Mitochondrial injury: an early event in cisplatin toxicity to renal proximal tubules

Abstract

Oxygen consumption (QO2) and net K+ transport were studied in rabbit proximal tubule suspensions to define the early effects of cisplatin on proximal tubule function. Cisplatin caused dose-dependent inhibition of QO2, which was delayed in onset. The concentration of cisplatin required for inhibition decreased as the duration of exposure was increased [40-min exposure, threshold concentration of 10(-4) M, inhibitor constant (Ki) of 10(-3) M; 4-h exposure, threshold concentration of 3 X 10(-5) M, Ki of 10(-4) M]. Both ouabain-sensitive and ouabain-insensitive QO2 were reduced, indicating inhibition of all adenosinetriphosphatases, including Na(+)- K(+)-ATPase activity. There was a parallel fall in ouabain-sensitive net K+ transport and cytosolic K+ content, confirming the latter observation. Na(+)-K(+)-ATPase activity was unchanged in cell membranes prepared by hypotonic lysis from cisplatin-treated tubules, indicating an indirect cytosol-dependent mechanism of enzyme inhibition. Nystatin-stimulated QO2 was reduced in cisplatin-treated tubules, excluding inhibition of Na+ entry as the mechanism of injury and suggesting mitochondrial injury. The latter was confirmed by measurement of carbonylcyanide-m-chlorophenylhydrazone (CCCP)-uncoupled QO2 in intact cells and ADP-stimulated (state 3) QO2 in digitonin-permeabilized tubules. Furthermore, by maximally stimulating mitochondrial respiration with CCCP and nystatin, it was possible to demonstrate mitochondrial injury at a time when basal QO2 and K+ transport were apparently normal. These data suggest that mitochondrial injury is a central event in cisplatin toxicity to the proximal tubule.

Cisplatin-Induced Toxicity Is Associated with Platinum Deposition in Mouse Kidney Mitochondria in Vivo and with Selective Inactivation of the α-Ketoglutarate Dehydrogenase Complex in LLC-PK1 Cells

“…The present finding that Pt bound to proteins is greater in renal mitochondria of mice injected with cisplatin than in the cytosolic fraction or the microsomal fraction (Figure 1) is consistent with the previous observation that cisplatin is a mitochondrial toxicant in confluent renal proximal tubule cells (50). Our finding leads to the question of how Pt is incorporated into renal mitochondrial proteins.…”

“…The LD 50 of cisplatin in LLC-PK 1 / mitAspAT cells was 126 µM with 95% confidence intervals ranging from 116 to 136 µM. The LD 50 of cisplatin in LLC-PK 1 /C1 cells was 182 µM with 95% confidence intervals ranging from 177 to 187 µM. There was a significant difference between the LD 50 of LLC-PK 1 /mitAspAT and LLC-PK 1 /C1 cells toward cisplatin (p <0.0001), and the slopes of the two dose curves were also significantly different (p = 0.0033).…”

“…The mean and standard deviation (SD) were computed for each treatment. LD 50 and its 95% confidence intervals were calculated with Prism sigmoidal dose-response (variable slope) curve fit. The two-tailed t-test was used to detect significant difference in cisplatin toxicity among the cell lines and to detect changes in enzyme activities in LLC-PK 1 cells overexpressing mitAspAT.…”

“…The LD 50 of cisplatin in dividing LLC-PK 1 /C1 cells was 25.7 µM with 95% confidence intervals ranging from 21.2 to 31.1 µM. The LD 50 of cisplatin in dividing LLC-PK 1 /mitAspAT cells was 28.2 µM with 95% confidence intervals ranging from 22.4 to 35.4 µM. There was no significant difference between the LD 50 of the dividing LLC-PK 1 /C1 and LLC-PK 1 /mitAspAT cells (p = 0.48).…”

Cisplatin Binding and Inactivation of Mitochondrial Glutamate Oxaloacetate Transaminase in Cisplatin-Induced Rat Nephrotoxicity

“…2,4,14,15) It is well established that mitochondria are target of cisplatin toxicity, and that mitochondrial injury is a very early event in cisplatininduced nephrotoxicity, 17,20,21,37) but why and how cisplatin causes mitochondrial dysfunction is not understood. The present study partially clarified the mechanisms of cisplatin-induced nephrotoxicity.…”