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
基本信息
- 批准号:10314433
- 负责人:
- 金额:$ 6.89万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 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:
- 关键词:AffectAgeAmino AcidsAnimal ModelAnionsBiologicalBiological ModelsBiophysicsCLC GeneCalciumCalcium OxalateCell Culture TechniquesCellular biologyCharacteristicsChloridesClinicalComplicationCrystal FormationCrystallizationCultured CellsDataDent DiseaseDependenceDevelopmentDialysis procedureDietDrosophila genusDrosophila melanogasterElectrophysiology (science)EndosomesEvaluationExcretory functionExhibitsExperimental ModelsFamilyFellowshipGeneticHealthHumanIn VitroIndividualIon TransportIonsKidneyKidney CalculiKidney FailureKidney TransplantationLocationMalabsorption SyndromesMalpighian TubulesMembrane Transport ProteinsMethodsMicrodissectionModelingMolecular WeightMonitorMutateMutationNephrocalcinosisNephronsPartner in relationshipPathogenicityPatientsPhenotypePhysiologyPropertyProteinsPublishingRenal tubule structureResearchRiskRoleSeveritiesSiteStudy modelsTestingTimeTissuesTrainingTransgenesTransgenic OrganismsTranslatingTubular formationVariantabsorptionantibody detectionbasebiophysical propertiescalcificationcareerdietaryexperimental studyfluorophoreflygenetic manipulationhypercalciuriain vivoin vivo evaluationknock-downmouse modelrenal calciumrenal epitheliumsensorurinaryvoltagevoltage clamp
项目摘要
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。
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。
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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