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This thesis includes three studies examining the role of the type I (NPT1) and type II (NPT2) renal sodium (Na+)-phosphate (Pi) cotransporter genes in inherited hypophosphatemias. In the first study, the chromosomal locations of the NPT1 and NPT2 genes in human and rabbit are determined by physical mapping techniques. The NPT1 and NPT2 genes map respectively to human chromosomes 6p22 and 5q35 and to rabbit chromosomes 12p11 and 3p11. The localization of the two cotransporter genes to autosomes excludes them as candidate genes for X-linked hypophosphatemia. In addition, these assignments agree with the previously reported homology between rabbit chromosome 12 and human chromosome 6 and provide the basis for the establishment of a conserved syntenic group between rabbit chromosome 3 and human chromosome 5. / The goal of the second study was to clone, sequence and characterize the structure of the human NPT2 gene in order to design intronic primers to amplify NPT2 exons from patient DNA. Parallel experiments were performed on the mouse Npt2 gene, so that a vector could be designed to knockout the mouse Npt2 gene. In both species, the type II renal Na+-Pi cotransporter gene is approximately 16kb in length and is comprised of 13 exons and 12 introns. This work provides a basis for the study of the regulation of NPT2 transcription and facilitates the screening of DNA samples from patients with autosomally inherited disorders of renal Pi reabsorption for mutations in the NPT2 gene. / In the third study, polymorphic markers flanking the NPT1 and NPT2 genes were typed in members of a Bedouin kindred segregating the autosomal disorder Hereditary hypophosphatemic rickets with hypercaloiuria (HHRH). Genotype data were examined for excess homozygosity and allele sharing among affected pedigree members. Data did not reveal excess allele sharing on either chromosome 6 or 5, where the NPT1 and NPT2 genes are located, but suggested chromosome 3p as a site for further investigation. Identification of a HHRH locus is the first step toward identifying a gene involved in the pathophysiology of this disorder.

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The X-linked Hyp mutation is characterized by a specific defect in phosphate (Pi) transport at the renal brush-border membrane (BBM). To understand the mechanism for the 50% decrease in Vmax of the high affinity Pi transport system in BBM of Hyp mice, we compared the effects of external Na$ sp+$ concentration, membrane potential, external pH, and Pi transport inhibitors on Pi uptake in BBM vesicles (BBMV) prepared from normal mice and Hyp littermates. / The apparent affinity for Na$ sp+$, the Na$ sp+{:}$Pi stoichiometry, the response to membrane potential and the response to external pH are similar in BBMV from both normals and mutants. / The Ki for phosphonoformic acid (PFA) inhibition of Na$ sp+$-Pi cotransport is lower in BBMV prepared from Hyp mice when compared to normal mice but not different in BBMV from Pi-deprived mice which are characterized by an increase in Vmax of the high affinity Na$ sp+$-Pi cotransport system. / We conclude that the decrease in Vmax of the high affinity Na$ sp+$-Pi cotransport system in the Hyp mouse is not the result of an inappropriate response of the transport system to Na$ sp+$, membrane potential or pH.

Na$ sp+$-Pi cotransport across the brush border membrane is the rate limiting step in renal Pi reabsorption. To determine its precise role in the maintenance of Pi homeostasis, we cloned and characterized the renal-specific Na$ sp+$-Pi cotransporter/Npt2 gene and generated a gene targeting vector for the creation of a knockout mouse. The gene was cloned by screening a genomic DNA library with a rat Npt2 cDNA probe. The Npt2 gene is approximately 17kb and contains 13 exons and 12 introns. A targeting construct was generated by inserting 5$ sp prime$ and 3$ sp prime$ homologous arms of 2.1 and 2kb, respectively, into the pPNT vector and replacing 7.7kb of Npt2 with a neomycin resistance gene. The vector also contained the herpes simplex virus thymidine kinase gene for negative selection. After electroporation into embryonic stem cells, clones were picked following selection in G418 and FIAU. Of 100 doubly-resistant clones that were screened by Southern analysis, 6 positive clones were detected giving a targeting frequency of 6%.

X-linked hypophosphatemia (XLH), the most prevalent form of inherited rickets in humans, is caused by mutations in the PHEX gene, which encodes a protein with high homology to the M13 family of type-II integral membrane zinc metallopeptidases. We created an online mutation database, PHEXdb (http://data.mch.mcgill.ca/phexdb), to catalogue PHEX mutations identified in XLH patients, and found that missense mutations account for 22% of the 157 mutations reported to date. We also undertook to examine the effects of eight missense mutations (C85R, D237G, Y317F, G579R, G579V, S711R, A720T, and F731Y) on synthesis, glycosylation, cellular trafficking, and catalytic activity of the recombinant proteins using several approaches. The wild-type protein was resistant to endoglycosidase H (endo H), indicating that it is fully glycosylated. In addition, biotinylation and immunofluorescence studies revealed that the wild-type protein resides at the cell surface. The D237G, Y317F and F731Y mutant PHEX proteins were also endo H resistant and thus terminally glycosylated. In contrast, endo H digestion demonstrated that C85R, G579R, G579V, S711R and A720T were not terminally glycosylated. Furthermore, immunofluorescence showed that C85R, G579R and S711R were sequestered in the endoplasmic reticulum (ER). A secreted form of wild-type and mutant PHEX (secPHEX) proteins was generated to examine catalytic activity, using a synthetic fluorogenic peptide substrate. For this purpose, rescue of ER-trapped mutant proteins was attempted by growing transfected cells at 26°C. Low temperature was able to rescue three of the five trapped mutant proteins (G579V, S711R and A720T). Residual catalytic activity was observed with four mutant proteins (D237G, Y317F, A720T and F731Y) relative to the wild-type. However, the rescued S711R mutant was devoid of catalytic activity. Finally, limited proteolysis with trypsin and endoproteinase Glu-c revealed that the mutations D237G and F731Y induce conform

This study shows that mice homozygous for the disrupted renal sodium-phosphate (Na+-Pi) cotransporter, Npt2, (Npt2 KO) failed to show an age-dependent decrease in renal Na+-Pi cotransport or an adaptive increase in renal Na+-Pi cotransport in response to dietary Pi restriction. None of the other known renal Na+ -Pi cotransporters could compensate for the loss of Npt2. Additionally, Npt2 gene ablation resulted in a marked decrease in osteoclast number that persisted with age. Although mineral apposition rate was normal at 25- and 115-days of age in Npt2 KO mice, bone formation rate was increased at 115-days of age. These data demonstrate that Npt2 gene expression is necessary for an age-dependent decrease in renal Na+-Pi cotransport and for the renal adaptive response to dietary Pi deprivation, and that Npt2 expression is essential for normal osteoclast function and influences bone formation.

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