Role of the renal sodium-phosphate co-transporter NaPi-IIc in phosphate homeostas

描述（由申请人提供）： HHRH是一种常染色体隐性遗传病。在一个受HHRH影响的大型贝都因家族中，我们最近描述了在编码NaPi-llc的基因SLC 34 A3中的纯合移码突变（c.228del）（Am J Genetics，78：179-192，2006）。对该家族的进一步分析表明，几个纯合子个体仅显示高钙尿症，而在调查时不存在与佝偻病一致的骨变化。同样，一些杂合子个体没有高钙尿症的证据。除了c.228del突变，几个不同的纯合或复合杂合错义，移码和缺失SLC 34 A3突变被发现在7个额外的HHRH激酶和零星的情况下，支持的结论，HHRH是一个单基因疾病。在这些病例中的一些病例中观察到肾结石。EGFP（增强的绿色荧光蛋白）标记的野生型和突变体NaPi-llc在负鼠肾（OK）细胞中的瞬时表达表明，插入刷状缘膜受到NaPi-llc突变G196 R、R468 VV和delL.527的存在的严重干扰，而其他NaPi-llc突变在顶端斑中显示正常表达。为了评估这些完全表达的NaPi-llc突变对磷酸盐摄取的影响，我们建立了非洲爪蟾卵母细胞mRNA注射模型（Aim 1a）。目的1b将扩展这些体外研究，以确定NaPi-llc与NaPi-lla相比在哺乳动物磷酸盐稳态中的相对重要性。此外，构建靶向载体以消除编码Npt-2c（NaPi-llc的鼠直系同源物）的基因。预计杂合动物仅发生特发性高钙尿症，而尽管存在Npt-2a，但预计纯合Npt-2c消融会导致HHRH（目的2）。由于SLC 34 A3突变的杂合和纯合个体的表型存在明显的变异性，因此确定哪些因素导致贝都因人家族中该突变的表达率将是重要的（目的3）。肾钙质沉着症、肾结石和慢性肾病（CKD）患者的肾脏钙和磷酸盐处理异常，这些疾病影响着当今大量美国人。NIH的K 08职业发展奖将帮助我进一步探索NaPi-llc在哺乳动物生物学中的重要性，并让我在成为独立科学家的道路上获得新的技能。

DESCRIPTION (provided by applicant): HHRH is an autosomal recessive disorder. In a large Bedouin kindred affected by HHRH, we recently described a homozygous frameshift mutation (c.228del) in SLC34A3, the gene encoding NaPi-llc (Am J Hum Genetics, 78:179-192, 2006). Further analysis of this family indicated that several homozygous individuals revealed only hypercalciuria, while bone changes consistent with rickets were absent at the time of investigation. Similarly, some heterozygous individuals showed no evidence for hypercalciuria. Besides the c.228del mutation, several different homozygous or compound heterozygous missense, frameshift and deletion SLC34A3 mutations were found in seven additional HHRH kindreds and in sporadic cases, supporting the conclusion that HHRH is a monogenic disorder. Renal stones were seen in some of these cases. Transient expression of EGFP (enhanced green fluorescence protein)-tagged wild-type and mutant NaPi-llc in Opossum kidney (OK) cells indicated that insertion into the brush border membrane is severely disturbed by the presence of NaPi-llc mutations G196R, R468VV, and delL.527, while other NaPi-llc mutations show normal expression in apical patches. To assess the impact of these fully expressed NaPi-llc mutations on phosphate uptake, we established a Xenopus oocyte mRNA injection model (Aim 1a). Aim 1b will expand these in vitro studies to determine relative importance of NaPi-llc compared to NaPi-lla in mammalian phosphate homeostasis. Furthermore, a targeting vector was constructed to ablate the gene encoding Npt-2c, the murine ortholog of NaPi-llc. Heterozygous animals are expected to develop only idiopathic hypercalciuria, while the homozygous ablation of Npt-2c is predicted to lead to HHRH, despite the presence of Npt-2a (Aim 2). Because of this apparent variability in phenotype of individuals who are either heterozygous and homozygous for mutations in SLC34A3, it will be important to determine which factors contribute to the expressivity of this mutation in the Bedouin kindred (Aim 3). Abnormal renal calcium and phosphate handling is seen in patients with nephrocalcinosis, nephrolithiasis and chronic kidney disease (CKD), disorders that affect a large number of Americans today. A K08 career development award by the NIH will help me to further explore the importance of NaPi-llc in mammalian biology, and allow me to acquire new skills on my way to becoming an independent scientist.