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Abstract: SA-PO580

Dent Disease in Drosophila: Homologous Functions Between the Fly and Human Chloride Transporters

Session Information

  • Genetic Diseases: Diagnosis
    November 05, 2022 | Location: Exhibit Hall, Orange County Convention Center‚ West Building
    Abstract Time: 10:00 AM - 12:00 PM

Category: Genetic Diseases of the Kidneys

  • 1102 Genetic Diseases of the Kidneys: Non-Cystic

Authors

  • Reynolds, Carmen J., Mayo Foundation for Medical Education and Research, Rochester, Minnesota, United States
  • Gillen, Christopher M., Kenyon College, Gambier, Ohio, United States
  • Dow, Julian A.t., University of Glasgow, Glasgow, Glasgow, United Kingdom
  • Romero, Michael F., Mayo Foundation for Medical Education and Research, Rochester, Minnesota, United States
Background

Mutations in the 2Cl-/H+ transporter CLC-5 cause Dent Disease type 1 (DD1) and lead to progressive renal failure by age 20-40. A major characteristic of DD1 is the renal mishandling of Ca2+ that increases urinary Ca2+ excretion, calcium oxalate (CaOx) kidney stones, and kidney calcification. Our laboratory has identified Drosophila Clc-c as the homolog of CLC-5 with conserved amino acids at DD1 mutation sites. We hypothesize that Clc-c shares functional similarities in Cl- transport and Ca2+ homeostasis.

Methods

To determine the function of Clc-c, we assessed Cl- transport by Clc-c and Clc-c with homologous DD1 mutations by voltage clamp assays. We then knocked-down expression of Clc-c in the renal Malpighian tubules of flies to assess evaluated crystal formation and cation secretion.

Results

Voltage clamp experiments in Xenopus oocytes show that Clc-c is electrogenic with similar outward-rectifying currents in Cl- solutions as observed with human CLC-5. Chloride transport was decreased by acidic (pH 6.0), but not by alkaline solution (pH 8.5) when compared to the standard solution of pH 7.5. Homologous DD1 mutations S393L, R494W, and Q777x impaired Cl- transport activity similar to previous observations for CLC-5 DD1 mutations S244L, R345W, and Q629x (Tang et al, Physiol Rep 4[8], 2016). RNAi knockdown of Clc-c (Clc-c-KD) in the Drosophila renal tubules (Malpighian tubules, MT) resulted in 50% Clc-c mRNA expression. Tubule Ca-oxalate crystals in adult anterior MT (7 days after eclosure) were present in all Clc-c-KD and were more abundant (15±4 crystals per fly) compared to 5±2 crystals in control flies (P= 0.02). Using MT-secretion experiments, cations secreted from MT of Clc-c KD flies contained higher concentrations of Ca2+ while all other ions (Na+, NH4+, K+, Mg2+), and volume were the same as WT.

Conclusion

In conclusion, Drosophila Clc-c has similarities to human CLC-5 including impaired function with voltage-gated Cl- transport, homologous DD1 mutations, increased CaOx crystal formation, and elevated Ca2+ in urine secretions. Thus, Clc-c has the potential to be an important model for future investigations on the effects Ca2+ homeostasis in DD1.

Funding

  • NIDDK Support