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

New findings: 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.

Abstract: 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.