Disturbed Ca2+-signaling by chloroacetaldehyde: A possible cause for chronic ifosfamide nephrotoxicity

Disturbed Ca2+-signaling by chloroacetaldehyde: A possible cause for chronic ifosfamide nephrotoxicity.BackgroundRenal damage following chemotherapy with ifosfamide is attributed to the metabolic activation of the drug and the generation of chloroacetaldehyde (CAA). Little is known about the mechanism by which CAA impairs renal function. In this study the effect of CAA on intracellular Ca2+ homeostasis in human renal proximal tubule cells (RPTEC) in primary culture was investigated.MethodsIntracellular Ca2+ was measured using the Ca2+-sensitive dye fura-2. Cell viability was determined by protein content and cell number. Oncotic and apoptotic cell death was assayed using trypan blue exclusion, caspase-3 activity, and 4′,6-diamino-2-phenylindole (DAPI) staining.ResultsCAA (1.5 to 150 μmol/L) induced sustained elevations of intracellular free calcium ([Ca2+]i) from 75 ± 3 nmol/L to maximal 151 ± 6 nmol/L. This effect was dependent on extracellular Ca2+, but not Ca2+ entry. The rise in [Ca2+]i mediated by CAA could be attributed to inhibition of Na+-dependent extrusion of intracellular Ca2+, indicating an inhibitory action of CAA on Na+/Ca2+ exchange. Modulation of protein kinase A (PKA), but not protein kinase C (PKC) blunted the effect of CAA. Thus, CAA seems to inhibit Na+/Ca2+ exchange by interaction with cyclic adenosine monophosphate (cAMP)-PKA-signaling. A 48-hour exposure to 15 μmol/L CAA significantly reduced cell number and protein content of RPTEC by induction of necrosis. This effect of 15 μmol/L CAA could be overcome by coadministration of the intracellular Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid acetoxymethyl ester (BAPTA-AM).ConclusionFirst, CAA inhibits the Na+/Ca2+-exchanger. Second, this effect is dependent on PKA. Third, CAA induces necrotic rather than apoptotic cell death. Finally, disturbed Ca2+ homeostasis via Na+/Ca2+ exchange contributes to the nephrotoxic action of CAA in RPTEC.