Correction of proximal tubule phosphate transport defect in Hyp mice in vivo and in vitro with indomethacin

Abstract

X-linked hypophosphatemia is the most prevalent inherited form of rickets. In this disorder, rickets results from hyperphosphaturia and inappropriately normal levels of 1,25(OH)2-vitamin D. Current therapy with oral phosphate and vitamin D improves the rickets, but has significant morbidity and does not significantly affect the short stature and hypophosphatemia. In the present study, we demonstrate that Hyp mice, which have a mutation homologous to that in patients with X-linked hypophosphatemia, have a 2-fold greater urinary prostaglandin E2 (PGE2) excretion than C57/B6 mice. To determine whether PGs were involved in the pathogenesis of this disorder, Hyp and C57/B6 mice received i.p. injections with vehicle or indomethacin (1 mg/kg of body weight twice daily for 4 days) and were studied approximately 12 h after the last dose of indomethacin. In the Hyp mice, indomethacin treatment decreased the fractional excretion of phosphate from 13.0 +/- 3.2% to 2.2 +/- 1.1% (P < 0.05), and increased serum phosphate from 2.9 +/- 0.2 mg/dl to 4.1 +/- 0.2 mg/dl (P < 0.05). There was no effect of indomethacin in C57/B6 mice. Indomethacin did not affect serum creatinine or inulin clearance, demonstrating that the normalization of urinary phosphate excretion was not caused by changes in glomerular filtration rate. Indomethacin treatment increased renal brush border membrane vesicle NaPi-2 protein abundance in Hyp mice to levels comparable to that of C57/B6 mice, but had no effect in C57/B6 mice. In vitro isolated perfused proximal tubule studies demonstrate directly that 10-6 M bath indomethacin normalized the phosphate transport defect in Hyp mice but had no effect on C57/B6 mice. In conclusion, there is dysregulation of renal PG metabolism in Hyp mice, and indomethacin treatment normalizes the urinary excretion of phosphate by a direct tubular effect.

Fig. 1.

Urinary PGE₂/creatine ratio in C57/B6 mice compared with Hyp mice. The same results were obtained when PG was normalized to urine volume. The number of measurements is given in parentheses in this and all figures. *, P < 0.05 vs. C57/B6.

Fig. 2.

Effect of indomethacin or vehicle treatment on FE_PO4 in C57/B6 and Hyp mice. Indomethacin treatment normalized the urinary phosphate excretion in Hyp mice but had no effect on C57/B6 mice. Urinary phosphate and creatinine were determined ≈12 h after the last dose of indomethacin. *, P < 0.05 vs. other groups.

Fig. 3.

Effect of indomethacin treatment on serum phosphate in C57/B6 and Hyp mice. Indomethacin treatment increased the serum phosphate levels in Hyp mice but had no significant effect on C57/B6 mice. Levels were determined ≈12 h after the last dose of indomethacin. *, P < 0.05 vs. other groups; +, P < 0.05 vs. Hyp.

Fig. 4.

Effect of indomethacin (INDO) treatment on glomerular filtration rate measured as inulin clearance in C57/B6 and Hyp mice.

Fig. 5.

(A) The immunoblot demonstrates the effect of vehicle or i.p. indomethacin (INDO) on NaPi-2 brush border membrane abundance in C57/B6 mice and Hyp mice. The last dose of indomethacin was administered ≈12 h before sacrifice. As is shown, there is no difference in brush border membrane NaPi-2 abundance in C57/B6 mice with indomethacin treatment, whereas there is an increase in brush border membrane NaPi-2 abundance in Hyp mice. (B) As is shown, C57/B6 male mice had significantly greater BBMV NaPi-2/β-actin abundance compared with Hyp mice treated with vehicle, but this difference was not present after indomethacin treatment. *, P < 0.05 vs. other groups.

Fig. 6.

Effect of 10^-6 M bath indomethacin or vehicle in isolated perfused proximal tubules from C57/B6 and Hyp mice. As can be seen, Hyp mice have a lower rate of phosphate transport than C57/B6 mice. Hyp proximal tubules with indomethacin in the bathing solution had rates of phosphate transport comparable to C57/B6 mice. *, P < 0.05 vs. other groups.