Differential actions of cisplatin on renal proximal tubule and inner medullary collecting duct cells
Brady, H.R.; Zeidel, M.L.; Kone, B.C.; Giebisch, G.; Gullans, S.R.
Journal of Pharmacology and Experimental Therapeutics 265(3): 1421-1428
1993
ISSN/ISBN: 0022-3565 PMID: 8389866 Document Number: 417369
Cisplatin (CP) nephrotoxicity in vivo is characterized by proximal tubule (PT) and collecting duct dysfunction. We reported previously that mitochondrial injury is an important early event in CP toxicity to PT cells and precedes inhibition of Na+,K+-ATPase activity and loss of cell K+. In the present study, we monitored oxygen consumption (QO-2) and net K+ fluxes in intact inner medullary collecting duct (IMCD) and PT cells in vitro, using O-2- and K+-sensitive electrodes, to determine if CP has similar effects on IMCD cells. Short-term exposure of IMCD cells to CP resulted in inhibition of spontaneous, ouabain-sensitive and ouabain-oversensitive QO-2, but to a lesser degree than in PT. Ouabain-sensitive K+ transport and cell K+ content were also reduced in intact IMCD cells in this setting, confirming inhibition of Na+,K+-ATPase activity. In contrast, Na+,K+-ATPase activity measured in IMCD cell lysates was not altered. These results suggested that CP inhibited Na+,K+-ATPase activity in intact IMCD cells indirectly either by blocking Na+ entry or by inhibiting mitochondrial oxidative phosphorylation. Nystatin (Na+ ionophore) and carbonyl cyanide m-chlorophenylhydrazone (CCCP, uncoupler of oxidative phosphorylation) were used to distinguish between these possibilities. Nystatin-stimulated and CCCP-uncoupled QO-2 were reduced in CP-treated IMCD cells by 34 +- 10% and 25 +- 5%, respectively, indicating mitochondrial injury. Again, the effects of CP on nystatin-stimulated and CCCP-uncoupled QO-2 in IMCD cells were significantly less dramatic than in PT cells. Taken together, these results suggested that CP inhibits mitochondrial function and thereby causes a fall in QO-2, ATPase activity and Na+ and K+ transport in both IMCD and PT cells, but that PT cells are more sensitive to the cytotoxic actions of this agent. Furthermore, prolonged exposure of PT to CP was associated with striking peroxidation of membrane lipids, as measured by malonyldialdehyde (MDA) levels, suggesting free radical-mediated injury. Lipid peroxidation was associated with inhibition of Na+,K+-ATPase activity in PT cell lysates and increased release of 51chromium, indicating loss of cell membrane integrity. In contrast, CP-treated IMCD cells had markedly lower MDA levels under these conditions, and the integrity of the cell membrane appeared to be maintained. Significantly less MDA, inhibition of Na+,K+-ATPase and QO-2, and release of 51chromium were observed in CP-treated PT the presence of catalase, a scavenger of hydrogen peroxide. These data suggest that PT cells are more sensitive than IMCD cells to CP cytotoxicity in vitro and that reactive oxygen species such as hydrogen peroxide or its metabolites, possibly derived from injured mitochondria, may be important mediators of CP injury in this setting.