Serum levels of phosphate (Pi) are kept within a constant range by regulated intestinal absorption, bone resorption/deposition, and renal reabsorption of Pi. These three processes are controlled e.g. by PTH, 1,25-dihydroxy-vitamin D, and FGF-23. In addition, various observations indicate that extracellular Pi itself might control above mentioned processes as well. The aim of this study is to identify a postulated Pi-sensor and to study its signaling mechanism in renal cells. For that purpose, we, initially, have used a proximal tubular cell line (OK cells)

OK cells were kept in a 0.1 mM Pi medium and then were exposed to 1 mM (up to 16 hours) and Na/Pi-cotransport activity and surface expression of NaPi-IIa were determined at several time points. Our results indicate a rapid (few hours) downregulation of Na/Pi-cotransport in response to high Pi. This down-regulation was not mimicked by phosphonoformic acid (PFA), a known inhibitor of Na/Pi-cotransporters of the Slc34 family. However, PFA did block the inhibitory effect of high Pi on Na/Pi cotranport. Several pathways (PKA, PKB, PKC and ERK) play a role in the PTH-induced down-regulation of Na/Pi cotransport. However, we found that these pathways are not involved in the response of OK cells to changes on extracellular Pi.

To further investigate the intracellular signaling pathways involved in the down-regulation of Na/Pi-cotransport by an increase of extracellular Pi, we next used a phospho-kinase array kit.. This analysis indicated that the phosphorylation of several kinases was reduced in OK cells treated with 1 mM Pi as compared with cells kept in low Pi media. However, we could not confirm this data by western blot analysis, mostly due to the limitation of available antibodies to recognize the protein from Opossum.

In preliminary (proof of principle) experiments, PTH mediated inhibition (down-regulation) of NaPi-IIa in single isolated perfused proximal tubules has been demonstrated. Thus, this in-vitro preparation might be suitable to investigate for a possible Pi sensing mechanism in intact proximal tubules.