Identification and expression analysis of type II and type III Pi transporters in the opossum kidney cell line.

The apical membrane of renal proximal tubular epithelial cells is the main controller of phosphate homeostasis, because it determines the rate of urinary Pi excretion. The opossum kidney (OK) cell line is a good model for studying this function, but only NaPiIIa (NaPi4) has been identified to date as a Pi transporter in this cell line. In this work, we have identified three additional Pi transporters that are present in OK cells: NaPiIIc, PiT1 and PiT2. All three sequences are similar to the corresponding orthologues, but PiT1 is missing the first transmembrane domain. Confluent cells exhibit characteristics of type II Pi transport, which increases with alkalinity and is inhibited by phosphonoformic acid (PFA), and they mainly express NaPiIIa and NaPiIIc, with a low abundance of PiT1 and PiT2. Proliferating cells show a higher expression of PiT1 and PiT2 and a low expression of NaPiIIa and NaPiIIc. Adaptation to a low Pi concentration for 24 h induces the expression of RNA from NaPiIIa and NaPiIIc, which is not prevented by actinomycin D. Small interfering RNA transfections revealed that PiT1 is not necessary for Pi transport, but it is necessary for adaptation to a low Pi , similar to NaPiIIa and PiT2. Our study reveals the complexity of the coordination between the four Pi transporters, the variability of RNA expression according to confluence and the heterogeneous correlation between Pi transport and RNA levels.