Modulating Pi, PTH, and FGF23 on Phosphatase

To study the effects of varying Pi concentrations on phosphatase expression, it was essential to control Pi concentration in the basal mineralizing medium. This ruled out the use of β-glycerophosphate (βGP) as the availability of Pi from βGP requires the action of TNAP (Huesa et al. 2015 (link)) which can itself be modulated by CKD-associated endocrine factors such as Pi, PTH, and FGF23 (Shalhoub et al. 2011 (link), Rendenbach et al. 2014 (link), Houston et al. 2016 (link)). Therefore, upon confluence (day 0), mineralization was induced by supplementing the growth medium (basal concentration: 1.8 mM Ca; 1 mM Pi) with 50 μg/mL l-ascorbic acid (AA) and 1.5 mM CaCl₂ to provide a final medium containing 3.3 mM Ca (Houston et al. 2016 (link)). Cultures were also supplemented with a range of Pi (1–5 mM), PTH (0–50 nM), and FGF23 (0–200 ng/mL) with or without klotho (50 ng/mL) (R&D Systems). Cells were maintained in a 5% CO₂ atmosphere at 37°C and mineralization media was changed every second/third day for 28 days.