Drosophila Clc-c model of Dent disease and human ClC-5 mutations

项目摘要/摘要 人类2Cl-/H+膜转运蛋白ClC-5的突变会导致临床上的Dent病 特点是肾脏低分子蛋白(LMWP)、钙离子(高钙尿)、 20-40岁时患肾结石、肾组织钙化和进行性肾功能衰竭的风险增加。 ClC-5在LMWP中的作用已被推测和验证，但它在肾脏钙离子错误处理中的作用是 由于缺乏合适的实验模型而未知。我推测果蝇是黑腹果蝇 (即果蝇)可能是研究这种氯离子转运蛋白的一个很好的模式生物，理由如下：(1) 果蝇天生表达与人类ClC-5 60%相同的氯离子转运体(Clc-c)，(2)钙晶体 苍蝇肾上皮细胞的形成重现哺乳动物/人肾结石，并易于诱导 实时定量，(3)果蝇的遗传操作简单、快速(即每7个人中就有一个交配和后代)。 10天)，并且特定于组织，以允许利用突变的天然或转基因来评估体内功能 蛋白质，以及(4)表达基因编码的荧光离子传感器(H+，Cl-，Ca~(2+))的苍蝇系允许 体内离子传输的评估。我们的初步评估和实验已经证实，所有已知的 ClC-5 Dent突变在ClC-c序列中是保守的，ClC-c和ClC-5具有相似的电压- 依存性和离子传输特性。此外，肾小管中ClC-c的基因敲除增加了钙离子。 草酸盐晶体形成与野生型小管的比较，类似于Dent肾结石形成的增加 疾病。在这项提议中，我假设果蝇Clc-c具有额外的生物物理和表型。 与CLC-5的特性相同，并且同样受到保守氨基酸残基突变的影响。我的第一次 目的利用电压钳技术比较ClC-c和ClC-5的生物物理特性。 评估，包括确定同源Dent突变对各自的活性的影响 传送器。其次，我打算在体内评价果蝇ClC-c。第二个目标将通过(1)进行测试 用抗体检测检测ClC-c的细胞和亚细胞定位，通过表达检测离子 基因编码的氯离子和氢离子离子传感器，以及(2)比较晶体形成和离子分泌 用体内和体外方法研究WT果蝇和CLC-c基因敲除果蝇的马氏管。这些 结果将决定Clc-c是否是研究肾钙生物学作用的有效模型。 和蛋白质吸收的这一氯-转运蛋白和Dent病。拟议的团契培训计划是 侧重于发展不同的技术视角和润色专业发展活动 这意味着在研究领域取得成功。

Project Summary/Abstract Mutations in the human 2Cl-/H+ membrane transporter, CLC-5, cause Dent disease, which is clinically characterized by increased renal excretion of low molecular weight protein (LMWP), Ca2+ (hypercalciuria), increased risk for kidney stones, calcification of the kidney tissue, and progressive renal failure by age 20-40. The involvement of CLC-5 in LMWP has been postulated and tested, but its role in renal Ca2+ mishandling is unknown due to the lack of an appropriate experimental model. I hypothesize that Drosophila melanogaster (i.e., fruit fly) may be an excellent model organism to study this Cl- transporter for the following justifications: (1) flies natively express a Cl- transporter (Clc-c) that is >60% identical to human CLC-5, (2) calcium crystal formation in fly renal epithelia recapitulate mammalian/human kidney stones and are easily induced for quantification in real time, (3) genetic manipulations in flies are easy, quick (i.e., one mating and progeny in 7- 10 days), and tissue-specific to allow for evaluation of in vivo function with mutated native or transgenic proteins, and (4) fly lines expressing genetically encoded fluorescent ion-sensors (H+, Cl-, Ca2+) allow assessment of in vivo ion transport. Our preliminary evaluations and experiments have identified that all known CLC-5 Dent mutations are conserved in the Clc-c sequence and that Clc-c and CLC-5 have similar voltage- dependent and ion-transport characteristics. In addition, knockdown of Clc-c in renal tubules increases calcium oxalate crystal formation compared to wildtype tubules, similar to increased kidney stone formation in Dent disease. In this proposal, I hypothesize that Drosophila Clc-c shares additional biophysical and phenotypical properties with those of CLC-5 and is similarly affected by mutations of conserved amino acid residues. My first aim proposes to compare the biophysical features of Clc-c to those of CLC-5 by using voltage clamp assessments, including determining the effect of homologous Dent mutations on the activity of the respective transporter. Second, I intend to evaluate Drosophila Clc-c in vivo. The second aim will be tested by (1) examining the cellular and subcellular localizations of Clc-c by antibody detection and of ions by expressing genetically-encoded ion sensors for Cl- and H+, and (2) comparing crystal formation and ion secretion in Malpighian tubules among WT flies and Clc-c knock-down flies by both in vivo and ex vivo methods. These results will determine if Clc-c is an efficient and effective model for studying the biological role in renal calcium and protein absorption of this Cl- transporter and Dent disease. The proposed fellowship training plan is focused on developing diverse technical perspectives and embellishing professional development activities that translate to a successful career in research.