ASN's Mission

To create a world without kidney diseases, the ASN Alliance for Kidney Health elevates care by educating and informing, driving breakthroughs and innovation, and advocating for policies that create transformative changes in kidney medicine throughout the world.

learn more

Contact ASN

1401 H St, NW, Ste 900, Washington, DC 20005

email@asn-online.org

202-640-4660

The Latest on X

Kidney Week

Abstract: FR-PO311

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

Session Information

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
  • Turin, Daniel Ryan, Mayo Clinic College of Medicine and Science, Rochester, Minnesota, United States
  • Holmes, Heather L., Mayo Clinic College of Medicine and Science, Rochester, Minnesota, United States
  • Reynolds, Carmen J., Mayo Clinic College of Medicine and Science, Rochester, Minnesota, United States
  • Arneson, Mariah L., Mayo Clinic College of Medicine and Science, Rochester, Minnesota, United States
  • Jayachandran, Muthuvel, Mayo Clinic College of Medicine and Science, Rochester, Minnesota, United States
  • Dow, Julian A.t., University of Glasgow, Glasgow, Glasgow, United Kingdom
  • Lieske, John C., Mayo Clinic College of Medicine and Science, Rochester, Minnesota, United States
  • Furrow, Eva, University of Minnesota Twin Cities College of Veterinary Medicine, Saint Paul, Minnesota, United States
  • Romero, Michael F., Mayo Clinic College of Medicine and Science, Rochester, Minnesota, United States
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