Aminoglycosides Increase Intracellular Calcium Levels and ERK Activity in Proximal Tubular OK Cells Expressing the Extracellular Calcium-Sensing Receptor

Abstract

Abstract. Aminoglycoside antibiotics (AGAs) are nephrotoxic, with most of the damage confined to the proximal tubule, but the mechanism for cellular toxicity is not clear. It has been previously shown that the extracellular-calcium sensing receptor (CaR) is expressed in intact rat proximal tubule and can be stimulated by the AGA neomycin. To investigate whether CaR could contribute to AGA-induced nephrotoxicity, the acute responses to various AGAs in the proximal tubule-derived opossum kidney (OK) cell line were examined. The presence in OK cells of CaR-related transcripts and protein was demonstrated by northern analyses, reverse transcriptase-PCR, immunocytochemistry, and immunoblotting. OK cells responded to elevated extracellular calcium (Ca 2ϩ o ) and neomycin but also to gentamicin and tobramycin with an increase in cytosolic [Ca 2ϩ

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

“…Unless otherwise stated, items were obtained from Sigma-Aldrich (Poole, Dorset, UK) or from sources described previously (9). Antisera were obtained from the following sources: phospho-Akt (Ser 473), phospho-Akt (Thr 308), pan-Akt 1/2/3, phospho-glycogen synthase kinase (GSK-3␤; Ser 9) and phospho-p38 mitogen-activated protein kinase (MAPK) were from Cell Signaling (Beverly, MA).…”

“…OK cells (used within 12 to 23 passages of purchase from the American Type Culture Collection, Rockville, MD) were cultured as described previously (9). HEK-293 cells, stably transfected with human parathyroid CaR (CaR-HEK) (11), were a gift from Dr. E.F. Nemeth (NPS Pharmaceuticals, Inc., Salt Lake City, UT), and empty vectortransfected HEK-293 cells were a gift from K. Croskery and Dr. R. Prince (University of Manchester, Manchester, UK) and were cultured as described previously (10).…”

“…However, chronic treatment of rats with low concentrations of gentamicin can elicit renal cortical apoptotic, rather than necrotic cell death (4). It is now well established that the extracellular calcium-sensing receptor (CaR) (5) is expressed on the apical surface of the PT (6 -9) and is responsive to micromolar concentrations of certain AGAs (5,9,10). Specifically, the AGAs stimulate the CaR with a rank order of potency that correlates positively with the number of their attached amino groups and indeed their nephrotoxicity.…”

“…As a G protein-coupled receptor, the CaR is capable of responding rapidly to AGA treatment, initiating signal transduction cascades that could theoretically alter both acute cell physiology and chronic cell fate. In this regard, we reported recently that in a well-characterized cell culture model of the PT, namely opossum kidney (OK) cells, exposure of the OK cells to elevated extracellular calcium levels, gentamicin, or neomycin caused intracellular Ca 2ϩ mobilization and activation of extracellular regulated protein kinase (ERK) 1,2 (9). In light of these circumstantial observations, to elucidate a possible cellular mechanism of AGA-induced toxicity, we investigated the consequences of acute and chronic aminoglycoside treatment on cell signaling in OK cells.…”

Drug-induced acid-base disorders

“…It has been hypothesized that gentamicin activates the CaSR in this area and in the distal convoluted tubule [168][169][170]. Under physiological conditions, the increased serum Ca 2+ levels stimulate this receptor and decrease the active reabsorption of Na + .…”