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影响的贝都因大家族中，我们最近描述了编码Napi-LLC的SLC34A3基因的纯合子移码突变(C.228del)(Am J Hum Genetics，78：179-192,2006)。对该家系的进一步分析表明，几个纯合子个体仅表现为高钙尿，而在调查时没有与软骨病一致的骨骼变化。同样，一些杂合子个体没有表现出高钙尿症的证据。除C.228del突变外，在另外7个HHRH家系和散发性病例中还发现了几种不同的纯合或复合杂合性错义、移码和缺失SLC34A3突变，支持HHRH是一种单基因疾病的结论。其中一些病例可见肾结石。EGFP标记的野生型和突变型Napi-LLC在负鼠肾脏细胞中的瞬时表达表明，Napi-LLC突变G196R、R468VV和delL.527的存在严重干扰了插入刷状缘膜，而其他Napi-LLC突变在根尖斑块中正常表达。为了评估这些完全表达的Napi-LLC突变对磷吸收的影响，我们建立了非洲爪哇卵母细胞mRNA注射模型(Aim 1a)。目的1b将扩大这些体外研究，以确定与napi-11a相比，napi-LLC在哺乳动物磷酸盐稳态中的相对重要性。此外，还构建了靶向载体来切割Napi-LLC的小鼠同源基因NPT-2c。杂合子的动物预计只会发生特发性高钙尿，而尽管NPT-2a的存在(目标2)，纯合子的NPT-2c消融被预测会导致HHRH。由于SLC34A3突变的杂合子和纯合子个体在表型上的这种明显差异，确定哪些因素影响这种突变在贝都因家族中的表达将是重要的(目标3)。肾钙素沉着症、肾结石和慢性肾脏疾病(CKD)患者会出现肾脏钙和磷的异常处理，这些疾病今天影响了大量美国人。NIH颁发的K08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，并让我在成为一名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.