Kidney Week

Abstract: TH-PO558

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

Session Information

• Bone and Mineral Metabolism: Basic
  November 07, 2019 | Location: Exhibit Hall, Walter E. Washington Convention Center
  Abstract Time: 10:00 AM - 12:00 PM

Category: Bone and Mineral Metabolism

• 401 Bone and Mineral Metabolism: Basic

Authors

• Turin, Daniel R., Mayo Clinic, Rochester, Minnesota, United States

Daniel R. Turin,

• Arneson, Mariah, Mayo Clinic, Rochester, Minnesota, United States

Mariah Arneson,

• Holmes, Heather L., Mayo Clinic, Rochester, Minnesota, United States

Heather L. Holmes,

• Cabrero, Pablo, University of Glasgow, Glasgow, United Kingdom

Pablo Cabrero,

• Dow, Julian A.t., University of Glasgow, Glasgow, United Kingdom

Julian A.t. Dow,

• Jayachandran, Muthuvel, Mayo Clinic, Rochester, Minnesota, United States

Muthuvel Jayachandran,

• Lieske, John C., Mayo Clinic, Rochester, Minnesota, United States

John C. Lieske,

• Furrow, Eva, University of Minnesota, St. Paul, Minnesota, United States

Eva Furrow,

• Romero, Michael F., Mayo Clinic, Rochester, Minnesota, United States

Michael F. Romero,

Background

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.

Methods

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.

Results

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.

Conclusion

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.

Funding

• NIDDK Support