Drosophila ClC-c Is a Homolog of Human CLC-5 and a New Model for Dent Disease Type 1

Figures

• 

  Figure 1. Sequence alignments. Segment of human ClC-5 (NP_000075.1, 746 a.a.; top rows) amino acid sequence stacked in alignment with Drosophila ClC-c (middle rows) and DD1 pathogenic variants (FBgn 0036466, 893 a.a.; bottom rows). Black lettering represents identical amino acids, whereas red represents different amino acids between human and Drosophila. Yellow highlighted amino acids represent locations of presently known DD1 point mutation sites.6,37,43,44,54–59 Cyan highlighted amino acids are involved in Cl− selectivity and/or proton gating.5,13,15,21,45 Boxed regions depict mutations used for electrophysiological experiments.

• 

  Figure 2. ClC-c expressed in Xenopus oocytes is electrogenic, voltage-gated, and pH sensitive. Voltage clamp experiments were conducted on oocytes injected with ClC-c cRNA or H2O perfused with ND96 solutions of pH 7.5 (A), pH 7.5 without Cl− (B), pH 6.0 (C), and pH 8.5 (D). *Significantly different from respective H2O-injected oocytes or as indicated by two-way ANOVA analysis with Bonferroni post-test. The conductance observed +80 mV compared across solutions by one-way ANOVA. Error bars represent mean±SEM. (E) Conductance from ClC-c–injected oocytes with the same α-notation (a, b, c) are statistically similar (P ≥ 0.05). *Significantly different conductance from H2O-injected oocytes for the respective solution (P < 0.05).

• 

  Figure 3. Chloride transport by ClC-c with homologous DD1 mutations is impaired. (A) Current responses to voltage clamp experiments for oocytes injected with wild-type ClC-c (WT), ClC-c with DD1 mutations (R494W, S393L, and Q777X), or H2O. *Significantly different from solution without Cl−’ by two-way ANOVA analysis with Bonferroni post-test (P < 0.05). (B) Conductances observed at 80 mV for each construct perfused by ND96 solutions with and without Cl−. Comparisons made by one-way ANOVA with Bonferroni comparisons. Error bars represent mean±SEM. Conductance from with Cl−’ solutions with the same α notation (a, b, c) are statistically similar (P ≥ 0.05). *Significantly different conductance between with Cl−’ and without Cl−’ solutions (P < 0.05).

• 

  Figure 4. ClC-c-GFP expressed in Malpighian tubules orient to the apical membrane and cytoplasm, but not the basolateral membrane. Apical localization is evident with both Uro-GAL4 and c507-GAL4 principal cell drivers (A4, C4, and E), as indicated by labeled arrows. Images include MTs from female UAS-ClC-c::eGFP (rows A, C, E) or control (w1118, rows B, D) crossed with Uro-GAL4 (rows A, B) and c507-GAL4 (rows C, D, E) drivers 7–14 days after eclosure. Confocal images of c507-Gal4 x UAS-ClC-c::eGFP in €(E) with labels for apical microvilli, cytosol, basolateral membrane, and trachea and rendered in 3D (E2). Dissected tubules were fixed with 4% paraformaldehyde (A–E), the basolateral membrane was stained with wheat germ agglutinin [WGA, (A–D)3, E1, E2], and nuclei are stained with DAPI in merged images at 40× magnification [(A–D)4]. Scale bars=20 µm. (F) Quantification of eGFP fluorescence (background corrected) from tubule traces in 40× images and analyzed by T-test comparisons with significant differences of *P < 0.001.

• 

  Figure 5. Knockdown of ClC-c in Drosophila in the Malpighian tubules, 7 days after eclosure. Knockdown of ClC-c was accomplished by crossing UAS-ClC-c-RNAi female flies with Uro-GAL4 male flies. Control crosses used female w1118 flies with Uro-GAL4 males. (A) qPCR expression of ClC-c mRNA in the tubules normalized to Rpl32 mRNA and relative to average of controls. (B) Concentration of protein secreted from the tubule ureter and compared by using the paired t-test. (C) Number of crystals in tubule pairs imaged by polarized light microscopy at 10× and quantified by ImageJ. *Significantly different by t-test, P < 0.05. Error bars represent mean±SEM. (D) Representative image of Clc-c-KD tubule pair with arrows depicting crystals. Scale bar=200 μm.

• 

  Figure 6. Crystals in Malpighian tubules induced by sodium oxalate. Ex vivo assessment of isolated MTs in 10 mM NaOx solution for 1 hour (A–C). Crystals from in MTs by 20 mM NaOx supplemented diet for 24 hours (D–F). Knockdown of ClC-c was accomplished by crossing UAS-ClC-c-RNAi female flies with Uro-GAL4 male flies. Control crosses used female w1118 flies with Uro-GAL4 males. Tubule pairs were imaged at 10× magnification by polarized light microscopy and quantified by ImageJ. Error bars depict mean±SEM. *Significantly different by t-test comparisons, P < 0.05.