Arneson, Mariah L.

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.

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