Roles of Drosophila subdued and anoctamin 4 in calcium oxalate stones

Abstract

Anoctamins typically function as Ca2+ 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 InformationSupport: Oxalosis & Hyperoxaluria Foundation, R25‐DK101405, Mayo FoundationDrosophila 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