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

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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.