Physiological and molecular mechanisms of inorganic phosphate handling in the toad Bufo bufo

Abstract

The aim of this study was to elucidate mechanisms of P(i) handling in toads (Bufo bufo). We introduced toads to experimental solutions of various [P(i)] and high P(i) diets and measured urine and lymph [P(i)]. Both lymph and urine [P(i)] increased with increasing P(i) loads, indicating P(i) 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 P(i) 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 P(i) transport in apical and basolateral membranes. Taken together, our results show that P(i) is handled by intestine, kidney and skin. The presently cloned NaPi-IIb is a likely candidate involved in P(i) absorption across these epithelia. In addition, electrophysiological experiments suggest that the collecting duct system plays an important role in P(i) homeostasis.