Variable phosphate-mediated regulation of vitamin D metabolism in the murine hypophosphatemic rachitic/osteomalacic disorders.

The familial hypophosphatemic (vitamin D-resistant) disorders are a variety of genetic and acquired syndromes that exhibit unexpected biochemical heterogeneity, manifest as variably abnormal or apparently normal regulation of 1,25-dihydroxyvitamin D [1,25-(OH)2D] production. Recently, we observed that Hyp and Gy mice, murine homologs of X-linked hypophosphatemic rickets, exhibit similarly disparate regulation of vitamin D metabolism. While Gy mice under basal conditions maintain an appropriate elevation (relative to hypophosphatemia) of renal 25-hydroxyvitamin D (25OHD)-1 alpha-hydroxylase, Hyp mice manifest only normal, not increased, enzyme activity. Whether such diversity results from maintenance of phosphate (P)-regulated 1,25-(OH)2D production in Gy mice and loss of this function in Hyp mice or from other variations remains unknown. Therefore, we examined the integrity of P-regulated enzyme activity in the Gy and Hyp mice by testing the effects of enzyme inhibition and alteration of the serum phosphorus concentration on 1,25-(OH)2D production. Initially, we discovered that inhibition of renal 25OHD-1 alpha-hydroxylase suppressed enzyme function in Hyp mice, but did not prevent expression of P-mediated activity in Gy mice. In this regard, while administration of a high calcium diet or 1,25-(OH)2D (0.4 ng/h, sc, for 48 h) resulted in a comparable inhibition of enzyme activity in Hyp (5.9 +/- 0.5 vs. 2.8 +/- 0.7 fmol/mg.min) and normal mice (4.4 +/- 0.6 vs. 2.0 +/- 0.2 fmol/mg.min), similar treatment did not effect complete inhibition of 25OHD-1 alpha-hydroxylase in Gy (10.3 +/- 0.6 vs. 4.9 +/- 0.3 fmol/mg.min) or P-depleted mice (10.2 +/- 0.5 vs. 5.1 +/- 0.4 fmol/mg.min). In accord with the apparent persistence of P-mediated stimulation of enzyme function in Gy mice, dietary P repletion in this mutant resulted in a serum phosphorus concentration similar to that of normal mice and decreased enzyme activity (4.0 +/- 0.8 fmol/mg.min) to a level no different from that expressed in controls (3.4 +/- 0.3 fmol/mg.min). However, in the absence of apparent P-mediated stimulation of enzyme activity, identical treatment of Hyp mice increased the serum phosphorus level comparably, but paradoxically enhanced 25OHD-1 alpha-hydroxylase (3.1 +/- 0.4 vs. 11.7 +/- 2.0 fmol/mg.min). Collectively, these data indicate that enhanced renal 25OHD-1 alpha-hydroxylase expressed in Gy mice and probably in related human diseases results from normally maintained P regulation of enzyme activity, an action absent or mutated in the genetically distinct Hyp mouse.

Normal regulation of calcitriol production in Gy mice. Evidence for biochemical heterogeneity in the X-linked hypophosphatemic diseases.

Phenotypic heterogeneity in X-linked hypophosphatemic rickets (XLH) is ascribed to variable penetrance of the genetic abnormality. However, studies of hypophosphatemic (Hyp) and gyrorotary (Gy) mice indicate that mutations at different loci along the X chromosome may underlie the genetically transmitted hypophosphatemic disorders. Thus, genetic heterogeneity may be a determinant of the phenotypic variability in XLH. To determine if such variance includes biochemical diversity, we examined whether Gy mice, similar to Hyp mice, exhibit abnormal regulation of renal 25-hydroxyvitamin D (25[OH]D)-1 alpha-hydroxylase. Serum phosphorus in Gy (4.7 +/- 0.3 mg/dl) and phosphate (P)-depleted mice (4.9 +/- 0.4) was significantly less than normal (8.4 +/- 0.5). Consistent with P depletion, the Gy mice exhibited enhanced renal 25(OH)D-1 alpha-hydroxylase activity (9.3 +/- 0.6 fmol/mg kidney per min), similar to that of P-depleted normals (9.1 +/- 1.5), but significantly greater than that of controls (3.1 +/- 0.3). Such normal enzyme responsiveness was confirmed upon PTH stimulation (1 IU/h s.c.), which revealed that Gy mice increased renal 1-hydroxylase (59 +/- 7.7) similarly to normals (65 +/- 7.7) and P-depleted animals (58.4 +/- 7.8). Calcitonin administration also enhanced enzyme function comparably in the animal models. Evidence confirming normally responsive calcitriol production in untreated Gy mice included increased serum 1,25-dihydroxyvitamin D levels, gastrointestinal calcium absorption, and urinary calcium. The normally regulated vitamin D metabolism in Gy mice indicates that biochemically diverse disease may result from mutations in the gene family regulating renal P transport and underlying X-linked hypophosphatemia. We suspect such heterogeneity is due to altered P transport at variable segments of the proximal convoluted tubule.

Abnormal adenosine 3'.5'-monophosphate stimulation of renal 1,25-dihydroxyvitamin D production in hyp mice: evidence that 25-hydroxyvitamin D-1 alpha-hydroxylase dysfunction results from aberrant intracellular function.

Previously we have established that abnormal regulation of renal 25-hydroxyvitamin D (25OHD)-1 alpha-hydroxylase in Hyp mice involves the PTH-adenylate cyclase component of enzyme activation. However, it remains unknown if the muted effects of PTH result from 1) abnormal second messenger production or 2) an intracellular defect limiting enzyme activation. To distinguish between these possibilities, we compared cAMP stimulation of renal 25OHD-1 alpha-hydroxylase in normal, phosphate-depleted normal, and Hyp mice. Administration of N6-monobutyryl cAMP iv (200 mg/kg/day) increased enzyme activity in normal (4.1 +/- 1.7 vs. 40.7 +/- 7.0 fmol/mg kidney.min) and phosphate-depleted mice (13.3 +/- 1.8 vs. 78.2 +/- 10.4) to a level significantly greater than that achieved in Hyp mice (7.4 +/- 1.1 vs. 22.7 +/- 3.6). Moreover, similar to our observations after PTH stimulation, the apparent abnormal cAMP effect did not result from an altered time course of enzyme activation or a rightward shift in the dose response. Collectively, these data indicate that abnormal regulation of 1,25-dihydroxyvitamin D production in Hyp mice results from aberrant intracellular regulation of 25OHD-1 alpha-hydroxylase, a defect probably related to deranged phosphate transport in the renal tubule.

Oral calcitriol and calcium: efficient therapy for uremic hyperparathyroidism.

Therapy with orally administered calcitriol often does not adequately control the biochemical manifestations of secondary hyperparathyroidism in uremic patients. This may be due to inadequate serum concentrations of 1.25(OH)2 vitamin D and/or to insufficient dietary calcium supplementation. In the present study, therefore, we examined the effect on parathyroid function of calcitriol and calcium carbonate, administered orally, in doses sufficient to normalize the serum 1.25(OH)2 vitamin D and calcium concentrations. After nine months of combined therapy, marked suppression of immunoreactive PTH occurred in the absence of hypercalcemia. Furthermore, prolonged therapy resulted in additional suppression of the PTH concentrations comparable in magnitude to that reported following intravenous calcitriol therapy and was associated with a mild degree of hypercalcemia similar to that which occurs with intravenous therapy. Euparathyroidism was achieved in 25% of the patients by 15 months of treatment. In conclusion, secondary hyperparathyroidism can be effectively controlled with combined oral therapy without significant hypercalcemia in selected patients with end-stage renal failure. This salutary effect may result from direct actions of 1.25(OH)2D on the parathyroid gland and/or gastrointestinal tract, or from an overall action of combined treatment to restore calcium homeostasis.