Kidney Week

Abstract: FR-PO311

Subdued Limits E. coli Infection and Ca-Oxalate Crystallization in Drosophila Renal Tubules

Session Information

• Bone and Mineral Metabolism: Basic
  November 03, 2023 | Location: Exhibit Hall, Pennsylvania Convention Center
  Abstract Time: 10:00 AM - 12:00 PM

Category: Bone and Mineral Metabolism

• 501 Bone and Mineral Metabolism: Basic

Authors

• Foresto-Neto, Orestes, Mayo Clinic College of Medicine and Science, Rochester, Minnesota, United States

Orestes Foresto-Neto,

• Turin, Daniel Ryan, Mayo Clinic College of Medicine and Science, Rochester, Minnesota, United States

Daniel Ryan Turin,

• Holmes, Heather L., Mayo Clinic College of Medicine and Science, Rochester, Minnesota, United States

Heather L. Holmes,

• Reynolds, Carmen J., Mayo Clinic College of Medicine and Science, Rochester, Minnesota, United States

Carmen J. Reynolds,

• Arneson, Mariah L., Mayo Clinic College of Medicine and Science, Rochester, Minnesota, United States

Mariah L. Arneson,

• Jayachandran, Muthuvel, Mayo Clinic College of Medicine and Science, Rochester, Minnesota, United States

Muthuvel Jayachandran,

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

Julian A.t. Dow,

• Lieske, John C., Mayo Clinic College of Medicine and Science, Rochester, Minnesota, United States

John C. Lieske,

• Furrow, Eva, University of Minnesota Twin Cities College of Veterinary Medicine, Saint Paul, Minnesota, United States

Eva Furrow,

• Romero, Michael F., Mayo Clinic College of Medicine and Science, Rochester, Minnesota, United States

Michael F. Romero,

Background

Anoctamins (ANO) are Ca²⁺-activated phospholipid scramblases, Ca²⁺-activated Cl^- channels or both. Pathogenic ANO4 changes have been identified in dogs with calcium oxalate (CaOx) stones, and ANO4 protein expression is decreased in urinary extracellular vesicles of human CaOx stone formers. In Drosophila Malpighian tubules (MTs), subdued (ANO4-homolog) has these functions and participates in host defense against gram-negative bacteria. Urinary inoculation of mice with uropathogenic E. coli (UPEC) increases intrarenal CaOx crystallization. Thus, we investigated the interaction of subdued and UPEC for promoting Drosophila MT CaOx crystallization.

Methods

C724:Gal4, Uro:Gal4, and CG10116:Gal4 flies were crossed with UAS:subdued-RNAi flies to knockdown (KD) MT subdued in MT-stellate cells (SC), MT-principal cells (PC), or midgut cells (MG), respectively, with or without UPEC:eGFP. Ex vivo, dissected MTs were submerged for 90 min in a 10mM NaOx+UPEC:eGFP solution. In vivo, flies were fed a diet supplemented with 20mM NaOx+UPEC:eGFP for 4 days.

Results

SC subdued-KD slightly increased UPEC infection but did not change ex vivo or in vivo crystallization. PC subdued-KD facilitated UPEC invasion and increased crystal formation during short-term ex vivo assays. Neither crystal formation nor aggregation were changed by PC subdued-KD alone despite prolonged NaOx feeding in vivo. However, when UPEC was introduced with the NaOx diet, larger CaOx crystals were developed in MT of subdued-KD compared to wild-type flies. MG subdued-KD substantially increased UPEC presence in the MT lumen, nonetheless, this did not change CaOx crystallization in feeding experiments.

Conclusion

Drosophila are a useful genetic tool to study bacterial infection and CaOx crystallization. subdued KD in PCs or SCs, or UPEC infection alone did not change MT CaOx crystallization. However, the combination of PC subdued-KD and UPEC feeding in vivo increased bacterial infection and MT crystal formation with aggregation. These data suggest a role for ANO4 in bacterial-related human lithiases. U54-DK100227, R01-DK092408, F32-DK128987, FAPESP (2022/01226-1), ULTR002494, Mayo Foundation.

Funding

• NIDDK Support