Recently, we reported cloning PHEX, the gene causal in the pathogenesis of XLH and potentially contributing to the regulation of P homeostasis and vitamin D metabolism. Whereas studies have clearly identified PHEX mutations as the genetic defect in XLH and documented inactivating mutations in affected subjects, the mechanism(s) by which these mutations influence bone mineralization, vitamin D metabolism and P transport remains unknown. Nevertheless, continued advances have provided the framework to improve our understanding of P balance and vitamin D metabolism, as well as the pathogenesis of XLH. These accomplishments should permit definition of the interrelationships between null mutations of PHEX, renal P wasting and 25(OH)D-1alpha-hydroxylase activity and the impact of these abnormalities on bone and cartilage mineralization. In the current application, we propose to investigate the factor(s) underlying the characteristic phenotypic abnormalities in the hyp-mouse and the dependency of disease expression on mutated Phex in the osteoblast/osteocyte. Using animals with transgenic overexpression of Npt2 in renal proximal convoluted tubules, we will determine the role of abnormal renal P transport and consequent hypophosphatemia in the aberrant bone mineralization and vitamin D metabolism of mutant mice. Studies of bone mineralization will encompass an investigation of P effects during embryogenesis, youth and adult life. Investigation of vitamin D metabolism will focus on the effects of normal renal P transport on 1,25(OH)2D production in adults. In parallel studies, we will use mice with conditional Phex knockout in osteoblasts/osteocytes to determine whether these cells are the physiologically relevant sites for the mutated gene. These investigations will explore the effects of abnormal Phex function in osteoblasts/osteocytes on bone mineralization, renal P transport and vitamin D metabolism. As a complement to these studies, we will perform experiments designed to further examine the abnormal vitamin D metabolism in hyp-mice, which is characterized by dissociation between mRNA expression and renal 25(OH)D-alpha-hydroxylase activity. We will emphasize exploring if diminished protein translation or increased protein turnover underlies the decreased enzyme function. Our studies are significant, as they will clarify the PHEX dependent aspects of the XLH phenotype, while enhancing our understanding of vitamin D metabolism and P homeostasis.