenic Pi transport in apical and basolateral membranes. Taken together, our results show that Pi is handled by intestine, kidney and skin. The presently cloned NaPi-IIb is a likely candidate involved in Pi absorption across these epithelia. In addition, electrophysiological experiments suggest that the collecting duct system plays an important role in Pi homeostasis.
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Acknowledgement
We thank Mai-Britt Andersen, Sofie Nehammer and Rikke Thorsteinsson, who were undergraduate students in our laboratory and performed spectrophotometrical Pi determinations. Dr. Jan Amstrup is warmly thanked for his help with RNA isolations and primer constructions and Dr. Aslak Jørgensen for providing DNA sequence facilities at the DBL–Institute for Health Research and Development, Charlottenlund, Denmark. We would also like to express our thanks to Dr. Berit Kristensen for introduction to Pi determination methods, Prof. Dr. Erik Hviid Larsen for helpful discussions and Ms. Alice Scheuer for taking care of the toads. Ms. Anni Olsen, Ms. Birthe Petersen, Mr. Thomas Sørensen and Mr. Arne Nielsen are kindly thanked for technical assistance.
The project was supported by the Carlsberg Foundation, the Danish Natural Science Research Council and Wellcome Trust (UK).
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Møbjerg, N., Werner, A., Hansen, S.M. et al. Physiological and molecular mechanisms of inorganic phosphate handling in the toad Bufo bufo . Pflugers Arch - Eur J Physiol 454, 101–113 (2007). https://doi.org/10.1007/s00424-006-0176-0
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DOI: https://doi.org/10.1007/s00424-006-0176-0