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

Abstract

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 Pi transport, but it is incomplete because only the NaPiIIa Pi transporter has been identified. What is the main finding and its importance?We have cloned and characterized the Pi transporters NaPiIIc, PiT1 and PiT2 from OK cells and have analysed the relevance of the four transporters to Pi transport. All four transporters are involved in the upregulated Pi transport of cells incubated using a low‐Pi 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 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.

PF-06869206 is a selective inhibitor of renal Pitransport: evidence from in vitro and in vivo studies

“…Of note, Na ϩ -independent transport accounts for 2-3% of P i transport in OK cells. This provides evidence, although indirectly, that the difference between PFA and Na ϩ -independent P i transport could be the contribution of PiT1, which has been described to contribute to P i transport in OK cells (27). PTH regulation of P i transport follows a temporal sequence (66).…”

“…So far, no specific Npt2a-I was available to determine this contribution. Of note, in addition to Npt2a, OK cells also express Npt2c, PiT1, and PiT2 (25,27). However, the exact contribution of each of these proteins for P i transport remains speculative.…”

“…In particular, OK cells have been extensively used to study P i transport (42). Expression analysis identified Npt2a and Npt2c at mRNA and protein levels (25,27). In addition, OK cells express functional PTH receptors and can respond to changes in P i content in the medium by modulating P i transport activity and Npt2a abundance (6,15).…”

Several phosphate transport processes are present in vascular smooth muscle cells

“…Furthermore, PiT1 and PiT2 show additional functions and subcellular organelle expression other than Pi transport and plasma membrane, respectively (5-7, 10, 26, 33). When PiT1 RNA is interfered, for example in the Opossum Kidney proximal tubular cell line, Pi transport is not affected, but when siRNA is directed to the much less abundant PiT2, the inhibition of expression dramatically reduces Pi uptake (17). In this case, PiT2 could have functions other than Pi transport, such as being a Pi sensor (6), a role that is compatible with the expression of an intracellular sensor of Pi (34) according to the expression location of these transporters (33).…”

“…The frequent simultaneous expression of PiT1 and PiT2 in the same cell constitutes an apparent redundancy, which led to the suspicion (and later proof) that these transporters have functions other than merely Pi transport, such as cell proliferation, apoptosis, signaling, etc. (5,10,17,26,29).…”