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

Visual Abstract


Introduction
2][3][4][5][6] The physiological role of ClC-5 is portrayed by the clinical characteristics of patients with Dent disease type 1 (DD1) who have a disease-causing variant in the X-chromosome gene, CLCN5.][9][10][11] The notion that an impaired Cl 2 transporter can cause Ca 21 dysregulation suggests an intricate relationship among these ions that remains unresolved because of the limitations of experimental models.Given the limited treatment options available for patients with DD1, achieving a better understanding of the underlying mechanisms of disease could lead to muchneeded therapeutic targets.
Current model systems to determine the mechanistic actions of ClC-5 are limited to cultured cells and knockout mice.][17][18][19][20][21] Cell culture, unfortunately, cannot appropriately recapitulate altered Ca 21 metabolism.3][24] To gain a better understanding on the dynamics of Ca 21 dysregulation, we need to use a model organism that demonstrates the Ca 21 dysregulation and allows for whole organism experimentation.
6][27] Insects use Malpighian tubules (MTs) to secrete solutes from the hemolymph and produce urine similarly to renal epithelia.Circulating hemolymph is filtered by nephrocytes equipped with slit diaphragm structures and proteins akin to mammalian podocytes. 28,29Solutes are removed by principal cells of the MT that are rich with luminal microvilli while fluid homeostasis is maintained by the MT stellate cells. 30Drosophila in particular have been used to study renal epithelial ion and water secretion, as well as models for several genetic kidney diseases. 31Urine secretions from the MT can be quantified per individual tubule if sensitive analytical equipment is available. 31Within MTs, crystals can form that are comparable with intratubular microlithiasis occurring in human kidneys.3][34] Not only are secretions and crystal formation by the MT quantifiable but the rapid fecundity of Drosophila lends well to genetic manipulations with the GAL4/UAS-directed expression or CRISPR/Cas9 editing methods.
Here, we introduce a Drosophila model for studying the mechanistic function of this Cl 2 transporter.6][37] In addition, we characterize the effect of ClC-c RNAi knockdown in Drosophila in replicating DD1 features.

Molecular Biology
Drosophila ClC-c was subcloned from MTs of w 1118 flies into pGEMHE Xenopus oocyte and into pUASTattB:eGFP Drosophila expression plasmids using primers described (Supplemental Table 1).Mutations were incorporated by site-directed mutagenesis for S393L, R494W, and Q777X into pGEMHE:ClC-c.The pGEMHE:ClC-c construct was amplified with respective mutagenesis primers by Phusion Hot Start II DNA Polymerase PCR (30 cycles of 98°C [2s]: 72°C [120s]; Thermo Fisher Scientific) and then treated with Dpn1 (New England Biolabs) at 37°C for 1 hour.PCR products were purified, transfected into confluent bacteria, and grown with ampicillin.Plasmids were isolated from bacteria by Qiagen Maxi-prep, and the concentrations were determined by nanodrop spectrometer.Mutations were confirmed by Sanger sequencing (Genewiz).

cRNA Synthesis
DNA constructs cloned into the pGEMHE expression vector were linearized by Not1 or Nhe1 (New England Biolabs, Ipswich, MA) restriction digest overnight at 37°C.cRNA was synthesized from the linear vector using the Invitrogen mMessage Machine T7 Transcription Kit (Thermo Fisher Scientific, Waltham, MA).

Xenopus laevis Oocytes
Frogs were maintained in accordance with and approved by the Institutional Animal Care and Use Committee of the Mayo Clinic College of Medicine & Science.Oocytes were collected by surgical laparotomy and follicular membranes digested as described previously. 38Cells were maintained in Leibovitz's 15 Media (Thermo Fischer, Waltham, MA) with 2.5% penicillin/streptomycin (Gibco, Thermo Fischer, Waltham, MA) and 1 mM HEPES at 16°C.One day after collection, oocytes were injected with 10 ng cRNA (in 50 nl H 2 O) or vehicle and then experiments were performed 3-6 days after injection.

Electrophysiology
Two-electrode voltage clamp experiments were performed on oocytes with an oocyte clamp amplifier (OC-725C, Warner Instruments, Hamden CT) using Heka software (Wiesenstrasse, Germany) as previously described. 36,37Briefly, electrodes filled with 3M KCl micropipette solution were inserted into oocytes submerged in ND96 solution (96 mM NaCl, 2 mM KCl, 1.8 mM CaCl 2 1 mM MgCl 2 , 5 mM HEPES, pH 7.5) at room temperature.Membrane voltage was maintained at 260 mV, and currents were recorded for 75 ms pulses from 280 mV to 180 mV in 120 mV increments.Experiments were repeated with alternate ND96 solutions at pH 6.0, pH 8.5 (pH adjusted by NaOH), or 0 Cl 2 (96 mM Na-gluconate, 2 mM Kgluconate, 1.8 mM Ca-gluconate 1 mM Mg-gluconate, 5 mM HEPES, pH 7.5).Conductance was calculated as ,conductance 5 current/voltage 5 amps/volts 5 Siemens.as current/voltage, or the average was determined by slope calculation of a best-fit linear equation.

Drosophila Crystallization Assays
Malpighian tubule crystal formation was induced in vivo, by a sodium oxalate (NaOx) diet, or ex vivo, by incubating tubules in medium containing NaOx.For in vivo assays, diets were prepared by mixing NaOx solution to melted standard diet to yield 20 mM solution and then allowing the diet to solidify at room temperature.Drosophila were transferred from vials containing standard diet to vials with 20 mM NaOx-enriched diet.After the designated time point, Drosophila were subjected to MT dissection in iPBS solution, and tubules were mounted on poly-L-coated slides in iPBS for polarized light microscopy of crystals.In ex vivo experiments, MTs from both control and ClC-c-KD flies were first dissected in Schneider's medium and mounted together on a poly-L-coated slide in iPBS.Images were collected by polarized light microscopy before and after incubation with 20 mM NaOx in iPBS for 1 hour.

Secretion Assays
Malpighian tubule secretions were collected following the Ramsay Assay protocol. 39Briefly, MTs were dissected in Schneider's Drosophila medium and submerged through mineral oil into a 10 ul bath containing 1:1 Schneider's insect medium: modified secretion saline (94 mM NaCl, 16 mM KCl, 1.6 mM MgCl 2 , 1.6 mM CaCl 2 , 6.9 mM HEPES, 0.45 mM NaH 2 PO 4 (H 2 O), 1.0 mM NaHCO 3 , 20 mM glucose, pH 6.8), with 10 mM of Drosophila kinin added as a diuretic.For protein secretion quantification, bovine serum albumin was added in the solution for a final concentration of 10 ug/ul.Protein from pooled secretions (4-6 tubules, 1 hour) was detected by Pierce Gold Assay and quantified by UV absorbance at 480 nM by Nanodrop ND-1000 (ThermoFischer Scientific, Waltham, MA).Ion chromatography was performed on individual ureter secretions collected for at least 50 minutes (mean6SEM; 9265 minutes).For most tubules (37/51), a second droplet was collected, and mean values of the first and second droplet are reported.Droplets were diluted in 500 ml ultrapure water using a Nanoject II (Drummond Scientific) and analyzed by cation chromatography.
Chromatography was performed on a Dionex Integrion HPIC (ThermoFisher Scientific, Waltham, MA) system with AS-AP autosampler, IonPac CG12A column, CDRS 600 suppressor, and 25 ml sample loop.Eluent was 25 mM methanesulfonic acid at 1 ml/min.Peak areas were converted to cation concentrations in secreted fluid on the basis of a 6point standard curve (Dionex six cation-II standard, Thermo-Fisher Scientific, Waltham, MA), accounting for the dilution.

Quantitative PCR
Expression of mRNA in MTs was assessed by quantitative PCR.MTs were dissected from flies in iPBS and placed in buffer RLT with 10% b-mercaptoethanol lysis buffer for RNA isolation.Once $50 tubules were pooled, RNA was isolated by Qiagen RNeasy Mini Kit (Qiagen Sciences Inc, Germantown, MD) with added DNase digestion step and eluted in 30 ul nuclease-free water.Total RNA was quantified by Nanodrop ND-1000.Then, cDNA was synthesized by Invitrogen Superscript III First Strand Synthesis (Ther-moFisher Scientific, Waltham, MA) with 1:1 Oligo dT and Random Heximer primers.PCR products were verified by RT-PCR (30 cycles 98 melting/55C annealing) in an Eppendorf Mastercycler Pro S thermocycler (Eppendorf AG, Hamburg, Germany) and then quantified with a Roche Lightcycler 480 SYBR Green 1 on a Q-PCR (Roche Diagnostics, Indianapolis, IN) for 40 cycles.

Microscopy
Images were collected by ZEISS Axio Observer 7 inverted wide-field or confocal microscopes equipped with Zen Blue 3.4 software (Carl Zeiss, Jena, Germany).Fluorescent images were collected using a Plan Neofluar 403/0.6 air objective as Z-stacks and processed by orthogonal projections or Plan Apochromat 633/1.4 oil objective with Airyscan Z-stacks processed in 3D.Fujii ImageJ was used for quantification of GFP fluorescence intensity of the whole tubule in 403 magnification with corrections made from background fluorescence.Tubule crystal images were collected by polarized light microscopy via a Plan Apochromat 103/0.45air observer with in-line light polarizers.Tubule images were collected in tiled fields and processed together as stitched.Crystal number and cross-sectional area were determined by Fuji ImageJ 3D Objects Counter.Total crystal area was calculated as the sum of crystal areas.All image thresholds were determined by control tissue images that were collected at the same time.

Statistics
Statistical comparisons were compiled in GraphPad Prism 9.5.1 (GraphPad Software, Boston, MA).Electrophysiology traces were compared by using two-way ANOVA with Bonferroni comparisons or one-way ANOVA with Bonferroni comparisons when comparing groups at single voltages.Comparisons between knockdown or GFP-labeled and control Drosophila were made by t-tests analysis and paired when date of collection was identified as an interaction.Differences were considered significant when P , 0.05.

Results
Sequence Homology of ClC-c to CLCN-5 The DIOPT Ortholog Prediction Tool (v9.1, flybase.org)shows that Drosophila ClC-c (FBgn 0036566) has greatest homology with CLCN3, CLCN4, and CLCN5 human sequences.Drosophila ClC-a and ClC-b also have homology with human CLCN sequences, but ClC-a is expressed in MT stellate cells and participates in fluid transport (Cabrero  2014), and ClC-b is not expressed in the MT.Both CLCN3 and CLCN4 have similar functions to CLCN5; however, these genes are associated with neural defects such as intellectual disability, epilepsy, and synaptic neural function more so than kidney dysfunction. 40,41Because DD1 affects the kidney, we have focused our investigation on ClC-c, which is highly expressed in the MT (Fly Cell Atlas scRNAseq).The amino acid sequence of Drosophila ClC-c (FBgn0036566) aligns to human ClC-5 (NM_000084) with 62.9% sequence identity and 78.6% sequence similarity (Smith Waterman Alignment, (SnapGene version 6.2) and 52% sequence identity by NCBI Blast Global Amino Acid    2).The currents generated by ClC-c were found to diverge significantly from controls at $ 140 mV (P , 0.001) under standard conditions (Figure 2A).When Cl 2 in the solution is substituted with gluconate, the currents are greatly diminished and do not differ significant from controls until $ 160 mV.The effect of bath Cl 2 suggests that the currents generated from ClC-c are related to the  inward flow of Cl 2 from the bath solution (Figure 2B).Because mammalian ClC-5 is recognized to exchange H 1 for Cl 2 , we tested both acidic and alkaline solutions on these oocytes.Increasing extracellular [H 1 ] (pH 6.0) impaired the current delaying the divergence from controls to $ 160 mV (Figure 2C).The alkaline solution of pH 8.5 had no apparent effect on the current with difference from controls remaining significant at $ 140 mV (Figure 2D).By comparing conductance at 180 mV, it is apparent that decreasing [Cl 2 ] and increasing [H 1 ] in the solution both impair the amplitude of the current while decreasing [H 1 ] does not (Figure 2E).These data demonstrate that Cl 2 /H 1 exchange occurs via Drosophila ClC-c.

Voltage Gating
The outward rectifying currents generated by ClC-c indicate that a voltage-gated mechanism drives activity.To determine the gating voltage (V g ), a segmented regression analysis was used that calculates the intersection point between two segments of a continuous line.The slopes of the current-voltage curve are indicated as conductance (G, in mS).The line segment ,V g corresponds to the lowactive state (G L ), whereas the line segment .V g is the active state (G a ).To verify gating, the gated two-line segment model was compared with a nongated linear model.H 2 Oinjected oocytes followed the linear model; therefore, V g and G a were not calculated.With ClC-c-injected oocytes, the V g was 3062 mV (95% confidence interval 26-33 mV) with no significant differences among treatments (P-values .0.5, not shown; Table 1).No differences were observed among G L of ClC-c-injected and/or H 2 O-injected controls.G a for ClC-c decreased with Cl 2 substitution and acidic solutions, but no effect was observed with the alkaline solution similar to Figure 2. All homologous DD1 mutations followed the line segment model, and V g were similar; however, the G a for mutations was significantly different from WT ClC-c.Furthermore, G a for mutations was different from their respective G L , indicating that the mutations affect Cl 2 transport rather than voltage gating.

ClC-c Appears Along the Apical Membrane of the Malpighian Tubules
Expression of UAS-ClC-c::eGFP was driven selectively in tubules with GAL4 drivers, Uro-GAl4 (expressed in the main segment, principal cells) and c507-GAl4 (expressed in the lower tubule segment and ureter, principal cells).Wild-type controls were used to assign thresholds for fluorescent channel intensities above autofluorescence in each channel.The Uro-GAl4 driver resulted in intense ClC-c-eGFP concentrated in a few principal cells at the midpoint of the tubule with lower expression distributed throughout the main segment.The c507 driver induced high expression in a distinct region of the tubule near the ureter.Regardless of driver, ClC-c localizes predominantly in the microvilli at the apical membrane of the MTs, with some GFP evident in the cytosol (Figure 4).No GFP was present at the basolateral membrane or in nuclei.ClC-c-KD in D. melanogaster UAS-ClC-c RNAi flies were crossed with the Uro-GAL4 driver to knockdown expression of ClC-c in MT principal cells.The resulting F1 generation had a 48% decrease in ClC-c mRNA expression in anterior MTs compared with controls 7 days after eclosure (P 5 0.03, n53 groups of approximately 50 pooled tubules; Figure 5A).On standard maintenance diet alone, tubule crystals were observed in all ClC-c KD flies, averaging 1564 crystals per anterior MT pair (n517), whereas crystals were observed in 71% of the control flies with fewer crystals than the KD flies (562 crystals per pair, n517, P 5 0.02; Figure 5C).Figure 5D illustrates birefringence of CaOx crystals observed in MT.

Malpighian Tubule Secretions
Ex vivo MT secretions were collected under kinin diuretic stimulation.Protein secretion via ClC-c-KD MTs (1.5360.30mg/ml protein) was significantly greater than control (1.1260.27mg/ml protein, P 5 0.038), when adjusting for date of collection by paired t-test (Figure 5B).Secretion rate and cations, including Na 1 , K 1 , NH 4 1 , Ca 21 , and Mg 21 , were measured by HPIC (Table 2).ClC-c-KD MTs had numerically lower secretion rate than controls, but this was not statistically different (P 5 0.13).Only Ca 21 had a notable difference such that ClC-c-KD MT secretions had a higher concentration of Ca 21 (6.360.8 mM Ca 21 ) than controls (4.160.5 mM Ca 21 , P 5 0.018).Mg 21 was also greater for ClC-c-KD, but this trend was not significant (P 5 0.082).Secretion of all other cations measured (Na 1 , K 1 , and NH 4 1 ) was not different between groups.
Sodium Oxalate-Induced Crystallization in the Malpighian Tubules ClC-c-KD flies had more crystals in the anterior MT than control flies; thus, KD of ClC-c may promote calcium oxalate (CaOx) precipitation and crystallization.Therefore, CaOx crystallization was induced both ex vivo in isolated tubules and in vivo by dietary supplementation.Tubules dissected from ClC-c-KD flies and submerged in 10 mM NaOx solution for 1 hour formed 204632 crystals (n59) and were similar to control flies (220627 crystals, n59, P 5 0.7; Figure 6A).Both average surface area (27.564.0 mm 2 , ClC-c KD versus 31.364.2mm 2 , control, P 5 0.5) and total crystal area (558061020 mm 2 ClC-c-KD versus 63006786 mm 2 control, P 5 0.6) were similar between groups (Figure 6B, 6C).ClC-c-KD and control flies that were fed a diet of 20 mM NaOx for 1 day produced similar number of crystals (432.5632crystals, ClC-c-KD versus 383.8641 crystals control; P 5 0.3), with similar areas (21.6561.7 mm 2 , ClC-c-KD versus 18.362.1 mm 2 , control; P 5 0.2; Figure 6D, 6E).However, the total crystal area (sum of crystal areas per pair of tubules) was significantly greater in the ClC-c-KD MTs (89346641 mm 2 ) than controls (65976785 mm 2 , P 5 0.03, Figure 6F).

Discussion
Hypercalciuria and Ca 21 dysregulation are prominent diagnostic criteria for DD1, but the relationship between this 2Cl 2 /H 1 transporter and Ca 21 metabolism remains unclear.In this study, we evaluated Drosophila ClC-c as a human ClC-5 homolog to establish an alternative model for deciphering the Cl 2 and Ca 21 connection.Drosophila models offer advantages which are unavailable or difficult to implement in mammalian models.Genetic manipulations are particularly useful as a variety of tools, such as RNAi knock-down and fluorescent ion sensors, are already developed, readily available, and can be expressed in specific tissues or cells.In addition, implementation of genetic constructs or breeding schemes can be accomplished rapidly (life cycle ,2 weeks) and cost effectively (,$50 USD for a line and almost no daily costs), particularly when compared with mammalian models.To justify using the Drosophila model, we start by asking, how well does this Drosophila ClC-c compare with mammalian ClC-5?
In silico, human ClC-5 and Drosophila ClC-c sequences align with only modest homology, but key amino acids remain conserved and predict equivalent functions.In addition, both ClC-5 and ClC-c are highly expressed in their respective renal organs.3][44] This electrogenic action of these transporters depends on Cl 2 (or other halide ion) in the extracellular bath solution and is hindered by acidic pH. 5,13,20,36,37,42,45With Drosophila ClC-c, similar experiments unveiled the same outward rectifying current gated at 131 mV.Ion transport by Drosophila ClC-c is dependent on [Cl 2 ] and decreased by acidic pH.The similarities in ion transport bolster the highly conserved role for this Cl 2 /H 1 exchanger in the renal organ throughout evolution.
Dent disease type 1 is caused by sequence variants in CLCN5 that alter ClC-5 protein functions.The mutations S244L, G261E, G333R, and R345W; in-frame deletion 523delV; and Q629X truncation have all shown impaired Cl 2 transport in vitro. 6,12,36,37,44Disease severity relates to the type of mutation such that patients with truncating mutations, such as Q629X, are prone to developing more severe phenotypes. 11Phenotypes associated with nontruncating mutations vary on the basis of the degree to which effective Cl 2 transport is altered.Most of the nontruncating DD1 pathogenic variants obstruct ion transport; however, the R345W variant allows voltage gating, but impairs only the amplitude of currents generated.On investigation, Chang and colleagues found that the human R345W construct is entrapped in the endoplasmic reticulum and cis-Golgi without translocation to endosomes and the plasma membrane. 36,37Chloride transport was similarly assessed for Drosophila ClC-c with DD1 homologs: S393L, R494W, and Q777X.As expected, Cl 2 transport, but not voltage gating, is affected by these mutations.Similar to the human R345W variant, the Drosophila R494W variant retained voltage gating, with a shift to 117.566.9 mV, but the amplitude of ion transport was decreased to 1/3 of its activity.One can reason that this Drosophila R494W mutation may also cause impaired trafficking in ClC-c and warrants additional investigations related to subcellular localization.Ultimately, homologous DD1 mutations in ClC-c do hinder ion transport and justify additional KD strategies to model DD1.
Identifying the cellular expression and localization of an epithelial transporter leads to understanding its physiological role in an organism.In the mammalian kidney, ClC-5 is expressed in proximal tubule and a-intercalated cells where solute reabsorption occurs.2,22,46 Drosophila and other insects use MTs as their analogous renal organ to maintain osmotic homeostasis of the hemolymph with principal cells providing cation secretion and stellate cells maintaining osmoregulation. 30,31,34,47Principal cells also use V-ATPase in a manner reminiscent of the proximal tubule and a-intercalated cells in mammals. 48,49Transcriptomics have shown that ClC-c is highly expressed in the adult MTs, and work by Cabrero and colleagues noted that ClC-c is more prominently expressed in principal cells than stellate cells. 34,50,51Here, GFP-labeled ClC-c expressed in principal cells localized to the apical microvilli and in the cytosolic regions along the apical membrane, suggesting that, like mammalian ClC-5, Drosophila ClC-c likely incorporates within endosomes.The similarities in localization suggest that the physiological comparisons between mammalian ClC-5 and Drosophila ClC-c are compatible and justify indepth assessments of the role of ClC-c in endosomal acidification and trafficking for future studies that exploit the variety of genetic tools of the Drosophila model.
Knockdown of ClC-c in Drosophila adult MTs reveals a striking resemblance to the phenotype described for patients with DD1.DD1 is recognized to be a rare kidney stone disease with the two most prevalent characteristics being low molecular weight proteinuria and hypercalciuria.With a modest 48% decrease in mRNA expression, the ClCc-KD flies exhibited increases in both Ca 21 and protein secretion.Not only did the KD MTs secrete more protein and Ca 21 , but they also had more crystals form spontaneously than control flies.In dissected MTs, ClC-c KD did not manifest as more CaOx crystal formation.However, when fed with a high sodium oxalate diet, total crystal area was greater with the KD although crystal number and size were not different.The differences in crystal formation between diet and isolated MTs suggest that the cause of crystal formation has a systemic aspect and is not resulting from mishandling of oxalates in principal cells.Because KD flies do have more spontaneous crystals, we should consider that the composition of these crystals may contain complex precipitates besides CaOx.Mixed composition crystals resemble the stones associated with DD1, which are composed of both CaOx and calcium phosphate. 7,8,52,53lthough CaOx crystals are relatively straightforward to assess using polarized light and birefringence, Caphosphate crystals require histologic stains for both Ca and phosphate and are not observed with birefringence.These staining requirements do not allow for coincident staining and also do not allow investigators to follow crystal formation as it is occurring.Nevertheless, the fly model reported here seems to recapitulate disease characteristics of human DD1 and may be an alluring complement to previous rodent models.
The model that we have described has inherent limitations and may limit a direct translation to human or DD1.The genetic contribution is an important limitation to consider.DD1 is X-linked and affects males more aggressively than female carriers.In Drosophila, ClC-c is on chromosome 2 and not sex-linked.Thus, male and female flies are assumed to be affected similarly.As for phylogenic differences, Drosophila do not require Ca 21 for bone formation and are not susceptible to extra-renal skeletal maladies, such as rickets or fracture.Female flies, however, require additional Ca 21 metabolism for egg production and only female flies were used in assessments and analytics.
In conclusion, we compared the functional attributes of Drosophila ClC-c with mammalian ClC-5 and have introduced the ClC-c-KD fly as an avatar to model DD1.This model recapitulates the two most prevalent features of DD1, with increased secretions of both protein and Ca 21 , as well as produces notable increases in spontaneous tubule crystal formations.Future investigations with this model will offer extensive tools for unraveling the relationship between this Cl 2 transporter and increased Ca 21 secretions and studying endosomal pH and Ca 21 transport, all of which are critical for improving management of patients with DD1.
Dents and Drosophila ClC-c, Reynolds et al.

Figure 2 . 17 Cl 2 Figure 3 .
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 H 2 O perfused with ND96 solutions of pH 7.5 (A), pH 7.5 without Cl 2 (B), pH 6.0 (C), and pH 8.5 (D).*Significantly different from respective H 2 O-injected oocytes or as indicated by two-way ANOVA analysis with Bonferroni post-test.The conductance observed 180 mV compared across solutions by one-way ANOVA.Error bars represent mean6SEM.(E) Conductance from ClCc-injected oocytes with the same a-notation (a, b, c) are statistically similar (P $ 0.05).*Significantly different conductance from H 2 O-injected oocytes for the respective solution (P , 0.05).

Figure 5 .
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 w 1118 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 103 and quantified by ImageJ.*Significantly different by t-test, P , 0.05.Error bars represent mean6SEM.(D) Representative image of Clc-c-KD tubule pair with arrows depicting crystals.Scale bar5200 mm.

Figure 6 .
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 w 1118 flies with Uro-GAL4 males.Tubule pairs were imaged at 103 magnification by polarized light microscopy and quantified by ImageJ.Error bars depict mean6SEM.*Significantly different by t-test comparisons, P , 0.05.
Dents and Drosophila ClC-c, Reynolds et al.
2' 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 2 .Comparisons made by one-way ANOVA with Bonferroni comparisons.Error bars represent mean6SEM.Conductance from with Cl 2 ' solutions with the same a notation (a, b, c) are statistically similar (P $ 0.05).*Significantly different conductance between with Cl 2 ' and without Cl 2 ' solutions (P , 0.05).
a Comparisons with respective control condition by one-way ANOVA (ClC-c in pH 7.5 solution).b Comparison between low active and active conductances to evaluate gating mechanism by t-test.c Significant difference, not significant (ns).that are not observed in H 2 O-injected controls (n510; Figure

Table 2 .
Concentration of ions secreted from individual Malpighian tubules a Statistically significant by t-test comparison between control and ClC-c-KD, P , 0.05.Dents and Drosophila ClC-c, Reynolds et al.