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Hypophosphatemia : cDNA cloning of a Na^+-dependent phosphate co-transporter from human kidney

低磷血症：从人肾中克隆 Na+ 依赖性磷酸盐协同转运蛋白的 cDNA

基本信息

批准号：
05670145
负责人：
MIYAMOTO Kenichi
金额：
$ 1.34万
依托单位：
Tokushima University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1993
资助国家：
日本
起止时间：
1993 至 1994
项目状态：
已结题

项目摘要

Hypophosphatemic vitamin D-resistant rickets is the most common form of hypophosphatemic rickets in humans. It is characterized by low renal tubular reabsorption of phosphate, low plasma phosphate, absence of elevated 1,25-dihydroxy-vitamine D despite the presence of hypophosphatemia and osteomalacic bone disease. In the present study, a cDNA (NPT-1) encoding a protein 69% identical in amino acid sequence to that of the Na/Pi cotransporter NaPi-1 was isolated from a human kidney library. Injected of RNA transcribed from this clone, NPT-1, into Xenopus oocuytes results in expression of a Na/Pi cotransport activity with high affinity for transport (Km=0.29mM). Kinetic characterization ([Pi], [Na]) showed that expressed transport activity has properties similar to that of oocytes injected juman kidney poly (A)^+RNA.Northern blotting showes that NPT-1 mRNA is expressed in kidney cortex, liver and brain but not in other tissues. Hybrid depletion with antisense oligonucleotide of NaPi-3 and NPT-1 completely inhibited poly(A)^+RNA-induced Na^+-dependent phosphate uptake in oocytes. These finding indicate that two high affinity Na/Pi cotransporter (NaPi-3 and NPT-1) are present in human kidney cortex.NPT-1 maps the location to human chromosome 6. NaPi-3 maps the location to human chromosome 5. The role of high affinity Na/Pi cotransporter in Hereditary Hypophosphatemic Rickets with Hypercarciuria (HHRH) which is autosomally inherited disorder and caused by defective reabsorption of Pi in the proximal tubule of the kidney is under study.

低磷性维生素d抗性佝偻病是人类最常见的低磷性佝偻病。其特点是肾小管对磷酸盐的重吸收低，血浆磷酸盐低，尽管存在低磷血症和骨疏松性骨病，但缺乏升高的1,25-二羟基维生素D。在本研究中，从人肾文库中分离到一个cDNA (NPT-1)，其编码的蛋白质与Na/Pi共转运蛋白NaPi-1的氨基酸序列相同69%。将该克隆转录的RNA NPT-1注入爪蟾卵母细胞，可表达Na/Pi共转运活性，具有高转运亲和力（Km=0.29mM）。动力学表征（[Pi], [Na]）表明，表达的转运活性与注射人肾poly (A)^+RNA的卵母细胞具有相似的性质。Northern blotting显示NPT-1 mRNA在肾皮质、肝和脑中表达，而在其他组织中不表达。NaPi-3和NPT-1的反义寡核苷酸杂交耗竭完全抑制poly(A)^+ rna诱导的Na^+依赖性磷酸盐摄取。这些发现表明两种高亲和力的Na/Pi共转运蛋白（NaPi-3和NPT-1）存在于人肾皮质中。NPT-1将其定位在人类6号染色体上。NaPi-3将其定位到人类5号染色体上。高亲和力Na/Pi共转运蛋白在遗传性低磷佝偻病伴高钙血症（HHRH）中的作用正在研究中。HHRH是一种常遗传性疾病，由肾近端小管对Pi的重吸收缺陷引起。