The principal investigator's interest has been the subject of calcium an phosphate transport across membranes during maturation. The P. I. has utilized in vivo and in vitro techniques to define the overall picture of the transport of phosphate and calcium in his first grant applicatio (1983-1986). In the competitive renewal (1986-1989) the P. I. extended the knowledge to studies which addressed the subcellular movements of calcium and phosphate and their regulation by vitamin D. In the competitive renewal (1989-1994) the P. I. proposed studies to explore, in depth, the process of phosphate transport and its abnormalities in th mouse model of the X-linked hypophosphatemic rickets. The studies were successful in characterization of the defect in the hypophosphatemic mouse (Hyp). The principal investigator has developed an expression for the phosphate transporter (Na+-Pi) in Xenopus oocytes and has cloned the mouse renal Na+-Pi transporter. The expression of the Na+-Pi transporte was significantly decreased in Xenopus oocytes injected with renal poly(A)+ RNA from (Hyp) mice compared to controls, whereas, the expression was similar in Xenopus oocytes injected with intestinal poly(A)+ RNA from (Hyp) and control mice. Northern blot analysis showed that the mRNA level encoding the renal Na+-Pi transporter was 3-fold lower in the (Hyp) mouse compared to its control. These findings were confirmed by in situ hybridization studies. The current proposal will extend knowledge acquired during the tenure of the grant to explore the molecular defect in the phosphate transporter and to determine the pathogenesis of the disorder X-linked hypophosphatemia. We now have the molecular tools necessary to make fundamental advances in this field.
The specific aims will be directed toward determination of the level of the defect in the (Hyp) mice by examining the Na+-Pi transporter at the protein level (post-translational), the mRNA level (translational), and at the gene level (transcriptional). To address the question of whether the defect in the (Hyp) is related to a humoral factor, we will establis primary cultures of the renal proximal tubules from the control and (Hyp mice and examine the expression of the Na+-Pi transporter in vitro. These studies will shed light on the molecular mechanisms involved in th X-linked hypophosphatemia which represents the most common form of vitamin D-resistant rickets in man.
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