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

What is the central question of this study? The opossum kidney (OK) cell line is the main in vitro model of proximal tubular P_i transport, but it is incomplete because only the NaPiIIa P_i transporter has been identified. What is the main finding and its importance? We have cloned and characterized the P_i transporters NaPiIIc, PiT1 and PiT2 from OK cells and have analysed the relevance of the four transporters to P_i transport. All four transporters are involved in the upregulated P_i transport of cells incubated using a low-P_i medium, and only PiT1 is not involved in basal transport.

The apical membrane of renal proximal tubular epithelial cells is the main controller of phosphate homeostasis, because it determines the rate of urinary P_i 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 P_i transporter in this cell line. In this work, we have identified three additional P_i 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 P_i 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 P_i 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 P_i transport, but it is necessary for adaptation to a low P_i , similar to NaPiIIa and PiT2. Our study reveals the complexity of the coordination between the four P_i transporters, the variability of RNA expression according to confluence and the heterogeneous correlation between P_i transport and RNA levels.