Mutational analysis of PHEX gene in X-linked hypophosphatemia.

Hypophosphatemic rickets is commonly an X-linked dominant disorder (XLH or HYP) associated with a renal tubular defect in phosphate transport and bone deformities. The XLH gene, referred to as PHEX, or formerly as PEX (phosphate regulating gene with homologies to endopeptidases on the X-chromosome), encodes a 749-amino acid protein that putatively consists of an intracellular, transmembrane, and extracellular domain. PHEX mutations have been observed in XLH patients, and we have undertaken studies to characterize such mutations in 46 unrelated XLH kindreds and 22 unrelated patients with nonfamilial XLH by single stranded conformational polymorphism and DNA sequence analysis. We identified 31 mutations (7 nonsense, 6 deletions, 2 deletional insertions, 1 duplication, 2 insertions, 4 splice site, 8 missense, and 1 within the 5' untranslated region), of which 30 were scattered throughout the putative extracellular domain, together with 6 polymorphisms that had heterozygosity frequencies ranging from less than 1% to 43%. Single stranded conformational polymorphism was found to detect more than 60% of these mutations. Over 20% of the mutations were observed in nonfamilial XLH patients, who represented de novo occurrences of PHEX mutations. The unique point mutation (a-->g) of the 5'untranslated region together with the other mutations indicates that the dominant XLH phenotype is unlikely to be explained by haplo-insufficiency or a dominant negative effect.

Sequence analysis of 139 kb in Xp22.1 containing spermine synthase and the 5' region of PEX.

Human Xp22.1 contains genes involved in mineral balance that are implicated in X-linked hypophosphatemia (XLH) in humans, its murine homologue (Hyp), and another distinct murine hypophosphatemic disorder (Gy). In XLH, a gene, PEX, has been found to be mutated in up to 83% of patients but the sequences of the promoter and 5' end have not been characterized. To further the understanding of this genomic region, 139,454 bp in Xp22.1 have been sequenced. Our analysis confirms the three most 5' published exons of PEX and extends through a putative PEX promoter region. The 5' untranslated sequence of PEX and the mouse and rat equivalents are very highly homologous, implying a conserved functional significance. In addition, we mapped and analyzed another gene 5' of PEX, spermine synthase (SpS), which encodes a ubiquitous enzyme of polyamine metabolism that may contribute to the pathophysiology of Gy. SpS consists of 11 exons spread over 54 kb. The definition of the locations of SpS and the putative promoter region of PEX will facilitate functional analysis of these genes.

Regulation of renal vitamin D-24-hydroxylase by phosphate: effects of hypophysectomy, growth hormone and insulin-like growth factor I.

Dietary phosphate (Pi) regulates the production of 1,25-dihydroxyvitamin D3. This control is blunted in hypophysectomized (HPX) rats but can be restored by growth hormone (GH) or IGF-I. The regulation of the vitamin D catabolism by Pi, GH and IGF-I is less clear. In the present study, we found that the activity and transcript levels of the catabolic enzyme, vitamin D-24-hydroxylase (24-OHase) were decreased 3- and 5-fold, respectively, during Pi restriction in normal rats, but this effect is greatly reduced in HPX rats. Examination of the serum chemistries revealed that HPX rats on the high Pi diet had lower serum Pi levels than normal rats on this diet, presumably due to the known defective reabsorption of Pi by HPX rats. Treatment of HPX rats, adapted to a 0.6% P diet, with GH (150 micrograms) or IGF-I (80 micrograms) suppressed 24-OHase mRNA levels by 88% and 64%, respectively, by 20 hours and these effects were preceded by decreases in serum Pi. Our findings show that the 24-OHase is regulated by dietary Pi and this control is modulated by hypophysectomy, GH and IGF-I.

Expression and cloning of the human X-linked hypophosphatemia gene cDNA.

X-linked hypophosphatemia (XLH), which is a heritable metabolic bone disease characterized biochemically by selective renal phosphate (Pi) wasting, is associated with mutations in the PEX (Phosphate-regulating gene with homologies to Endopeptidases on the X-chromosome) gene. To further explore the physiologic role of PEX and define its effect in XLH we have determined the expression and tissue distribution. Northern analysis found abundant PEX mRNA in a restricted pattern, predominantly in adult ovary and fetal lung. In addition, PEX expression was also found in adult lung and fetal liver. A PEX cDNA of 2550 basepairs, which contains the full PEX coding region, was isolated from a human ovary cDNA library. The PEX cDNA shows high homology to other membrane-bound zinc metallopeptidases. The presence of PEX in nonosseous tissues strongly suggests features of a systemic role, rather than a unique function in bone development.

New insights into X-linked hypophosphatemia.

X-linked hypophosphatemia is a heritable form of rickets characterized biochemically by phosphaturia and abnormal bioactivation of vitamin D. Recent advances include the observation, using kidney cells from the X-linked hypophosphatemia mouse model (Hyp), that in-vitro renal phosphate transport is normal yet bone mineralization may be intrinsically abnormal. Of special interest is the identification of a gene (PEX) that is mutated in X-linked hypophosphatemic patients.

Renal calcitriol synthesis and serum phosphorus in response to dietary phosphorus restriction and anabolic agents.

Calcitriol [1,25-(OH)2D3] synthesis by the renal 25-hydroxyvitamin D3-1alpha-hydroxylase (1alpha-hydroxylase) is induced in rats on a low phosphorus diet, but not in the hypophysectomized (HPX) or diabetic rat. However, the normal response is restored by the administration of growth hormone (GH) or insulin-like growth factor-I (IGF-I), or insulin, respectively. To further characterize this in vivo phenomenon, the acute effects of GH, IGF-I, and insulin were studied in the HPX rat. In the HPX rat the low phosphorus diet alone did not significantly alter serum phosphorus or 1alpha-hydroxylase activity, but treatment with GH resulted in a marked decrease in serum phosphorus that was associated with a fivefold induction of enzyme activity. Time course studies showed that by 6 hours after GH administration, hepatic IGF-I mRNA had increased 10-fold while renal IGF-I mRNA had increased by only 52%. Between 6 and 12 hours, serum phosphorus decreased dramatically and 1alpha-hydroxylase activity increased twofold. Treatment of phosphorus-restricted HPX rats with IGF-I resulted in a decrease in serum phosphorus by 2 hours that preceded a fourfold increase in enzyme activity between 6 and 10 hours. Treatment of phosphorus-restricted HPX rats with insulin produced similar results. This is the first demonstration of hypophosphatemia preceding induction of the 1alpha-hydroxylase after administration of IGF-I or insulin to the HPX rat on a low phosphorus diet. Although these growth factors may have a direct effect on the 1alpha-hydroxylase, these data suggest that the influence of GH, IGF-I, and insulin on transcellular phosphorus flux may have an independent effect on enzyme activity. Furthermore, the much greater induction of hepatic compared with renal IGF-I mRNA in response to GH suggests that systemic, rather than the local, IGF-I may be required for induction of the 1alpha-hydroxylase. This effect may be mediated by either the insulin or the IGF-I receptor.

Expression of the renal 25-hydroxyvitamin D-24-hydroxylase gene: regulation by dietary phosphate.

1,25-Dihydroxyvitamin D3 [1,25(OH)2D3] plays a key role in phosphate (Pi) homeostasis through its phosphatemic actions on intestine and bone. In turn, dietary Pi restriction increases serum 1,25(OH)2D3 by stimulating its production, but its effect on vitamin D catabolism is less clear. Here we have examined the effects of dietary Pi on the expression of the renal vitamin D-24-hydroxylase (24-OHase), the first enzyme in the catabolic pathway for vitamin D compounds. Rats fed a low Pi (0.02% P) diet showed a fivefold decrease in renal 24-OHase mRNA compared with rats fed a normal Pi (0.67% P) diet. 24-OHase mRNA and 24-OHase activity decreased within 24 h of Pi restriction, reached a minimum by 48 h, and remained low through 14 days. Decreased 24-OHase mRNA was observed with more moderate Pi restriction (0.2% P), but higher Pi (1.2% P) did not increase 24-OHase mRNA over the 0.8% P diet. 24-OHase mRNA correlated well with plasma Pi (r = 0.862, P < 0.001). In conclusion, renal 24-OHase expression is regulated by dietary phosphate at the mRNA level.

Cyclosporine-induced elevation in circulating endothelin-1 in patients with solid-organ transplants.

The long-term use of cyclosporine is associated with significant complications, including hypertension and renal failure. Recent data from animal experiments suggest that alterations in renal function induced by high-dose CsA may be mediated by endothelin-1 (ET-1), a potent endothelium-derived vasoconstrictor and mitogenic peptide. The aim of the present study was to determine the effect of oral CsA on circulating levels of ET-1 in patients receiving standard immunosuppressive therapy following solid-organ transplantation (13 renal, 7 heart, 1 heart-lung, 1 liver). Plasma levels of ET-1 were measured by radioimmunoassay over a 24-hr period beginning with the oral administration of a single daily dose of CsA in 18 patients (5.6 +/- 0.5 mg/kg), or similar immunosuppressive therapy without CsA in 4 patients. Blood levels of CsA (parent compound and metabolites) were measured in 10 of the patients by RIA. Predose levels of ET-1 were similar in the two groups (1.73 +/- 0.32 and 1.29 +/- 0.9 pg/ml, respectively). Patients not receiving CsA showed no change in plasma ET-1 over the 24-hr period. In contrast, in the CsA-treated group there was a significant increase in plasma ET-1, reaching a peak at 6 hr (2.45 +/- 0.56 pg/ml, P < 0.03) that followed the peak increase in CsA parent compound and preceded the peak increase in metabolites. No significant differences were found between peak and trough levels of ET-1 in patients with moderate renal dysfunction (creatinine (Cr) > or = 150 mumol/L) compared with those with near-normal renal function, or patients receiving renal compared with nonrenal grafts. However, patients with long-term grafts (> 60 days) showed an exaggerated response to CsA, with a fractional increase in plasma ET-1 of 3.67 +/- 0.52 (n = 8) compared with 2.16 +/- 0.28 (n = 10) for patients with more recent grafts (P < 0.05). Therefore, oral administration of CsA causes an increase in circulating ET-1 in patients with solid-organ transplants that might contribute to CsA-associated nephrotoxicity and hypertension, particularly during long-term immunosuppressive therapy.

The mechanism for the disparate actions of calcitriol and 22-oxacalcitriol in the intestine.

22-Oxacalcitriol (OCT) is one of several new analogs of vitamin D that retain many of the therapeutically useful properties of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], but have much less calcemic activity. In the present study we examined the actions of OCT on intestinal calcium absorption and calbindin D9k mRNA in vitamin D-deficient rats. After ip injection of OCT (1 microgram/kg), calcium absorption increased significantly by 2 h and was maximal at 4 h (2.5-fold above control), but returned to pretreatment levels by 16 h. In contrast, the same dose of 1,25-(OH)2D3 caused a 3-fold increase in calcium absorption, which lasted more than 48 h. The transient effect of OCT on calcium absorption was also observed when the analog was infused at a dose of 1 micrograms/kg.day for 3 days. At the end of the infusion period, calcium absorption was 3-fold higher than that in vehicle-infused controls, but fell to pretreatment levels by 24 h after removing the minipumps. The time courses for induction of calbindin D9k mRNA were similar for OCT and 1,25-(OH)2D3, with no change observed until more than 4 h after injection. However, calbindin mRNA levels returned to pretreatment values more rapidly in the OCT-treated rats. Consistent with these findings, we observed that a 1 microgram/kg dose of [3H] OCT was completely cleared by 4-6 h after injection. This was paralleled by a loss of [3H]OCT associated with the intestinal vitamin D receptor. The rapid clearance of OCT is probably due to its low affinity for the serum vitamin D-binding protein. This low affinity would also be expected to allow greater accessibility to target cells. In support of this, we found that higher amounts of OCT than 1,25-(OH)2D3 were associated with the intestinal vitamin D receptor after the injection of several doses of these tritiated ligands. In summary, our results indicate that the pharmacokinetic properties of OCT are responsible at least in part for its low calcemic activity. Furthermore, comparison of the transient elevation of calcium absorption by OCT with its more prolonged effects on PTH and calbindin D9k indicates that each action of vitamin D compounds has a distinct biological half-life. The short circulating half-life of OCT can exploit these differences to provide a therapeutic advantage in the treatment of vitamin D-responsive diseases.

Calcium metabolism in acute renal failure due to rhabdomyolysis.

We report a patient with drug and hyperthermia induced rhabdomyolysis who developed acute renal failure. During the oliguric phase of 22 days, there was profound hypocalcemia (lowest ionized calcium of 0.34 mmol/l), associated with appropriately elevated intact PTH levels and high normal 1,25(OH)2D levels. Massive calcification in necrotic muscle occurred during this time. In the recovery phase, hypercalcemia was present lasting 33 days (maximum ionized calcium of 1.99 mmol/L), associated with suppression of PTH secretion, low 1,25(OH)2D3 levels, decreased bone resorption and mobilization of the muscle calcium deposits. This case report illustrates that the changes in serum calcium in rhabdomyolysis-associated acute renal failure are explicable by the deposition or removal of mineral into or from necrotic muscle with the parathyroid and vitamin D changes occurring secondarily.

22-oxacalcitriol suppresses 25-hydroxycholecalciferol-1 alpha-hydroxylase in rat kidney.

22-oxacalcitriol can decrease the serum level of 1,25-dihydroxycholecalciferol by increasing its metabolic clearance rate and decreasing its production rate. To determine whether 22-oxacalcitriol suppressed the renal 25-hydroxycholecalciferol-1 alpha- hydroxylase we treated rats with 200 ng of 22-oxacalcitriol daily for 1 week. Enzyme activity was measured in vitro by measurement of production of 1,25-dihydroxycholecalciferol by renal slices incubated with 25-hydroxycholecalciferol. 22-oxacalcitriol significantly decreased the 25-hydroxycholecalciferol-1 alpha-hydroxylase activity from 797 +/- 208 pg of 1,25-dihydroxycholecalciferol/g of kidney/h in the control group to 257 +/- 150 pg of 1,25-dihydroxycholecalciferol/g of kidney/h (p less than 0.05). This is the first demonstration of suppression of the renal 25-hydroxycholecalciferol-1 alpha-hydroxylase by the vitamin D3 analog, 22-oxacalcitriol.

Detection of miliary tuberculosis by Ga-67 scintigraphy.

Two patients are presented in whom pulmonary accumulation of Ga-67 led to an early diagnosis of miliary tuberculosis and its prompt treatment. Their chest radiographs were unremarkable and thus noncontributory to the diagnostic process.