This project is going to address the question whether the sodium-phosphate co-transporter NaPi-IIc forms dimers or multimers when expressed in Xenopus oocytes and mammalian cell lines and whether human mutations in NaPi-IIc interfere with sodium-phosphate co-transport by the formation of non-functional heterodimers. Hereditary hypophosphatemic rickets with hypercalciuria (HHRH) is caused by homozygous or compound heterozygous missense or deletion mutations in SLC34A3/NaPi-IIc. HHRH is a disorder that persists life-long and heterozygous relatives of individuals affected by HHRH often have hypercalciuria and renal calcifications due to elevated 1,25(OH)2D levels as a result of renal phosphate losses. In contrast, mice lacking SLC34A3/NaPi-IIc show only mild hypercalcemia and absorptive hypercalciuria at an early age due to increased 1,25(OH)2D levels, but these animals do not develop hypophosphatemia, and heterozygous animals are normal. While haploinsufficiency could explain the hypercalciuria observed in heterozygous human carriers of NaPi-IIc mutations, it is therefore possible that some mutations, particularly those associated with a more severe phenotype such as renal stones, lead to dominant negative effects on wild-type NaPi-IIc or other sodium-phosphate co-transporters. In the past two years since the start of my KO8 """"""""Role of the renal sodium-phosphate co-transporter NaPi-IIc in phosphate homeostasis"""""""" I have established all techniques required for functional analysis of SLC34A3/NaPi-IIc mutations, which were discovered in patients referred with hereditary hypophosphatemic rickets with hypercalciuria (HHRH) and related disorders. When co-expressed in Xenopus oocytes, the mutants T137M and V446Stop impair wild-type NaPi-IIc function in a dominant-negative fashion, while NaPi-IIa function was unaffected. Using cross-linking and co-immunoprecipitation techniques, I have shown dimerization of wild-type NaPi-IIc after over-expression in Xenopus oocytes. These experiments were carefully controlled for non-specific protein aggregation effects. My plan is to repeat these studies in OK, HEK293 and LLCPK-1 cells to show that dimerization also occurs in mammalian proximal tubular cell lines. I also plan to establish the FRET technology, which does not require cross-linking and purification of dimers as an independent method to show that dimerization occurs. I will furthermore use adenoviral transduction of mutant and wild-type NaPi-IIc to show the dominant negative effect in OK, HEK293 and LLCPK-1 cells. The proposed in vitro studies will provide first proof that dimerization of NaPi-IIc is important for it's function. If successful, these data will support a systematic evaluation of the human heterozygous carriers of T137M and V446Stop in metabolic ward studies, and the use of transgenic or knock-in mouse models to test the hypothesis whether these mutations can have a dominant negative effect and lead to kidney stone formation and/or nephrocalcinosis in vivo.

Public Health Relevance

SLC34A3/NaPi-IIc is a renal sodium-phosphate co-transporter, which helps reclaiming phosphate from the urine to increase blood phosphate levels. Homozygous mutations in NaPi-IIc cause hereditary hypophosphatemic rickets with hypercalciuria (HHRH) in humans, but it is unclear why also heterozygous carriers of NaPi-IIc mutations sometimes have a relatively severe phenotype such as renal stones. This proposal is going to test the hypothesis that some mutations may interfere with wild-type co-transporters by the formation of non-functional heterodimers thereby explaining the relatively severe phenotype of heterozygous carriers.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Small Research Grants (R03)
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Diabetes, Endocrinology and Metabolic Diseases B Subcommittee (DDK)
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Rankin, Tracy L
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Massachusetts General Hospital
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Bergwitz, Clemens; Jüppner, Harald (2012) FGF23 and syndromes of abnormal renal phosphate handling. Adv Exp Med Biol 728:41-64
Yu, Y; Sanderson, S R; Reyes, M et al. (2012) Novel NaPi-IIc mutations causing HHRH and idiopathic hypercalciuria in several unrelated families: long-term follow-up in one kindred. Bone 50:1100-6
Phulwani, Priya; Bergwitz, Clemens; Jaureguiberry, Graciana et al. (2011) Hereditary hypophosphatemic rickets with hypercalciuria and nephrolithiasis-identification of a novel SLC34A3/NaPi-IIc mutation. Am J Med Genet A 155A:626-33
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