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Aminoglycosides Induce Acute Cell Signaling and Chronic Cell Death in Renal Cells that Express the Calcium-Sensing Receptor

Donald T. Ward^*, David Maldonado-Pérez^*,, Laura Hollins^* and Daniela Riccardi^*,

^* Faculty of Life Sciences, The University of Manchester, Manchester, United Kingdom; and Cardiff School of Biosciences, University of Cardiff, Cardiff, United Kingdom

Address correspondence to: Dr. Donald Ward, G38 Stopford Building, The University of Manchester, Oxford Road, Manchester, M13 9PT United Kingdom. Phone: 011-44-161-275-5459; Fax: 011-44-161-275-5600; d.ward{at}manchester.ac.uk

Dr.Daniela Riccardi, Cardiff School of Biosciences, Cardiff University, Museum Avenue, Cardiff CF10 3US, United Kingdom. Phone: 011-44-29208-79132; Fax: 011-44-29208- 74117; riccardid{at}cardiff.ac.uk

Received for publication August 3, 2004. Accepted for publication January 31, 2005.

The aminoglycoside antibiotics (AGAs) are calcium-sensing receptor (CaR) agonists that are toxic to the renal proximal tubule. Proximal tubule-derived opossum kidney (OK) cells express CaR-like proteins and respond to AGAs with intracellular Ca²⁺ mobilization and extracellular regulated protein kinase (ERK) phosphorylation. To examine the possible cellular basis of AGA toxicity, acute and chronic responses to AGA treatment in OK cells and in CaR stably transfected HEK-293 cells (CaR-HEK) were studied. Changes in cell-fate signaling, proliferation, and cell death were detected by semiquantitative Western blotting, Hoechst staining, cell counting, and FACS analysis. Confocal microscopy was used to study the relative internalization of fluorophore-labeled gentamicin in CaR-transfected and -nontransfected cells. Here it is reported that the AGA neomycin and gentamicin elicit acute, phosphatidylinositol-3 kinase-dependent phosphorylation of Akt, glycogen synthase kinase 3, and p38 mitogen-activated protein kinase. After 24 h of gentamicin treatment, OK cell proliferation was observed, whereas after 4 d, the OK cells underwent cell death, an effect that was mimicked by the CaR agonists spermine and polyarginine. Furthermore, gentamicin elicited substantially more cell death in CaR-HEK cells than in nontransfected HEK-293 cells. The pan-specific caspase inhibitor Z-VAD significantly inhibited cell death in both OK and CaR-HEK cells. Finally, the intracellular uptake of Texas Red-labeled gentamicin was equivalent in both CaR-transfected and vector-transfected HEK-293 cells, suggesting that the CaR does not enhance drug uptake. Together, these observations demonstrate that the AGAs induce both acute and chronic cell fate changes in OK cells and CaR-HEK cells and that the proximal tubular CaR is likely to contribute to signaling underlying the renal toxicity of the AGAs.

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Copyright © 2008 by the American Society of Nephrology. Online ISSN: 1533-3450 Print ISSN: 1046-6673