Ubiquitin COOH-terminal hydrolase L1 deletion is associated with urinary α-klotho deficiency and perturbed phosphate homeostasis.

Loss of ubiquitin COOH-terminal hydrolase L1 (UCHL1), a deubiquitinating enzyme required for neuronal function, led to hyperphosphatemia accompanied by phosphaturia in mice, while calcium homeostasis remained intact. We therefore investigated the mechanisms underlying the phosphate imbalance in Uchl1-/- mice. Interestingly, phosphaturia was not a result of lower renal brush border membrane sodium-phosphate cotransporter expression as sodium-phosphate cotransporter 2a and 2c expression levels was similar to wild-type levels. Plasma parathyroid hormone and fibroblast growth factor 23 levels were not different; however, fibroblast growth factor 23 mRNA levels were significantly increased in femur homogenates from Uchl1-/- mice. Full-length and soluble α-klotho levels were comparable in kidneys from wild-type and Uchl1-/- mice; however, soluble α-klotho was reduced in Uchl1-/- mice urine. Consistent with unchanged components of 1,25(OH)2D3 metabolism (i.e., CYP27B1 and CYP24A1), sodium-phosphate cotransporter 2b protein levels were not different in ileum brush borders from Uchl1-/- mice, suggesting that the intestine is not the source of hyperphosphatemia. Nonetheless, when Uchl1-/- mice were fed a low-phosphate diet, plasma phosphate, urinary phosphate, and fractional excretion of phosphate were significantly attenuated and comparable to levels of low-phosphate diet-fed wild-type mice. Our findings demonstrate that Uchl1-deleted mice exhibit perturbed phosphate homeostasis, likely consequent to decreased urinary soluble α-klotho, which can be rescued with a low-phosphate diet. Uchl1-/- mice may provide a useful mouse model to study mild perturbations in phosphate homeostasis.

FGF23 acts directly on renal proximal tubules to induce phosphaturia through activation of the ERK1/2-SGK1 signaling pathway.

Fibroblast growth factor-23 (FGF23) is a bone-derived endocrine regulator of phosphate homeostasis which inhibits renal tubular phosphate reabsorption. Binding of circulating FGF23 to FGF receptors in the cell membrane requires the concurrent presence of the co-receptor αKlotho. It is still controversial whether αKlotho is expressed in the kidney proximal tubule, the principal site of phosphate reabsorption. Hence, it has remained an enigma as to how FGF23 downregulates renal phosphate reabsorption. Here, we show that renal proximal tubular cells do express the co-receptor αKlotho together with cognate FGF receptors, and that FGF23 directly downregulates membrane expression of the sodium-phosphate cotransporter NaPi-2a by serine phosphorylation of the scaffolding protein Na(+)/H(+) exchange regulatory cofactor (NHERF)-1 through ERK1/2 and serum/glucocorticoid-regulated kinase-1 signaling.

Decreased bone density and increased phosphaturia in gene-targeted mice lacking functional serum- and glucocorticoid-inducible kinase 3.

Insulin and growth factors activate the phosphatidylinositide-3-kinase pathway, leading to stimulation of several kinases including serum- and glucocorticoid-inducible kinase isoform SGK3, a transport regulating kinase. Here, we explored the contribution of SGK3 to the regulation of renal tubular phosphate transport. Coexpression of SGK3 and sodium-phosphate cotransporter IIa significantly enhanced the phosphate-induced current in Xenopus oocytes. In sgk3 knockout and wild-type mice on a standard diet, fluid intake, glomerular filtration and urine flow rates, and urinary calcium ion excretion were similar. However, fractional urinary phosphate excretion was slightly but significantly larger in the knockout than in wild-type mice. Plasma calcium ion, phosphate concentration, and plasma parathyroid hormone levels were not significantly different between the two genotypes, but plasma calcitriol and fibroblast growth factor 23 concentrations were significantly lower in the knockout than in wild-type mice. Moreover, bone density was significantly lower in the knockouts than in wild-type mice. Histological analysis of the femur did not show any differences in cortical bone but there was slightly less prominent trabecular bone in sgk3 knockout mice. Thus, SGK3 has a subtle but significant role in the regulation of renal tubular phosphate transport and bone density.

Akt2/PKBbeta-sensitive regulation of renal phosphate transport.

AIM: The protein kinase B (PKB)/Akt is known to stimulate the cellular uptake of glucose and amino acids. The kinase is expressed in proximal renal tubules. The present study explored the influence of Akt/PKB on renal tubular phosphate transport. METHODS: The renal phosphate transporter NaPi-IIa was expressed in Xenopus oocytes with or without PKB/Akt and Na(+) phosphate cotransport determined using dual electrode voltage clamp. Renal phosphate excretion was determined in Akt2/PKBbeta knockout mice (akt2(-/-)) and corresponding wild-type mice (akt2(+/+)). Transporter protein abundance was determined using Western blotting and phosphate transport by (32)P uptake into brush border membrane vesicles. RESULTS: The phosphate-induced current in NaPi-IIa-expressing Xenopus oocytes was significantly increased by the coexpression of Akt/PKB. Phosphate excretion [micromol per 24 h per g BW] was higher by 91% in akt2(-/-) than in akt2(+/+) mice. The phosphaturia of akt2(-/-) mice occurred despite normal transport activity and expression of the renal phosphate transporters NaPi-IIa, NaPi-IIc and Pit2 in the brush border membrane, a significantly decreased plasma PTH concentration (by 46%) and a significantly enhanced plasma 1,25-dihydroxyvitamin D(3) concentration (by 46%). Moreover, fractional renal Ca(2+) excretion was significantly enhanced (by 53%) and bone density significantly reduced (by 11%) in akt2(-/-) mice. CONCLUSIONS: Akt2/PKBbeta plays a role in the acute regulation of renal phosphate transport and thus contributes to the maintenance of phosphate balance and adequate mineralization of bone.

Delayed transport of tissue-nonspecific alkaline phosphatase with missense mutations causing hypophosphatasia.

Hypophosphatasia is a rare genetic disease characterized by diminished bone and tooth mineralization due to deficient activity of tissue-nonspecific alkaline phosphatase (TNSALP). The disease is clinically heterogeneous due to different mutations in the TNSALP gene. In order to determine whether mutated TNSALP proteins may be sequestered, degraded, or subjected to delay in their transport to the cell membrane, we built a plasmid expressing a YFP-TNSALP fluorescent fusion protein allowing the observation of cellular localization in live cells by fluorescence confocal microscopy at different time points after transfection. We studied five mutants (c. 571G>A, c. 653T>C, c. 746G>T, c. 1363G>A and c. 1468A>T) exhibiting various levels of in vitro residual enzymatic activity. While the wild-type protein reached the membrane within the first 24h after transfection, the mutants reached the membrane with delays of 24, 48 or 72 h. For all of the tested mutations, accumulation of the mutated proteins, mainly in the Golgi apparatus, was observed. We concluded that reduced ALP activity of these TNSALP mutants results from structural disturbances and delay in membrane anchoring, and not from compromised catalytic activity.

Serum hyaluronidase aberrations in metabolic and morphogenetic disorders.

Hyaluronidases are endo-glycosidases that degrade both hyaluronan (hyaluronic acid) (HA) and chondroitin sulfates. Deficiency of hyaluronidase activity has been predicted to result in a phenotype similar to that observed in mucopolysaccharidosis (MPS). In the present study, we surveyed a variety of patients with phenotypes similar to those observed in MPS, but without significant mucopolysacchariduria to determine if some are based on aberrations in serum hyaluronidase (Hyal-1) activity. The study included patients with well-characterized dysmorphic disorders occurring on genetic basis, as well as those of unkown etiology. The purpose of the study was to establish how wide spread were abnormalities in levels of circulating Hyal-1 activity. A simple and sensitive semi-quantitative zymographic procedure was used for the determination of activity. Levels of both beta-N-acetylglucosaminidase and beta-glucuronidase whose activities contribute to the total breakdown of hyaluronan (HA) were also measured, as well as the concentration of circulating HA. Among 48 patients with bone or connective tissue abnormalities, low levels of Hyal-1 activity were found in six patients compared to levels in 100 healthy donors (2.0-3.2 units/microL vs 6(+/- 1 SE) units/microL). These six patients exhibited a wide spectrum of clinical abnormalities, in particular shortened extremities: they included three patients with unknown causes of clinical symptoms, one patient with Sanfilippo disease, one of the seven patients with achondroplasia, and one with hypophosphotemic rickets. Normal levels of serum Hyal-1 activities were found in patients with Morquio disease, GM1 gangliosidosis, I cell-disease, 6 of the 7 patients with achondroplasia, Marfan's-syndrome and Ehlers-Danlos syndrome. No patient totally lacked serum Hyal-1 activity. Serum HA concentration was elevated in patients with Sanfilippo A and I-cell disease. Determination of serum and leukocyte Hyal-1 and serum HA may be useful to evaluate patients with metabolic and morphogenetic disorders.

Evidence for abnormal translational regulation of renal 25-hydroxyvitamin D-1alpha-hydroxylase activity in the hyp-mouse.

Hyp-mice exhibit abnormal regulation of 25-hydroxyvitamin D [25(OH)D]-1alpha-hydroxylase activity. Previous observations suggest such aberrant modulation is posttranscriptional. To investigate this possibility further, we examined whether hyp-mice manifest abnormal translation of 25(OH)D-1alpha-hydroxylase mRNA. We compared phosphate, parathyroid, and calcitonin effects on renal 25(OH)D-1alpha-hydroxylase protein as well as mRNA and enzyme activity in normal and hyp-mice. We assayed protein by Western blots, mRNA by real-time RT-PCR, and enzyme activity by measuring 1,25-dihydroxyvitamin D production. Although phosphate-depleted mice exhibited enhanced enzyme function, with significantly increased mRNA and protein expression, hyp-mice comparably increased mRNA but failed to augment enzyme activity, concordant with an inability to increase protein expression. PTH stimulation increased mRNA and protein expression as well as enzyme activity in normal mice but in hyp-mice, despite effecting mRNA enhancement, did not increment enzyme function or protein. The inability of hypophosphatemia and PTH to increase 25(OH)D-1alpha-hydroxylase activity and protein expression in hyp-mice was not universal because calcitonin stimulation was normal, suggesting proximal convoluted tubule localization of the defect. These data, in accord with absent undue enhancement of protein expression in hyp-mice treated with protease inhibitors, establish that abberrant regulation of vitamin D metabolism results from abnormal translational activity.

Disordered regulation of renal 25-hydroxyvitamin D-1alpha-hydroxylase gene expression by phosphorus in X-linked hypophosphatemic (hyp) mice.

X-linked hypophosphatemic (Hyp) mice exhibit hypophosphatemia, impaired renal phosphate reabsorption, defective skeletal mineralization, and disordered regulation of vitamin D metabolism: In Hyp mice, restriction of dietary phosphorus induces a decrease in serum concentration of 1,25-dihydroxyvitamin D and renal activity of 25-hydroxyvitamin D-1alpha-hydroxylase (1alpha-hydroxylase), and induces an increase in renal activity of 25-hydroxyvitamin D-24-hydroxylase (24-hydroxylase). In contrast, in wild-type mice, phosphorus restriction stimulates renal 1alpha-hydroxylase gene expression and suppresses that of 24-hydroxylase. To determine the molecular basis for the disordered regulation of vitamin D metabolism in Hyp mice, we determined renal mitochondrial 1alpha-hydroxylase activity and the renal abundance of p450c1alpha and p450c24 mRNA in wild-type and Hyp mice fed either control, low-, or high-phosphorus diets for 5 d. In wild-type mice, phosphorus restriction increased 1alpha-hydroxylase activity and p450c1alpha mRNA expression by 6-fold and 3-fold, respectively, whereas in the Hyp strain the same diet induced changes of similar magnitude but opposite in direction. Phosphorus supplementation was without effect in wild-type mice, whereas in Hyp mice the same diet induced 3-fold and 2-fold increases, respectively, in enzyme activity and p450c1alpha mRNA abundance. In wild-type mice, both renal 1alpha-hydroxylase activity and p450c1alpha mRNA abundance varied inversely and significantly with serum phosphorus concentrations, whereas in Hyp mice the relationship between both renal parameters and serum phosphorus concentration was direct. In Hyp mice, phosphorus restriction induced a significant increase in renal p450c24 mRNA abundance, in contrast to the lack of effect observed in wild-type mice. The present findings demonstrate that regulation of both the p450c1alpha and p45024 genes by phosphorus is disordered in Hyp mice at the level of renal 1alpha-hydroxylase activity and renal p450c1alpha and p450c24 mRNA expression.

Structure and function of disease-causing missense mutations in the PHEX gene.

The PHEX gene that is mutated in patients with X-linked hypophosphatemia (XLH) encodes a protein homologous to the M13 family of zinc metallopeptidases. The present study was undertaken to assess the impact of nine PHEX missense mutations on cellular trafficking, endopeptidase activity, and protein conformation. Secreted forms of wild-type and mutant PHEX proteins were generated by PCR mutagenesis; these included C85R, D237G, Y317F, G579R, G579V, S711R, A720T, and F731Y identified in XLH patients, and E581V, which in neutral endopeptidase 24.11 abolishes catalytic activity but not plasma membrane localization. The wild-type and D237G, Y317F, E581V, and F731Y proteins were terminally glycosylated and secreted into the medium, whereas the C85R, G579R, G579V, S711R, and A720T proteins were trapped inside the transfected cells. Growing the cells at 26 C permitted the secretion of G579V, S711R, and A720T proteins, although the yield of rescued G579V was insufficient for further analysis. Endopeptidase activity of secreted and rescued PHEX proteins, assessed using a novel internally quenched fluorogenic peptide substrate, revealed that E581V and S711R are completely inactive; D237G and Y317F exhibit 50-60% of wild-type activity; and A720T and F731Y retain full catalytic activity. Conformational analysis by limited proteolysis demonstrated that F731Y is more sensitive to trypsin and D237G is more resistant to endoproteinase Glu-c than the wild-type protein. Thus, defects in protein trafficking, endopeptidase activity, and protein conformation account for loss of PHEX function in XLH patients harboring these missense mutations.

Up-regulation of liver glucose-6-phosphatase in x-linked hypophosphatemic mice.

We previously showed that a phosphate-deficient diet resulting in hypophosphatemia upregulated the catalytic subunit p36 of rat liver glucose-6-phosphatase, which is responsible for hepatic glucose production. A possible association between phosphate and glucose homeostasis was now further evaluated in the Hyp mouse, a murine homologue of human X-linked hypophosphatemia. We found that in the Hyp mouse as in the dietary Pi deficiency model, serum insulin was reduced while glycemia was increased, and that liver glucose-6-phosphatase activity was enhanced as a consequence of increased mRNA and protein levels of p36. In contrast, the Hyp model had decreased mRNA and protein levels of the putative glucose-6-phosphate translocase p46 and liver cyclic AMP was not increased as in the phosphate-deficient diet rats. It is concluded that in genetic as in dietary hypophosphatemia, elevated glucose-6-phosphatase activity could be partially responsible for the impaired glucose metabolism albeit through distinct mechanisms.

Modulation of potassium channels in the hearts of transgenic and mutant mice with altered polyamine biosynthesis.

Inward rectification of cardiac I(K1)channels was modulated by genetic manipulation of the naturally occurring polyamines. Ornithine decarboxylase (ODC) was overexpressed in mouse heart under control of the cardiac alpha -myosin heavy chain promoter (alpha MHC). In ODC transgenic hearts, putrescine and cadaverine levels were highly elevated ( identical with 35-fold for putrescine), spermidine was increased 3.6-fold, but spermine was essentially unchanged. I(K1)density was reduced by identical with 38%, although the voltage-dependence of rectification was essentially unchanged. Interestingly, the fast component of transient outward (I(to,f)) current was increased, but the total outward current amplitude was unchanged. I(K1)and I(to)currents were also studied in myocytes from mutant Gyro (Gy) mice in which the spermine synthase gene is disrupted, leading to a complete loss of spermine. I(K1)current densities were not altered in Gy myocytes, but the steepness of rectification was reduced indicating a role for spermine in controlling rectification. Intracellular dialysis of myocytes with putrescine, spermidine and spermine caused reduction, no change and increase of the steepness of rectification, respectively. Taken together with kinetic analysis of I(K1)activation these results are consistent with spermine being a major rectifying factor at potentials positive to E(K), spermidine dominating at potentials around and negative to E(K), and putrescine playing no significant role in rectification in the mouse heart.

Molecular cloning of a novel human UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase, GalNAc-T8, and analysis as a candidate autosomal dominant hypophosphatemic rickets (ADHR) gene.

The UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase (ppGaNTase) family of enzymes initiates mucin-like O-glycosylation of specific proteins. Using exon-prediction analysis on genomic sequence from human chromosome 12p13.3, we identified novel exons that shared significant homology with the ppGaNTases. cDNA library screening and RT-PCR produced the complete coding sequence of a novel human ppGaNTase family member, designated GalNAc-T8. The open reading frame (ORF) of GalNAc-T8 codes for a 637 amino acid, type-II membrane protein that is 45-60% identical to the other mammalian ppGaNTases. GalNAc-T8 shares high homology within the functional regions of the known ppGaNTases; however, the enzyme possesses a novel residue substitution within a characteristic motif of the catalytic domain. Northern analysis of multiple human tissue mRNAs demonstrated that the 5.0 and 2.1kb GalNAc-T8 transcripts are widely expressed. The metabolic disorder autosomal dominant hypophosphatemic rickets (ADHR) was previously mapped to the region of chromosome 12p13.3 in which GalNAc-T8 resides. Using a positional-candidate strategy for identifying the ADHR gene, GalNAc-T8 was subjected to mutational analysis in DNA from ADHR individuals. We detected multiple polymorphisms in the human GalNAc-T8 ORF, but did not find ADHR mutations. In summary, these studies identified the human GalNAc-T8 gene, as well as multiple genomic polymorphisms that will be useful for further understanding the structure-function relations of the ppGaNTases.

Comparison of total alkaline phosphatase and three assays for bone-specific alkaline phosphatase in childhood and adolescence.

We compared serum levels of total alkaline phosphatase (TAP) and bone-specific alkaline phosphatase (BAP) as determined by three different assays (lectin affinity electrophoresis, immunoradiometric assay, enzyme-linked immunosorbent assay) in subjects aged 5-20 years suffering from X-linked hypophosphatemic rickets (n = 14), chronic renal failure (n = 10) and chronic cholestatic liver disease (n = 16). Results were compared to controls of the same age and were expressed as standard deviation scores (SDS). TAP correlated significantly with BAP (r > 0.9 for each assay; p < 0.001) in controls. In children with cholestatic diseases, TAP (median SDS + 2.0) was elevated, but BAP, as measured by the electrophoretic assay, was within the reference range for most patients (median SDS: -0.4; p = 0.003 for the difference between the median SDS of TAP and BAP). In contrast, results for BAP as determined by the two immunoassays were not significantly different from TAP in any of the three patient groups (p > 0.05 in each group for both assays). In this study, the two immunoassays did not have a detectable advantage over lectin affinity electrophoresis in the determination of BAP.

A gene (PEX) with homologies to endopeptidases is mutated in patients with X-linked hypophosphatemic rickets. The HYP Consortium.

X-linked hypophosphatemic rickets (HYP) is a dominant disorder characterised by impaired phosphate uptake in the kidney, which is likely to be caused by abnormal regulation of sodium phosphate cotransport in the proximal tubules. By positional cloning, we have isolated a candidate gene from the HYP region in Xp22.1. This gene exhibits homology to a family of endopeptidase genes, members of which are involved in the degradation or activation of a variety of peptide hormones. This gene (which we have called PEX) is composed of multiple exons which span at least five cosmids. Intragenic non-overlapping deletions from four different families and three mutations (two splice sites and one frameshift) have been detected in HYP patients, which suggest that the PEX gene is involved in the HYP disorder.

Sodium-phosphate transport in the kidney and intestine of the hypophosphatemic mouse.

X-linked hypophosphatemic vitamin D-resistant rickets is the most common inherited form of vitamin D-resistant rickets in man. The current studies were designed to characterize the defect in the sodium (Na+)-phosphate transporter in the (Hyp) mouse model. The slope of initial rate of phosphate uptake was significantly decreased in the kidney but not in intestinal brush border membranes of the (Hyp) mice compared with genetically matched controls. Phosphate uptake by the basolateral membranes of the intestine and kidney was similar in the (Hyp) and control mice. Kinetic analysis of phosphate uptake by renal brush border membranes showed a Vmax of 0.32 +/- 0.06 and 1.6 +/- 0.1 nmol/mg protein per 15 s (P < 0.01) and Km of 0.07 +/- 0.06 and 0.39 +/- 0.05 mM in (Hyp) and control mice respectively (P < 0.05). Vmax and Kmax of jejunal uptake of phosphate were similar in (Hyp) and control mice. To confirm these findings, we expressed the Na(+)-phosphate transporter in Xenopus laevis oocytes. Na(+)-dependent phosphate uptake in the oocytes was expressed 6 days after renal and intestinal poly(A)+ RNA injection, however, uptake values were significantly lower in oocytes injected with renal poly(A)+ RNA from the (Hyp) mice compared with controls (P < 0.01). No differences were noted in phosphate uptake by oocytes injected with poly(A)+ RNA from the jejunum of the (Hyp) or control mice. These studies suggest that the defect in the (Hyp) mice is localized to the kidney and is secondary to diminished activity and/or function of the Na(+)-phosphate transporter.

25-Hydroxyvitamin D-24-hydroxylase in phytohemagglutinin-stimulated lymphocytes: intermediate bioresponse to 1,25-dihydroxyvitamin D3 of cells from parents of patients with vitamin D-dependent rickets type II.

A method for assay of 25-hydroxyvitamin D-24-hydroxylase (24-hydroxylase) activity in phytohemagglutinin (PHA)-stimulated lymphocytes was applied to determine whether vitamin D-dependent rickets type II (VDDR II) is hereditary. In normal lymphocytes incubated with PHA for 3 days, maximal and half-maximal responses of 24-hydroxylase were observed after exposure to 10(-8) mol/L and (1.3 +/- 0.4) x 10(-9) mol/L 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], respectively. These responses were similar to those of cultured skin fibroblasts. In contrast, after exposure to 10(-8), 10(-7), and 10(-6) mol/L 1,25-(OH)2D3, no 24-hydroxylase activity was detected in cells from patients with VDDR II, and intermediate activity was observed in cells from their parents. These findings indicated the presence of an intracellular receptor-effector system for 1,25-(OH)2D3 in peripheral lymphocytes. Heterozygotes of VDDR II could be identified, and autosomal recessive inheritance of the disease was demonstrated. Detection of heterozygotes of this disease was not possible by assay of inhibition of thymidine incorporation, another marker of the function of 1,25-(OH)2D3 in PHA-stimulated lymphocytes. Therefore, assay of 24-hydroxylase induction reflected the receptor status more closely than assay of inhibition of DNA biosynthesis. The assay of 24-hydroxylase activity in PHA-stimulated lymphocytes described here will be useful for diagnosis of VDDR II and study of families of patients with this disease.

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.

Abnormal parathyroid hormone stimulation of 25-hydroxyvitamin D-1 alpha-hydroxylase activity in the hypophosphatemic mouse. Evidence for a generalized defect of vitamin D metabolism.

Abnormal regulation of vitamin D metabolism is a feature of X-linked hypophosphatemic rickets in man and of the murine homologue of the disease in the hypophosphatemic (Hyp)-mouse. We previously reported that mutant mice have abnormally low renal 25-hydroxyvitamin D-1 alpha-hydroxylase (1 alpha-hydroxylase) activity for the prevailing degree of hypophosphatemia. To further characterize this defect, we examined whether Hyp-mouse renal 1 alpha-hydroxylase activity responds normally to other stimulatory and inhibitory controls of enzyme function. We studied stimulation by parathyroid hormone (PTH) using: (a) a calcium-deficient (0.02% Ca) diet to raise endogenous PTH; or (b) 24-h continuous infusion of 0.25 IU/h bovine PTH via osmotic minipump. In both cases enzyme activity of identically treated normal mice increased to greater levels than those attained by Hyp-mice. The relative inability of PTH to stimulate 1 alpha-hydroxylase activity is not a function of the hypophosphatemia in the Hyp-mouse since PTH-infused, phosphate-depleted normal mice sustained a level of enzyme activity greater than that of normal and Hyp-mice. In further studies we investigated inhibition of enzyme activity by using: (a) a calcium-loaded (1.2% Ca) diet to suppress endogenous PTH; or (b) 24-h continuous infusion of 0.2 ng/h 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). The 1 alpha-hydroxylase activity of normal and Hyp-mice was significantly reduced to similar absolute levels following maintenance on the calcium-loaded diet. Further, infusion of 1,25(OH)2D3 caused a comparable reduction of 1 alpha-hydroxylase activity in normal, Hyp-, and phosphate-depleted normal mice. These observations indicate that the inhibitory control of 1 alpha-hydroxylase by reduced levels of PTH or increased 1,25(OH)2D3 concentrations is intact in the mutants. However, the inability of PTH and hypophosphatemia to stimulate enzyme activity in a manner analogous to that in normal and phosphate-depleted mice indicates that a generalized defect of 1 alpha-hydroxylase regulation is manifest in Hyp-mice.