614.20 - Gentamicin induces calciuresis by blocking TRPV5

Wouter van Megen (University of Southern Denmark), Megan Beggs (University of Alberta, Womens and Childrens Health Institute, University of Alberta), Sung-Wan An (University of Texas Southwestern Medical Center), Patrícia Ferreira (University of Southern Denmark), Justin Lee (University of Alberta), Matthias Wolf (University of Texas Southwestern Medical Center), R. Alexander (University of Alberta, Womens and Childrens Health Institute, University of Alberta, University of Alberta ), Henrik Dimke (University of Southern Denmark, Odense University Hospital)

Background

Treatment with the aminoglycoside antibiotic gentamicin can be associated with severe side effects including renal Ca2+wasting. The underlying mechanism is unknown but proposed to involve activation of the Ca2+-sensing receptor (CaSR) in the thick ascending limb (TAL), which would increase expression of claudin-14 (CLDN14) and limit Ca2+ reabsorption. However, no direct evidence for this hypothesis has been presented. 

Methods

The effect of gentamicin on renal Ca2+ handling was studied in vivo using mouse models with impaired Ca2+ reabsorption in the proximal tubule and TAL. The effects of gentamicin in CaSR activation were studied in vitro using a Cldn14 promoter luciferase-reporter assay, while the effect of gentamicin on the distal nephron Ca2+ channel transient potential receptor vanilloid 5 (TPRV5) activity was determined by patch-clamp in HEK293 cells.

Results

Gentamicin increased urinary Ca2+ excretion in wildtype mice following acute and chronic administration. This calciuretic effect was unaltered in mice with genetic CaSR overactivation and remained present in furosemide-treated animals. Moreover, gentamicin-induced calciuresis was not significantly different in Cldn14-/- mice or mice with impaired proximal tubular Ca2+reabsorption (Cldn2-/- mice), compared to wildtype animals. In vitro, gentamicin failed to activate the CaSR. In contrast, patch-clamp analysis revealed that gentamicin inhibited rabbit and human TRPV5 channel currents in a dose-dependent manner. In accordance with a direct effect of gentamicin on TRPV5, chronic gentamicin administration downregulated gene expression of distal nephron Ca2+ transporters in mice. 

Conclusion

We show here that gentamicin does not cause hypercalciuria via activation of the CaSR-CLDN14 pathway or by interfering with proximal tubular CLDN2-dependent Ca2+ reabsorption. Instead, gentamicin blocks distal Ca2+ reabsorption by direct inhibition of the Ca2+ channel TRPV5. These findings offer new insights into Ca2+ wasting in patients treated with gentamicin.

This work was supported by grants from Erasmus+ 2018/E+/4458087 awarded to WH van Megen, the Canadian Institutes for Health research to RT Alexander and the Novo Nordisk Foundation, the Beckett Foundation, the Carlsberg Foundation, and Independent Research Fund Denmark awarded to H. Dimke. Dr. R. Todd Alexander is the Canada Research Chair in Renal Tubular Epithelial Transport Physiology and is a Distinguished Researcher of the Stollery Childrenamp;rsquo;s Hospital Foundation. Matthias T Wolf is supported by the Department of Defense (W81XWH1910205), the National Institute of Health (R01DK119631, DK079328-11), and the Childrenamp;rsquo;s Clinical Research Advisory Committee (CCRAC), Childrenamp;rsquo;s Health System, Dallas.