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An anoctamin 4 (ANO4) mutation was identified in dogs with calcium oxalate (CaOx) stones via GWAS, and the content of ANO4 is decreased in urinary extracellular vesicles of human CaOx stone formers. ANO proteins are Ca2+-activated Cl- channels and/or Ca2+-activated phospholipid scramblases. In addition to these functions, the homolog of ANO4 in Drosophila, subdued, has been shown to play a role in host defense against gram-negative bacteria. Uropathogenic E. coli (UPEC) increases intrarenal CaOx crystallization in mammals. Thus, we investigated the interaction of subdued and UPEC for promoting CaOx crystallization in Drosophila Malpighian tubules (MTs).
The driver lines Uro:Gal4, C724:Gal4, and CG10116:Gal4 were crossed with UAS:subdued-RNAi flies to knockdown (KD) subdued in MT-principal cells (PC), MT-stellate cells (SC), or midgut cells (MG), respectively. For ex vivo experiments, dissected MTs were submerged for 90 minutes in a 10mM NaOx+UPEC:eGFP solution. For in vivo crystallization, flies were fed a diet supplemented with 20mM NaOx+UPEC:eGFP for 4 days. HEK cells were transfected with wild-type subdued-eGFPN1 or mutation Q→H subdued-eGFPN1 and underwent whole-cell patch clamp experiments.
KD of subdued in PC facilitated UPEC infection, resulting in increased crystal formation in ex vivo assays. Neither crystal formation nor aggregation were affected by the KD of subdued in PC alone during NaOx feeding. However, when UPEC was added to the NaOx diet, crystal number and crystal size were increased in MT of subdued-KD flies compared to wild-type flies. KD of subdued in SC slightly increased UPEC infection but did not change ex vivo or in vivo crystallization. KD of subdued in MG largely increased UPEC presence in the MT, nonetheless, this increased the expression of subdued and did not change crystallization in MT in vivo. Cl- currents from Q→H subdued-transfected cells were less than currents from wild-type subdued-transfected cells.
Alone, the KD of subdued in PCs or SCs, or UPEC infection did not interfere with crystallization. However, the combination of PC subdued-KD and presence of UPEC increased CaOx crystal formation with aggregation in the MTs. Changes in Cl- currents may be involved in reduced protection against bacterial infection and increased crystallization in subdued mutation/KD cells. These data suggest a role for ANO4 in bacterial-related human and canine lithiases. U54-DK100227, F32-DK128987, R01-DK092408, ULTR002494, FAPESP 2022/01226-1, Mayo Foundation.