*3.2.* α*M's Protection is Associated to the Preservation of Mitochondrial Bioenergetics*

The main nephrotoxic mechanism of CDDP is mitochondrial damage [13,14]. Therefore, we determined if the preservation of mitochondrial bioenergetics was related to protection by αM. So, we evaluated the respiratory states in the whole cells. As shown in Figure 3, CDDP-treatment reduced the Routine respiration, the Leak of respiration, the maximum capacity of the electron transfer system (E) and the respiration associated to oxidative phosphorylation (P) at 24 h. Interestingly, due to both Routine and Leak respiration reduced, the respiratory control (RC) did not change with CDDP-treatment (Figure 3E). All respiratory state alterations were reversed by αM co-treatment. Importantly αM alone did not alter any respiratory parameters at this concentration (Figure 3).

**Figure 2.** Changes in oxidative stress markers in LLC-PK1 cells treated with αM, CDDP or both. After 24 h of αM-, CDDP-treatment or both, the oxidative stress was evaluated by the lipoperoxidative markers (**A**) malondialdehyde (MDA) and (**C**) 4-hydroxynonenal (4HNE). (**A**) and (**C**) show the densitometry data and (**B**) and (**C**) show the representative blots. The data are presented as mean ± SD, *n* = 6–7. \*\* *p* < 0.01 vs. control. ## *p* < 0.01 vs. CDDP. α-Tub = alpha-tubulin.

**Figure 3.** αM co-treatment prevents CDDP induced a decrease in respiratory parameters at 24 h. (**A**) Cellular routine respiration = Routine, (**B**) leak of respiration, (**C**) maximum capacity of electron transfer system = E, (**D**) respiration associated to oxidative phosphorylation = P, (**E**) respiratory control <sup>=</sup> RC. The data are presented as mean <sup>±</sup> SD, *<sup>n</sup>* <sup>=</sup> 3–5. \*\* *<sup>p</sup>* <sup>&</sup>lt; 0.01 and \* *<sup>p</sup>* <sup>&</sup>lt; 0.05 vs. control. ### *<sup>p</sup>* <sup>&</sup>lt; 0.001 and # *p* < 0.05 vs. CDDP.

The decrease in all the respiratory states without changes in RC, suggested that the mitochondrial bioenergetics alterations were related to a reduction in the activity of the whole electron transport system (ETS). So, we evaluated the CI-linked respiration, the CIV activity and the percentage of routine respiration attributable to CI. The CDDP-treatment diminished CI-linked respiration but had no effect on CIV activity and the percentage of basal respiration attributable to CI was not reduced (Figure 4). These results, together with the decrease in E, imply a reduction in whole ETS activity induced by CDDP, which could be caused by a reduction in mitochondrial mass.

**Figure 4.** Determination of respiratory parameters at 24 h in LLC-PK1 cells treated with αM, CDDP or both. (**A**) Complex I (CI)-linked respiration, (**B**) percentage of respiration attributable to CI and (**C**) activity of complex IV (CIV). The data are presented as mean ± SD, *n* = 4–6. \* *p* < 0.05 vs. control. ## *p* < 0.01 vs. CDDP.
