Aminoglycoside antibiotics (AGAs) are important in the treatment of life-threatening, Gram-negative infections. However, their clinical usefulness is limited by their otoxicity and nephrotoxicity. To better understand the molecular mechanisms underlying AGA toxicity, we studied their actions in proximal tubule-derived opossum kidney (OK) cells and in human embryonic kidney (HEK) cells stably transfected with the calcium-sensing receptor (CaR). Previously, we have demonstrated that OK cells express the CaR endogenously and that their acute exposure to high extracellular Ca²⁺ levels or to the AGAs neomycin and gentamicin results in intracellular Ca²⁺ mobilisation and phosphorylation of extracellular signal-regulated kinase (ERK) and Akt (Ward et al. 2002a,b). Since such responses are associated with altered cell fate, here we have examined the effect of chronic AGA treatment on OK cell proliferation and cell death.

AGA treatment (500 µM gentamicin) increased OK cell proliferation modestly, but significantly within 24 h as determined by cell counting (+8 ± 2% mean ± S.E.M., n = 7, P < 0.01 by Student’s paired t test) and XTT assay (+9 ± 1% n ²ge³ 3, P < 0.001 by ANOVA). This is consistent with the acute signalling reported previously (Ward et al. 2002a,b). However, after 4 days’ gentamicin treatment there was no evidence of increased cell number, or of G2/M arrest, but instead OK cells underwent dose-dependent cell death as determined using trypan blue (control, 5.5 ± 0.9 % cell-death; gentamicin, 12.6 ± 1.0% n = 7, P < 0.01 by ANOVA) and propidium iodide (flow cytometry). Furthermore, gentamicin elicited significantly more cell death in CaR-HEK cells (control, 2.3 ± 0.1 % cell death; 200 µM gentamicin, 4 ± 0.3% 500 µM gentamicin, 6 ± 0.8% n ²ge³ 6, P < 0.01 by ANOVA) than in non-transfected HEK-293 cells (control, 1.9 ± 0.1% 200 µM gentamicin, 1.8 ± 0.1% 500 µM gentamicin, 2.9 ± 0.9% n ²ge³ 6, NS). The size and granularity profiles of the gentamicin-treated cells were comparable for both cell types and neomycin elicited similar effects to gentamicin.

Together these data demonstrate that chronic AGA treatment induces cell death in proximal tubule-derived OK cells. In addition, the presence of the CaR in HEK cells significantly increases their susceptibility to gentamicin-induced cell death raising the possibility that the CaR may itself contribute to AGA nephrotoxicity.

This work was supported by the National Kidney Research Fund (TF6/2002).