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Abstract: TH-PO558

Subdued, an Anoctamin 4 Homolog, Changes Calcium Oxalate Crystal Formation in Drosophila Renal Structures Revealing Potential Roles in Mammals

Session Information

Category: Bone and Mineral Metabolism

  • 401 Bone and Mineral Metabolism: Basic


  • Turin, Daniel R., Mayo Clinic, Rochester, Minnesota, United States
  • Arneson, Mariah, Mayo Clinic, Rochester, Minnesota, United States
  • Holmes, Heather L., Mayo Clinic, Rochester, Minnesota, United States
  • Cabrero, Pablo, University of Glasgow, Glasgow, United Kingdom
  • Dow, Julian A.t., University of Glasgow, Glasgow, United Kingdom
  • Jayachandran, Muthuvel, Mayo Clinic, Rochester, Minnesota, United States
  • Lieske, John C., Mayo Clinic, Rochester, Minnesota, United States
  • Furrow, Eva, University of Minnesota, St. Paul, Minnesota, United States
  • Romero, Michael F., Mayo Clinic, Rochester, Minnesota, United States

Anoctamins function as Ca2+ activated Cl- channels (CaCC) or as phospholipid scramblases. Using a miniature schnauzers GWAS, we discovered a risk locus for recurrent calcium oxalate (CaOx) stones harboring an anoctamin 4 (ANO4) variant. Human stone formers have decreased ANO4 protein in urinary vesicles. In Drosophila Malpighian tubules (MTs) the ANO4-homolog, subdued, functions as a CaCC and scramblase. Subdued also functions in bacterial defense, so we knocked it down to determine if bacterial infection changes crystal growth.


Subdued knockdown (KD) in MT principle cells was used a MT-promotor and a subdued-RNAi. CaOx crystallization experiments were conducted on ex-vivo MTs by 1h addition ofsodium oxalate (NaOx); while prolonged feeding (4d) assays used food with NaOx. With DIC optics, we imaged CaOx birefringence then counted crystals using ImageJ. Uropathic E. coli (UPEC) were fed to flies or added to NaOx solution before an overnight time-lapse.


The canine ANO4 variant has a 0.01 allele frequency at a conserved residue (humans, canines and Drosophila). Rapid crystallization experiments show that subdued-KD MTs have less CaOx crystals than controls. However, feeding assays showed that MT subdued-KD produces enlarged crystals. These assays also show clustering and biofilms that change crystal appearance with patterning similar to human kidney stones. UPEC facilitates CaOx crystal aggregates.


The Drosophila avatar illustrates that subdued (ANO4) aggregates CaOx. An apparent pathogenic variant is conserved in mammals and flies; subdued’s role in fly bacterial infection indicates a possible additional role of ANO4 in kidney stones. Examining antibacterial treatments or other aspects of CaOx crystal formation with UPEC may allow an antibiotic approach to more efficiently eliminate kidney stones. Future experiments will determine the localization of ANO4 in mammalian kidneys to better understand the cell specific pathology. Finally, these studies indicate that this simple kidney stone avatar- the fly – continues to provide new insights to the mechanistic role of specific genes and infection.


  • NIDDK Support