Assay development of inducible human renal phosphate transporter Npt2A (SLC34A1) in Flp-In-Trex-HEK293 cells.
Hyperphosphatemia is associated with severe decline of renal function in chronic kidney disease and elevates cardiovascular mortality. Type II sodium dependent phosphate transporter 2A (Npt2A) plays a major role in renal phosphate reabsorption and could be explored as a target for anti-hyperphosphatemia therapy. Human Npt2A transporter activity was examined upon transfection into CHO, MDCK, HEK293, Flp-In-CHO and Flp-In-HEK293 cells. Only kidney-derived cells expressed functional Npt2A. HEK293 and Flp-In-HEK293 cell lines stably transfected with hNpt2A could be selected, but these cells were inactive in phosphate transport. This suggests that high-level, constitutive Npt2A expression has deleterious effects on the cell. By using the conditional promoter in the Flp-In-Trex vector, functional expression of Npt2A was achieved by doxycycline induction in HEK293 cells. The EGFP tagged and non-tagged, inducible stable hNpt2A-HEK293 cell lines afforded development of a robust phosphate uptake assay mediated by hNpt2A, which can be used to screen hNpt2A inhibitors and inducers of hNpt2A expression. Using this assay, the small molecule LC-1 was identified as a potent inhibitor of hNpt2A, suggesting that it is feasible to develop potent specific hNpt2A inhibitors to control phosphate overloading for hyperphosphatemia therapy.
Biology Department, Sundia MediTech Company, Ltd., Shanghai 201203, China; Metabolic Disease Franchise, ChemPartern Research Company, Ltd., Shanghai, China. Electronic address: Thomaswhz@hotmail.com.
Hongzhong
H
Wu
Chonghong
C
Mao
Georg
G
Duenstl
Wan
W
Su
Su
S
Qian
D001681:Biological Assay / Q000379:methods*; D001692:Biological Transport / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D004318:Doxycycline / Q000494:pharmacology; D004353:Drug Evaluation, Preclinical; D057809:HEK293 Cells; D006801:Humans; D010710:Phosphates / Q000378:metabolism; D012717:Sesquiterpenes / Q000494:pharmacology; D012964:Sodium / Q000494:pharmacology; D050613:Sodium-Phosphate Cotransporter Proteins, Type IIa / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D013997:Time Factors; D014162:Transfection
C524858:LC-1 compound; D010710:Phosphates; C493554:SLC34A1 protein, human; D012717:Sesquiterpenes; D050613:Sodium-Phosphate Cotransporter Proteins, Type IIa; D012964:Sodium; D004318:Doxycycline
Assay development; Hyperphosphatemia; Npt2A; Phosphate uptake; SLC34A1; Specific inhibitor