Phosphate transport by proximal renal tubuies is regulated by the function and the apical membrane expression of sodium-phosphate cotransporter proteins. The purpose of this study was to determine if PTH decreased expression of NaPi-4 in OK cells, a model for proximal renal tubules. Polyclonal antibodies were raised in rabbits against the C-terminal 12 amino acid sequence of NaPi-4. Immunoblots of crude OK cell membrane preparations using these antisera identified two bands, 60-65 kDa, corresponding closely to the predicted molecular weight of NaPi-4. OK cells grown in medium with or without PTH~7 for 24 h were fixed, incubated in NaPi-4 peptlde antisera, count erst ai ned with a rhodamine-conjugated secondary antibody, and visualized by laser confocal microscopy. Three-dimensional reconstruction of the fluorescence showed apical distribution of antibody binding which was markedly reduced in PTH-treated cells. A time course study of the effect of PTH on fluorescence showed a progressive decrease in the fluorescence in cells treated with PTH for up to 2 h. Thus, the antisera recognize a protein of the size and cellular distribution characteristic of NaPi-4. PTH reduces antibody binding with a time course paralleling the effect of PTH on phosphate uptake. We conclude that PTH inhibits phosphate uptake by decreasing the membrane expression of NaPi-4 in OK cells.
|Original language||English (US)|
|State||Published - Dec 1 1996|
ASJC Scopus subject areas
- Molecular Biology