Biochemical measurements of calcium, phosphate, PTH, and FGF-23 (A–E), and NaPi2a immunohistochemistry in various mouse mutants (F). Serum calcium (A) levels in control (n = 15), Fgf-23−/− (n = 6), Fgf-23−/−/1α(OH)ase−/− (n = 8), and 1α(OH)ase−/− (n = 11) animals; serum PTH (B) levels in control (n = 8), Fgf-23−/− (n = 4), Fgf-23−/−/1α(OH)ase−/− (n = 3), and 1α(OH)ase−/− (n = 9) animals; serum phosphate (C) levels in control (n = 22), Fgf-23−/− (n = 11), Fgf-23−/−/1α(OH)ase−/− (n = 7), 1α(OH)ase−/− (n = 14), Fgf-23−/−/NaPi2a−/− (n = 3), and NaPi2a−/− (n = 3) animals; urinary phosphate (D) control (n = 9), Fgf-23−/− (n = 5), Fgf-23−/−/1α(OH)ase−/− (n = 5), and 1α(OH)ase−/− (n = 9) animals; and serum Fgf-23 (E) levels in control (n = 8), 1α(OH)ase−/− (n = 5), Hyp (light blue, n = 1), and Coll I-FGF23 (red, n = 2) animals were measured in 5- to 12-week-old mice. Statistical significance: **P < 0.01, ***P < 0.001, ****P < 0.0001. Immunostaining of NaPi2a in the kidney sections prepared from wild-type, Fgf-23−/−, Fgf-23−/−/1α(OH)ase−/−, and 1α(OH)ase−/− animals using a polyclonal antibody (F). Please note that in contrast to the increased expression of NaPi2a in Fgf-23−/− mice, there is significantly less expression of NaPi2a protein in the Fgf-23−/−/1α(OH)ase−/− mice, similar to the expression in mice that lack the 1α(OH)ase gene [1α(OH)ase−/−].