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

Dent 病和人类 ClC-5 突变的果蝇 Clc-c 模型

• 批准号: 10314433
• 负责人: Carmen Reynolds
• 金额: $ 6.89万
• 依托单位: MAYO CLINIC ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-29 至 2022-07-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10314433
• 关键词: Affect; Age; Amino Acids; Animal Model; Anions; Biological; Biological Models; Biophysics; CLC Gene; Calcium; Calcium Oxalate; Cell Culture Techniques; Cellular biology; Characteristics; Chlorides; Clinical; Complication; Crystal Formation; Crystallization; Cultured Cells; Data; Dent Disease; Dependence; Development; Dialysis procedure; Diet; Drosophila genus; Drosophila melanogaster; Electrophysiology (science); Endosomes; Evaluation; Excretory function; Exhibits; Experimental Models; Family; Fellowship; Genetic; Health; Human; In Vitro; Individual; Ion Transport; Ions; Kidney; Kidney Calculi; Kidney Failure; Kidney Transplantation; Location; Malabsorption Syndromes; Malpighian Tubules; Membrane Transport Proteins; Methods; Microdissection; Modeling; Molecular Weight; Monitor; Mutate; Mutation; Nephrocalcinosis; Nephrons; Partner in relationship; Pathogenicity; Patients; Phenotype; Physiology; Property; Proteins; Publishing; Renal tubule structure; Research; Risk; Role; Severities; Site; Study models; Testing; Time; Tissues; Training; Transgenes; Transgenic Organisms; Translating; Tubular formation; Variant; absorption; antibody detection; base; biophysical properties; calcification; career; dietary; experimental study; fluorophore; fly; genetic manipulation; hypercalciuria; in vivo; in vivo evaluation; knock-down; mouse model; renal calcium; renal epithelium; sensor; urinary; voltage; voltage clamp

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.

项目概要/摘要 人类 2Cl-/H+ 膜转运蛋白 CLC-5 的突变会导致临床上常见的 Dent 病 其特点是低分子量蛋白 (LMWP)、Ca2+（高钙尿症）的肾脏排泄增加， 20-40 岁时，肾结石、肾组织钙化和进行性肾衰竭的风险增加。 CLC-5 在 LMWP 中的参与已被假设和测试，但其在肾 Ca2+ 处理不当中的作用尚不清楚 由于缺乏合适的实验模型，未知。我推测果蝇 （即果蝇）可能是研究这种 Cl-转运蛋白的绝佳模式生物，理由如下：(1) 果蝇天然表达与人类 CLC-5 相同 >60% 的 Cl-转运蛋白 (Clc-c)，(2) 钙晶体 果蝇肾上皮细胞的形成再现了哺乳动物/人类肾结石，并且很容易诱导 实时定量，（3）果蝇的基因操作简单、快速（即一次交配并在 7- 10 天），并且具有组织特异性，可以评估突变的天然或转基因的体内功能 蛋白质和 (4) 表达基因编码荧光离子传感器（H+、Cl-、Ca2+）的蝇系允许 体内离子转运的评估。我们的初步评估和实验表明，所有已知的 CLC-5 Dent 突变在 Clc-c 序列中是保守的，并且 Clc-c 和 CLC-5 具有相似的电压- 依赖性和离子传输特性。此外，肾小管中 Clc-c 的敲低会增加钙 与野生型肾小管相比，草酸盐晶体形成，类似于 Dent 肾结石形成增加 疾病。在这个提案中，我假设果蝇 Clc-c 具有额外的生物物理和表型特征 与 CLC-5 的特性相同，并且类似地受到保守氨基酸残基突变的影响。我的第一个 goal 建议使用电压钳比较 Clc-c 与 CLC-5 的生物物理特征 评估，包括确定同源 Dent 突变对各自活性的影响 运输者。其次，我打算在体内评估果蝇 Clc-c。第二个目标将通过 (1) 进行测试 通过抗体检测检查 Clc-c 的细胞和亚细胞定位，通过表达检查离子的细胞和亚细胞定位 Cl- 和 H+ 的基因编码离子传感器，以及 (2) 比较晶体形成和离子分泌 通过体内和离体方法观察 WT 果蝇和 Clc-c 敲低果蝇中的马氏小管。这些 结果将确定 Clc-c 是否是研究肾钙生物学作用的有效模型 以及这种 Cl-转运蛋白的蛋白质吸收和 Dent 病。拟议的奖学金培训计划是 专注于发展多样化的技术视角和完善专业发展活动 这转化为研究事业的成功。