Dow, Julian A. T.

Key Points Drosophila can be a model for Dent Disease type 1. Drosophila Clc-C mutations function similar to human CLC-5 Dent 1 mutations. Background Drosophila serve as exceptional alternative models for in vivo and ex vivo research and may provide an avenue for in-depth investigation for human ClC-5 and Dent disease type 1 (DD1). The Drosophila ClC-c (CG5284) has sequence homology with human ClC-5 and is hypothesized to encompass similar functional and phenotypical roles with ClC-5 and variants that cause DD1. Methods Ion transport function and activity of Drosophila ClC-c and homologous DD1 variants were assessed by voltage clamp electrophysiology. Membrane localization was demonstrated in Drosophila expressing a GFP-labeled construct of ClC-c. Genetic expression of an RNAi against ClC-c mRNA was used to generate a knockdown fly that serves as a DD1 disease model. Tubule secretion of cations and protein were assessed, as well as the crystal formation in the Malpighian tubules. Results Voltage clamp experiments demonstrate that ClC-c is voltage-gated with Cl − -dependent and pH-sensitive currents. Inclusion of homologous DD1 mutations pathogenic variants (S393L, R494W, and Q777X) impairs ClC-c ion transport activity. In vivo expression of ClC-c-eGFP in Malpighian tubules reveals that the membrane transporter localizes to the apical membrane and nearby cytosolic regions. RNAi knockdown of ClC-c (48% decreased mRNA expression) causes increased secretion of both urinary protein and Ca 2+ and increased occurrence of spontaneous tubule crystals. Conclusions Drosophila ClC-c shows orthologous function and localization to human ClC-5. Thus, Drosophila and ClC-c regulation may be useful for future investigations of Cl − transport, Ca 2+ homeostasis, and urinary protein loss in DD1.

Introduction: Dent Disease type 1 (DD1) results from X-linked, mutations in CLC-5 (2Cl-/H+ exchanger) and results in progressive renal failure by 20-40. DD1 is characterized by kidney Ca2+ mishandling, and subsequent hypercalcuria, calcium oxalate (CaOx) kidney stones, and kidney calcification as well as proteinuria. We identified Drosophila Clc-c as the CLC-5 homolog with conserved amino acids at DD1 mutation sites. We hypothesize that Clc-c shares functional similarities in Cl- transport, Ca2+ homeostasis and protein processing. Methods and Results: By voltage clamping Xenopus oocytes expressing Clc‑c and CLC-5, we find that both are electrogenic and have the same outward-rectifying current in Cl- solutions. Cl- transport (current at +80mV) was lowered by acidity (2.37±0.55 μA, pH 6.0, P=0.06), but not by alkaline solution (4.45±0.96 μA, pH 8.5, P= 0.7) when compared to the standard solution of pH 7.5 (4.97±1.12 μA). Similarly, Cl- transport was lowered by homologous DD1 mutations S393L (0.67±0.098 μA, P<0.0001), R494W (2.9±0.46 μA, P =0.006), and Q777x (1.13±0.28 μA, P =0.0001) vs. WT (7.4±1.2 μA) as observed previously for CLC-5 DD1 mutations: S244L, R345W, and Q629x (Tang et al, Physiol Rep 4[8], 2016). To determine cell membrane and possible intracellular localization of DD1 mutations, HA-tagged Clcc and mutations (identical placement as in CLC-5-HA, Tang 2016) were made. Likewise, transgenic Drosophila expressing Clc-c-GFP were made to localize Clc-c in cell and intracellular membranes of renal tubules (Malpighian tubules, MT) in vivo (Judd-Mole, U Monash, Dissertation, 2016). RNAi MT knockdowns of Clc-c (Clc-c-KD) were made and resulted in 50% decrease in Clc-c mRNA expression. CaOx crystals in anterior MT (7 days after eclosure) were present in all Clc-c-KD and were more abundant (15±4 crystals/fly) than w1118 controls (5±2 crystals/fly, P= 0.02). MT secretions showed that Clc-c KD flies contained higher [Ca2+] while all other cations (Na+, NH4+, K+, Mg2+), and volume were the same as w1118 . Similarly, urinary protein concentration (4-6 pooled flies per n) appears elevated in DD1 flies (1.41 ± 0.14 mg/mL, n=3) vs w1118 (1.16 ± 0.08 mg/mL, n=5). Conclusion: Drosophila Clc-c has similarities to human CLC-5 including impaired function of voltage-gated Cl- transport, homologous DD1 mutations, increased CaOx crystal formation, and elevated urinary [Ca2+] and protein. Our results indicate that the Drosophila Clc-c avatar may help to provide more direct insights to the cellular and organismal pathophysiology for future investigations of Cl- transport, Ca2+ homeostasis and urinary protein loss in DD1. F32 DK128987, T32 DK007013 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Anoctamins (ANO) are activated by intracellular Ca2+ and function as phospholipid scramblases and/or Cl- channels. ANO4 is mutated in miniature schnauzers with calcium oxalate stones (CaOx), and ANO4 protein in urinary extracellular vesicles is decreased in human CaOx stone formers. In Drosophila Malpighian tubules (MT), subdued (ANO4-homolog) functions as Ca2+-activated Cl- channel, scramblase, and plays a role in host defense against gram-negative bacteria. Increasing evidence shows the participation of bacteria in urinary stone formation in humans. We used a Drosophila avatar to investigate whether subdued changes CaOx crystallization and if crystallization increases with bacterial infection.Uro:Gal4 flies were crossed with UAS:subdued-RNAi flies to knockdown (KD) subdued in MT principal cells (PC) or with w1118 flies to generate normal controls (WT). F1 generation underwent CaOx crystallization in association or not with uropathogenic E. coli (UPEC) infection. In vivo, flies were fed on diet supplemented with 20mM sodium Ox (NaOx) + UPEC:eGFP for 4 days. For ex vivo assays, dissected MTs were submerged in solution containing 10mM NaOx+ UPEC:eGFP for 90 minutes. subdued-KD in PC reduced subdued mRNA by 56% in MT. Neither crystal number nor size were changed by subdued-KD after prolonged NaOx feeding. However, when UPEC is introduced with the NaOx diet, subdued-KD flies exhibited a large bacterial presence in MT lumen and produced enlarged CaOx crystals compared to WT flies. Similar augmented presence of UPEC in subdued-KD MTs was observed during ex vivo assays. Short-term exposure to NaOx+UPEC resulted in increased number of crystals in subdued-KD MTs compared to WT MTs, but no difference was observed in crystal size.Fly cell atlas ( flycellatlas.org ) revealed that subdued is also found in MT stellate cells (SC; water and Cl- secretion). C724:Gal4>UAS:subdued-RNAi flies were used to assess the effect of subdued-KD in SC. This s ubdued-KD slightly increased bacterial infection but did change CaOx crystallization in MTs after 4 days of NaOx+UPEC feeding.The presence of subdued in PC limits CaOx crystals formation and aggregation through reducing bacterial invasion in the MT lumen. These Drosophila results suggest a role for ANO4 in bacterial-related human and dog kidney stone formation. Future experiments will determine whether other functional aspects of subdued affect CaOx crystallization. U54-DK100227, R01-DK092408, FAPESP (2022/01226-1), Mayo Foundation. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

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 Ca 2+ that increases urinary Ca 2+ excretion, calcium oxalate (CaOx) kidney stones, and kidney calcification. Interestingly, none of the DD1 symptoms include or relate to Cl ‐ homeostasis despite impaired transport function. Thus, how does a Cl ‐ transporter cause Ca 2+ dysregulation? Our laboratory has identified Drosophila Clc‐c to be 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 Ca 2+ homeostasis. To test our hypothesis, we used electrophysiology to measure ion transport, and then assessed CaOx crystal formation in adult fly renal tubules (Malpighian tubules, MTs) of Drosophila . Voltage clamp experiments with Xenopus oocytes show that Clc‐c is electrogenic with the same outward‐rectifying current in Cl ‐ solutions as observed with human CLC‐5. Next, knockdown flies (Clc‐c‐KD) were generated by crossing Uro‐Gal4 (MT driver) with UAS‐Clc‐c‐RNAi flies, resulting in 50% Clc‐c mRNA expression in the MTs. Crystals in adult MT (7 days after eclosure) were present in 100% of Clc‐c‐KD compared to 71% of control flies. The average number of crystals in adult tubule pairs was significantly greater in Clc‐c‐KD flies (15±4 crystals per pair) than control flies (5±2 crystals, P= 0.02). When fed a diet with 2mM NaOx ClcC‐KD for 4 days, Clc‐c‐KD flies had a significantly greater number of crystals in each tubule (24±5 crystals) than controls (4±2 crystals, P= 0.005). However, the KD flies did not produce more crystals than control flies when fed a supraphysiological concentration of sodium oxalate (20 mM NaOx diet) for 1 or 4 days and in ex vivo assays of dissected MTs submerged in solution containing 10 mM NaOx for 1 hour. In conclusion, Drosophila Clc‐c has both identical sequences at DD1 mutation sites and similar biophysical functions to human CLC‐5. Furthermore, Clc‐c‐KD increases incidence of CaOx crystals which corresponds to the increased risks for kidney stones in DD1 patients. With this fly‐DD1 model, we will be able to assess the relationship of Clc‐c Cl ‐ transport to renal Ca deposits. Future experiments will evaluate renal pathophysiology of DD1 mutations and their relationship to Ca 2+ transport in Clc‐c transgenic flies.

Background Kidney stones (nephrolithiasis) are an expensive and painful health problem with a complex etiology with ~70% of stones composed of calcium oxalate (CaOx). The Slc26 gene family contains several proteins that transport oxalate in the gut and kidney. Slc26a6 and its homologs exchange Cl − for oxalate (ox 2− ), as well as SO 4 2− , HCO 3 − and formate. Slc26a6 −/− mice have hyperoxaluria and CaOx stones illustrating a physiologic collaboration between the gut and kidney. We developed a Drosophila genetic model for CaOx stones (dPrestin, Slc26a5/6, manipulation) allowing rapid gut, renal and whole‐body physiological assessment (min/h v. mo/y). Methods (Tissue) First, Malpighian tubules (MT, renal tubule) were dissected from wild type and dPrestin knockdown mutants and soaked in ox 2− solutions. CaOx stones were visualized by DIC microscopy. Second, we generated Drosophila expressing eYFP in the MT, MT+gut or whole body. eYFP is a halide (Cl − ) sensor, allowing quantification of Cl − /ox 2− exchange by native dPrestin. (Whole body) Wild type and dPrestin knockdown flies were fed ox 2− enriched food, and flies imaged (CaOx quantified) by micro computed tomography (MicroCT). Results Exposing dPrestin‐knockdowns to bath ox 2− elicited almost no CaOx crystals. However, exposing wt‐MT to ox 2− elicited many CaOx crystals in 20 min. Increased CaOx crystal formation was also coupled with an eYFP intensity increase, matching dPrestin Cl − /ox 2− exchange characteristics. MicroCT showed more CaOx crystals in flies that fed on ox 2− longer. Conclusions Our observations indicate that Drosophila and dPrestin manipulation can be used for rapid, detailed studies of gut, renal and whole organism oxalate metabolism & CaOx stone formation. [DK83007]

Background Kidney stones are an expensive and painful health problem with calcium oxalate (CaOx) being 70% of cases. We developed a Drosophila model of CaOx stones [Hirata et al., AJP‐Renal Physiol, 2012]. This work showed that dPrestin (Slc26a5/a6) exchanges Cl − for oxalate (Ox). Thiosulfate (S 2 O 3 ) and tannic acid (TA) are implicated to inhibit CaOx crystals in supersaturated urine. Here, we report transporter, renal tubule and whole body effects of S 2 O 3 and TA on crystal formation. Methods S 2 O 3 and TA transport effects were examined by voltage clamping of dPrestin‐expressing Xenopus oocytes. Cis ‐inhibition of oxalate transport was also examined in (a) dissected Malpighian tubules (MT) or flies fed ox‐enriched food plus S 2 O 3 or TA. MT CaOx crystals were viewed by DIC, or whole flies by microCT. Results dPrestin transports S 2 O 3 but not TA, yet both inhibited Ox transport (K i (S 2 O 3 )~10mM; K i (TA)~100μM). S 2 O 3 competitively inhibits while TA blocks dPrestin. MT treated with S 2 O 3 or TA ↓ crystal formation compared to MT soaked in Ox only. Flies fed with S 2 O 3 or TA also showed ↓ CaOx in MTs. Conclusions S 2 O 3 and TA are both inhibitors of CaOx crystallization. These experiments show that these inhibitors have distinct mechanisms of action. Our Results indicate that (a) this Drosophila model can be used as a chemical screening assay for CaOx stone prevention and (b) TA, due to low Ki, might be a reasonable therapy for certain CaOx stones patients.[ DK83007, DK92408]

Background : Zn is implicated as having a role in the formation of calcium oxalate (CaOx) kidney stones. Our studies indicate that Slc39a10 (Zip10) may be important in this process. Zip10 has identifiable sequences in human, dog and fly ( Drosophila ). As we are interested in a translational model of kidney stones, we examined Zip10 from all 3 species. Methods: Zip10 cDNAs were cloned from human & dog (kidney) and fly (whole fly) using gene specific primers and RT‐PCR. Clones were put into a Xenopus oocyte expression vector so that Zip10 function could be measured. Three days post‐injection, oocytes were pre‐incubated in ND96, followed by 30 min in ND96 + 10 μM ZnCl + the PET isotope 63 Zn, with varying pHs and ion concentrations. 63 Zn uptake was measured in each oocyte using a gamma counter. Zip10 oocytes were also checked using electrophysiology (ion selective electrodes & voltage clamping). Results: Zip10 mRNA & protein are found in gut and kidney. In mouse kidney, Zip10 antibodies stained the apical membrane of proximal tubules. All clones transported 63 Zn (nmol/oocyte/hr): hZip10 = 70±8; cZip10= 44 ±7; dZip10= 64 ±8; water = 0.4 ±0.1. These clones seem to be most active at pH 7.5 as uptakes decreased at 5.5 and 8.5. Replacement of either all Cl ‐ (with HCO 3 舉 ) or all Na + (with choline) reduced Zn‐uptake. Electrophysiology revealed no evidence of I or V changes with addition of Zn (electroneutral). Conclusions: Our results indicated that all three Zip10 clones transport Zn 2+ . Understanding the detailed mechanisms of these Zn transporters in oocytes presents the opportunity to better use our Drosophila model in the study of CaOx stone formation and absorption. Grant Funding Source : Supported by DK83007, DK92408

Anoctamins typically function as Ca 2+ activated Cl − channels (CaCC) or as specific, phospholipid scramblases. GWAS of miniature schnauzers (>40% with recurrent calcium oxalate (CaOx) kidney stones) revealed a risk locus including an anoctamin 4 (ANO4) variant. ANO4 in mammals was initially reported as a phosphatidylserine (PS) scramblase. Urinary extracellular vesicles (EV) show decreased ANO4 protein in human stone formers. However, in Drosophila renal structures (Malpighian tubules, MTs), subdued (the ANO4‐homolog), functions as both a CaCC and scramblase. Since subdued also functions to defend against gram‐negative bacteria, it was knocked down to determine whether CaOx crystals were altered by bacterial infection. Subdued was knockdown (KD) in MT principle cells by crossing Uro:Gal4 flies with UAS: subdued ‐RNAi flies. F1‐flies were evaluated using CaOx crystallization in dissected MTs as well as after 4d NaOx feeding protocols. To evaluate infection, uropathic E. coli (UPEC):eGFP were fed to flies or added to NaOx solution before an overnight time‐lapse. Interestingly, the canine ANO4 variant has a 0.01 allele variant frequency at a residue conserved in humans, canines and Drosophila . Rapid crystallization experiments show that MTs with subdued‐KD have fewer CaOx crystals than control MTs. However, prolonged NaOx feeding assays showed that subdued‐KD in MTs produces enlarged CaOx crystals. These feeding assays also showed an unexpected phenotype: crystal clustering and matting structures (e.g., bacterial biofilms). These structures have similar patterning to human kidney stones [Sivaguru et al., PMID: 30213974]. Following a four day feeding of NaOx, when UPEC + NaOx is introduced overnight ( ex vivo ), a three hour bacterial event occurs, causing crystals to more tightly pack. Finally, when UPEC is introduced during a rapid crystallization experiment, the bacteria heavily localize in subdued‐KD MT lumens while control MTs have little eGFP‐fluorescence. Thus, UPEC facilitates CaOx crystal aggregates in subdued‐KD MTs while the nature of this interaction is unknown. In human kidney, ANO4 localizes in thick ascending limb (NKCC2 + ), while in murine kidney localizes more proximal in the Loop of Henle (UMOD + ). As mice are resistant to CaOx stones, these slight location differences may reveal altered TAL function by ANO4. This Drosophila avatar indicates that subdued (ANO4) normally limits CaOx crystal aggregation and limits UPEC growth. As an apparent pathogenic amino acid is conserved in mammals and flies; subdued’ s role in fly bacterial infection suggests an additional ANO4 role in human kidney stone formation. These results indicate that examining antibacterial treatments with UPEC infection may allow an antibiotic approach to more efficiently eliminate kidney stones. Future experiments determine what functional aspect of subdued alters CaOx crystallization. Moreover, this simple kidney stone avatar‐the fly – continues to provide new and mechanistic insights to CaOx stone formation. Support or Funding Information Support: Oxalosis & Hyperoxaluria Foundation, R25‐DK101405, Mayo Foundation Drosophila MT (Uro:Gal4 x UAS‐ subdued ‐RNAi) viewed with (A) DIC for CaOx birefringence, (B) fluorescence to visualize UPEC:eGFP and (C) merged to show coincidence of UPEC with CaOx crystals. The red line indicates the length of the CaOx/UPEC mass. This is not observed in control MTs. Figure 1

Significance The tiny insect renal (Malpighian) tubule can transport fluid at unparalleled speed, suggesting unique specializations. Here we show that strategic allocation of major intrinsic proteins (MIPs) to specific cells within the tubule allows the separation of metabolically intense active cation transport from passive chloride and water conductance. This specialized renal architecture is general to at least many higher insects, providing a clue to the unique success of the class Insecta in colonizing an extraordinary range of ecological niches.