Defective adaptation to a low phosphate environment by cultured renal tubular cells from X-linked hypophosphatemic (Hyp) mice

Effects of parathyroid hormone (PTH), low phosphate environment, and 12-O-tetradecanoyl phorbol-13-acetate (TPA) on the phosphate reabsorption by the renal tubular cells from mutant hemizygous hypophosphatemic (Hyp/Y) mice and their littermates (+/Y) were studied using a phosphate accumulation system which had been developed recently. This system mimics phosphate transport at the renal tubules. When cultured in a normal phosphate medium, the characteristics of the phosphate accumulation by Hyp cells was almost identical with that by normal cells; a PTH-induced inhibition and a TPA-induced stimulation of phosphate accumulation. However, when preincubated in a low phosphate medium, the accumulation of phosphate by normal cells increased significantly, while that by Hyp cells did not. These results indicate that the adaptation to the low phosphate environment is defective in Hyp cells and it may be one of the cause of renal phosphate leakage in the Hyp mouse.     

Primary cultures of renal epithelial cells from X-linked hypophosphatemic (Hyp) mice express defects in phosphate transport and vitamin D metabolism.

Mutation in a gene (symbol Hyp) on the X chromosome causes hypophosphatemia in the mouse. The murine phenotype is a counterpart of X-linked hypophosphatemia in man. Both exhibit impaired renal reabsorption of phosphate in vivo. In vitro studies in the Hyp mouse have shown decreased Na+-dependent phosphate transport at the brush border membrane and abnormal mitochondrial vitamin D metabolism. To determine whether the mutant renal phenotype is intrinsic to the kidney or dependent upon putative extrinsic humoral factor(s) for its expression, we established primary cultures of renal epithelial cells from normal and Hyp male mouse kidneys. The cells are derived from proximal tubule. Initial uptake rates of phosphate and alpha-methyl-D-glucopyranoside (alpha-MG), a metabolically inert analogue of D-glucose, were measured simultaneously in confluent monolayers exhibiting epithelial polarity and tight junctions. The mean phosphate/alpha-MG uptake ratio in Hyp cultures was 82% of that in normal cells (P less than 0.01, n = 96). Moreover, the production of 24,25-dihydroxyvitamin D3 was significantly elevated in confluent cultures of Hyp cells relative to normal cells. These results imply that the Hyp gene is expressed in situ in renal epithelium and suggest that humoral factors are not necessary for the mutant renal phenotype in X-linked hypophosphatemia of mouse and man.     

In vivo Response of X-linked hypophosphatemic mice to calcitonin

Other investigations, using the Hyp mouse as an animal model for the human disease X-linked hypophosphatemia (XLH), have demonstrated renal hypersensitivity to calcitonin (CT) using in vitro single renal tubule adenylate cyclase microassays. Renal hypersensitivity to CT may explain the elevated fractional excretion of phosphate (FE-P) and urinary 3'-5'-cyclic AMP (UcAMP) present in Hyp mice. This current study was designed as the in vivo counterpart to the in vitro experiments. CT dose-dependent hypocalcemia when data were constructed in normal and Hyp mice 18 hours after thyroparathyroidectomy (TPTX). Exogenous CT administered to TPTX normal mice resulted in dose-dependent hypocalcemia when data were expressed as the percent change from pretreatment. However, CT did not elicit a hypocalcemic response in TPTX Hyp mice at any dose. Only TPTX normal mice responded to CT with significant hypophosphatemia. FE-P and UcAMP were not significantly changed by CT in either genotype. In a different experiment, a large pharmacologic dose of CT was given to TPTX mice. This dose resulted in a significant but similar elevation of FE-P 1 hour after injection in both Hyp and normal TPTX mice. While UcAMP also rose significantly in both genotypes, the percent increase compared to controls was greater in Hyp mice. In summary, results from these in vivo experiments indicate that Hyp mice are not hypersensitive to physiologic doses of CT, and in fact they seem to be resistant to the hypocalcemic effect of the hormone. The greater increase in UcAMP in TPTX Hyp mice after a pharmacologic dose of CT may be the basis for the earlier in vitro results reported by others. We conclude that renal hypersensitivity to CT does not play a role in the etiology of XLH in the Hyp mouse.     

Metabolism of 25-hydroxyvitamin D3 in renal slices from the X-linked hypophosphatemic (Hyp) mouse: abnormal response to fall in serum calcium

The effect of the X-linked Hyp mutation on 25-hydroxyvitamin D3 (25-OH-D3) metabolism in mouse renal cortical slices was investigated. Vitamin D replete normal mice and Hyp littermates fed the control diet synthesized primarily 24,25-dihydroxyvitamin D3 (24,25-(OH)2D3); only minimal synthesis of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) was detected in both genotypes and 1,25-(OH)2D3 formation was not significantly greater in Hyp mice relative to normal littermates, despite hypophosphatemia and hypocalcemia in the mutants. Calcium-deficient diet fed to normal mice reduced serum calcium (p less than 0.01), increased renal 25-hydroxyvitamin D3-1-hydroxylase (1-OHase) activity (p less than 0.05), and decreased 25-hydroxyvitamin D3-24-hydroxylase (24-OHase) activity (p less than 0.05). In contrast, Hyp littermates on the calcium-deficient diet had decreased serum calcium (p less than 0.01), without significant changes in the renal metabolism of 25-OH-D3. Both normal and Hyp mice responded to the vitamin D-deficient diet with a fall in serum calcium (p less than 0.01), significantly increased renal 1-OHase, and significantly decreased renal 24-OHase activities. In Hyp mice, the fall in serum calcium on the vitamin D-deficient diet was significantly greater than that observed on the calcium-deficient diet. Therefore the ability of Hyp mice to increase renal 1-OHase activity when fed the vitamin D-deficient diet and their failure to do so on the calcium-deficient diet may be related to the resulting degree of hypocalcemia. The results suggest that although Hyp mice can respond to a disturbance of calcium homeostasis, the in vivo signal for the stimulation of renal 1-OHase activity may be set at a different threshold in the Hyp mouse; i.e. a lower serum calcium concentration is necessary for Hyp mice to initiate increased synthesis of 1,25(-OH)2D3.     

X-linked adrenoleukodystrophy mice demonstrate abnormalities in cholesterol metabolism

The neurodegenerative disorder X-linked adrenoleukodystrophy (X-ALD) is caused by ABCD1 mutations and characterized by very long-chain fatty acid (VLCFA) accumulation. Cholesterol-lowering normalized VLCFA in fibroblasts and plasma of X-ALD patients. We show that in cultured cells, cholesterol-loading induces ABCD1. In X-ALD mice, plasma cholesterol is elevated and not further increasable by cholesterol-feeding, whereas hepatic HMG-CoA reductase and Abcd2 are downregulated. Upon cholesterol modulation, brain VLCFA increased in X-ALD mice, but decreased in controls. In murine X-ALD fibroblasts, cholesterol-lowering did not normalize VLCFA. Thus, ALDP-deficiency and VLCFA are linked to cholesterol but species differences complicate evaluating cholesterol-lowering drugs in X-ALD mice.     

Bone mineral density of the spine and radius shaft in children with X-linked hypophosphatemic rickets (XLH)

X-linked hypophosphatemic rickets (XLH) is characterized by inadequate skeletal mineralization. The bone mineral density (BMD) of the radius shaft and the lumbar spine was determined in 13 children with XLH. Ten patients were on treatment, whereas three patients had discontinued treatment 20–32 months prior to this study. Two of them had radiological evidence of rickets.The radius shaft BMD was significantly diminished: Z score was −1.33 ± 0.89 (P < 0.001), while the BMD of lumbar spine was significantly augmented (Z score + 1.95 ± 1.17, P < 0.001). A positive correlation was found between the Z scores for the BMD of the radius shaft and spine. The two patients with overt rickets had lower radius shaft BMD values and a lesser increment of BMD of the spine. The BMD deficit of cortical bone may be related to the lack of efficacy of the treatment and/or to an intrinsic defect of the bone on this disease. On the other hand, the augmented BMD of the lumbar spine might reflect the overabundance of partially mineralized osteoid.The determination of the BMD of the radius shaft by SPA was a sensitive method for detecting abnormalities of the bone mass in XLH patients under treatment without radiological signs of rickets.     

Craniometric measurements of craniofacial malformations in the X-linked hypophosphatemic (Hyp) mouse on two different genetic backgrounds: C57BL/6J and B6C3H.

This study reports data on craniometric measurements in the X-linked hypophosphatemic (Hyp) mouse on two different genetic backgrounds: C57BL/6J and B6C3H. Heads of normal females "+/+," normal males "+/Y," heterozygous mutant females "Hyp/+," and hemizygous mutant males "Hyp/Y" for each genetic background were examined. Data were collected via skull measurements. On a C57BL/6J background, the neurocranium of mutants "Hyp/+" and "Hyp/Y" was shorter and slightly higher than in normal counterparts. On a B6C3H background, mutant mice "Hyp/+" and "Hyp/Y" were shorter in neurocranial length than in normal counterparts. Viscerocranial height was larger in "Hyp/Y" than in normal counterparts. No differences in neurocranial and mandibular height were found. Mutant mice on a C57BL/6J background were compared to mutant mice on a B6C3H background. No differences in neurocranial length were found. Cranial length was shorter in "Hyp/Y" on C57BL/6J than in "Hyp/+" on B6C3H. Facial length parameters were shorter in "Hyp/Y" on C57BL/6J than in "Hyp/Y" and "Hyp/+" mutant mice on B6C3H. Mandibular length was shorter in "Hyp/Y" on C57BL/6J than in "Hyp/+" on C57BL/6J and both mutant mice ("Hyp/Y" and "Hyp/+") on a B6C3H background. The results of this study indicate that craniofacial growth is less affected in mutant mice on a B6C3H genetic background than in mutant mice on a C57BL/6J genetic background.     

Mutational analysis of the PEX gene in patients with X-linked hypophosphatemic rickets.

X-linked hypophosphatemic rickets (HYP) is a dominant disorder characterized by renal phosphate wasting and abnormal vitamin D metabolism. PEX, the gene that is defective in HYP and is located on Xp22.1, is homologous to members of the neutral endopeptidase family. However, the complete coding sequence of the PEX cDNA, the structure of the PEX gene, and the role that PEX plays in phosphate transport remain unknown. We determined the genomic structure of the published PEX gene, which was found to be composed of 18 short exons, and demonstrated that the genomic organization of PEX shares homology to members of the family of neutral endopeptidases. Primer sets were designed from the intron sequence, to amplify each PEX exon from genomic DNA of HYP patients. Mutations in PEX were identified in 9/22 unrelated HYP patients, confirming that defects in PEX are responsible for HYP. The mutations detected included three nonsense mutations, a 1-bp deletion leading to a frameshift, a donor splice-site mutation, and missense mutations in four patients. Although the entire PEX gene has not been identified and some mutations may have been missed, the lack of detection of mutations in the remaining 13 patients, especially in 1 patient who has an apparently balanced, de novo 9;13 translocation, implies that there may be other loci involved in the generation of the HYP phenotype.     

Craniofacial and mucopolysaccharide abnormalities in Kniest dysplasia

Serial roentgencephalograms of a male patient with Kniest dysplasia were obtained between 1 7/12 and 11 3/12 years of age and were analyzed and compared to cephalometric normative data. The patient displayed macrocephaly with increased size of the neurocranium in all three dimensions. The cranial base angle was significantly flattened, partly as a result of anterior displacement of the sella turcica. The odontoid process was short and wide. At 11 years of age there was bony fusion between the anterior arch of the atlas and the odontoid process as well as between the posterior arch of the atlas and the cranial base. The facial skeleton, including the nasal bones, infra-orbital rims, maxilla and mandible, was retropositioned relative to the anterior cranial base. The mandibular retrognathia was pronounced at an early age but improved with growth. At age 11 years the patient had a straight facial skeletal profile. Examination of the patient's 24-hour urinary excretion of keratan sulfate revealed values markedly elevated for his age. Three additional patients with Kniest dysplasia demonstrated similarly increased excretion of this glycosaminoglycan. The diagnosis of Kniest dysplasia can usually be made from roentgenograms of the extremities, the spine, and the pelvis. However, the morphologic characteristics of the head, as shown by cephalometric analysis, and the increased urinary excretion of keratan sulfate add confirmatory evidence useful in differential diagnosis.     

New intragenic deletions in the Phex gene clarify X-linked hypophosphatemia-related abnormalities in mice

X-linked hypophosphatemic rickets (XLH) in humans is caused by mutations in the PHEX gene. Previously, three mutations in the mouse Phex gene have been reported: Phex^Hyp, Gy, and Phex^Ska1. Here we report analysis of two new spontaneous mutations in the mouse Phex gene, Phex^Hyp-2J and Phex^Hyp-Duk. Phex^Hyp-2J and Phex^Hyp-Duk involve intragenic deletions of at least 7.3 kb containing exon 15, and 30 kb containing exons 13 and 14, respectively. Both mutations cause similar phenotypes in males, including shortened hind legs and tail, a shortened square trunk, hypophosphatemia, hypocalcemia, and rachitic bone disease. In addition, mice carrying the Phex^Hyp-Duk mutation exhibit background-dependent variable expression of deafness, circling behavior, and cranial dysmorphology, demonstrating the influence of modifying genes on Phex-related phenotypes. Cochlear cross-sections from Phex^Hyp-2J/Y and Phex^Hyp-Duk/Y males reveal a thickening of the temporal bone surrounding the cochlea with the presence of a precipitate in the scala tympani. Evidence of the degeneration of the organ of Corti and spiral ganglion also are present in the hearing-impaired Phex^Hyp-Duk/Y mice, but not in the normal-hearing Phex^Hyp-2J/Y mice. Analysis of the phenotypes noted in Phex^Hyp-Duk/Y an Phex^Hyp-2J/Y males, together with those noted in Phex^Ska1/Y and Phex^Hyp/Y males, now allow XLH-related phenotypes to be separated from non-XLH-related phenotypes, such as those noted in Gy/Y males. Also, identification of the genetic modifiers of hearing and craniofacial dysmorphology in Phex^Hyp-Duk/Y mice could provide insight into the phenotypic variation of XLH in humans. *Bothauthorscontributedequallytothisresearch.     

Craniofacial abnormalities induced by ectopic expression of the homeobox gene Hox-1.1 in transgenic mice

Hox-1.1 is a murine homeobox-containing gene expressed in a time- and cell-specific manner during embryogenesis. We have generated transgenic mice that ectopically express Hox-1.1 from the chicken beta-actin promoter. In these mice Hox-1.1 expression was changed to an almost ubiquitous pattern. Ectopic expression of Hox-1.1 leads to death of the transgenic animals shortly after birth and is associated with multiple craniofacial anomalies, such as cleft palate, open eyes at birth, and nonfused pinnae. This phenotype is similar to the effects seen after systemic administration of retinoic acid during gestation. This suggests that retinoic acid embryopathy and the specific developmental defects caused by ectopic expression of a potential developmental control gene share a common pathogenic mechanism.     

The defect in transcellular transport of phosphate in the nephron is located in brush-border membranes in X-linked hypophosphatemia (Hyp mouse model)

We purified renal cortex brush-border membranes from mutant hemizygous hypophosphatemic (Hyp/Y) mice and male control (+/Y) littermates. Tenfold purification of mutant and wild-type membranes was obtained. Phosphate enters +/Y brush-border membrane vesicles by a saturable Na+-dependent arsenate-inhibited component and also by a diffusional component observed in the presence of a potassium gradient. Phosphate is not bound or incorporated significantly by mouse brush-border membrane vesicles. Parallel studies with rat renal cortex brush-border membrane vesicles revealed that phosphate and D-glucose transport in rat and mouse vesicles are similar and have the characteristics reported by other workers. Brush-border membrane vesicles prepared from Hyp/Y renal cortex have significant (p less than 0.001) partial loss of phosphate transport on the Na+-dependent arsenate-inhibited component. D-Glucose transport is not affected. Our previous studies reveal that other components of transcellular phosphate flux in kidney are normal. Therefore, we conclude that the mutant gene product in the Hyp mouse is confined to the brush-border membrane. Stability of the X-chromosome in mammalian evolution implied that the same gene product is involved in the classic human disease, familial 'vitamin D 'resistant' X-linked hypophosphatemia.     

B lymphocyte production in the bone marrow of mice with X-linked immunodeficiency (xid)

CBA/N mice carry an X-linked recessive immunodeficiency (xid) gene manifested by the absence of a B lymphocyte subpopulation, but the manner in which the xid gene exerts its effect on B lymphocyte development is unknown. The production of B lymphocytes in the bone marrow of CBA/N mice has now been compared with that of normal CBA/J mice by using two in vivo assays: immunofluorescence stathmokinetic studies measured pre-B cell proliferation, whereas radioautographic [3H]thymidine labeling was used to evaluate small lymphocyte turnover. Although the total cellularity of CBA/N mouse bone marrow was greater than normal, the absolute number of marrow small lymphocytes, pre-B cells, and B lymphocytes were all similar to those in CBA/J controls. Furthermore, in the bone marrow of CBA/N mice, the proliferation rate of pre-B cells, calculated from their rate of entry into mitosis, and the turnover rate of small lymphocytes, derived from their rate of [3H]thymidine labeling, were not significantly different from those seen in nondefective mice. The present findings that pre-B cell proliferation and small lymphocyte production proceed at similar rates in the bone marrow of xid and normal mice suggest that the xid gene does not act at the level of primary B cell genesis in the bone marrow. The findings are in accord with the view that the xid gene produces a maturation block or a functional abnormality among B lymphocytes in the peripheral lymphoid tissues rather than the deletion of a sublineage of B lymphocytes in the bone marrow.     

Co-existence of X-linked hypophosphatemic rickets (XLH) and primary hyperparathyroidism: case report and review of the literature

X-linked hypophosphatemic rickets (XLH) is a dominant disorder characterized by hypophosphatemia due to impaired renal tubular reabsorption of inorganic phosphate. Cardinal manifestations include defective calcification of cartilage and bone, growth retardation and resistance to phosphorus and vitamin D therapy. Although secondary hyperparathyroidism (HPT) is a common complication of treatment, autonomous HPT is rare, especially in the absence of previous phosphate therapy. We report a case of an adult untreated male XLH patient with primary HPT and give a brief review of the literature regarding the prevalence and pathophysiology of this complication.