*3.3. Mitochondrial Respirometry Alterations are Associated to Mitochondrial Mass Decrease Related to Mitochondrial Biogenesis Reduction*

To determine if the reduction in the respiration states was linked to a decrease in mitochondrial mass, the protein levels of VDAC and some mitochondrial complex subunits were evaluated by WB. We evaluated mitochondrial subunits using an cocktail contained antibodies to labile mitochondrial subunits. So, they allow to evaluate the changes in the mitochondrial proteins of the ETS in the membrane only. Furthermore, several models of acute and chronic kidney damage have previously reported a reduction in protein levels of these subunits as well as in levels of their messengers, which has been related to a reduction in mitochondrial biogenesis and bioenergetics in such models [30–35]. Figure 5 shows that CI-NDUFB8 (Figure 5A), CII-SDHB (Figure 5B) and CIV-MTCO1 (Figure 5C) subunits were decreased with CDDP-treatment, whereas the CV-ATP5A subunit was unchanged (Figure 5D). Moreover, we found a decrease in VDAC protein levels with CDDP-treatment (Figure 5F,G), which was partially prevented by αM-treatment. To corroborate these results, we obtained representative images of the mitochondrial mass using MTG, a selective mitochondrial fluorescent label [36] and also MFN2, fusion marker. Figure 6A shows the images of CDDP treated cells, where a fragmented mitochondrial network was observed, with loss of continuity and fluorescence focal points. In addition, we found the MNF2 level in CDDP treatment that was avoided by αM co-treatment suggests fusion diminishing (Figure 6B,C). Together, these results support the idea that CDDP increases mitochondrial mass reduction and fragmentation, while αM can partially preserve it.

To determine if the observed reduction in mitochondrial mass was related to alterations in mitochondrial biogenesis, we evaluated the levels of mitochondrial biogenesis proteins PCG-1α, NRF1, and TFAM. Interestingly NRF1 levels were upregulated in all treatments (Figure 7B), although we expected a reduction. The implications of these changes are deliberated in the discussion section. Furthermore, although we did not observe changes in PGC-1α (Figure 7A) CDDP-treatment downregulated TFAM levels (Figure 7C) and αM co-treatment prevented it. These results suggest that mitochondrial mass reduction is partially attributable to the decrease in mitochondrial biogenesis induced by CDDP.

**Figure 5.** CDDP-induced toxicity is related to a decrease in mitochondrial proteins. LLC-PK1 cells were treated with αM, CDDP or both for 24 h. After treatment, western blotting of total oxidative phosphorylation (OXPHOS) cocktail in the whole cell (**A**–**D**,**F**) voltage-dependent anion channel (VDAC) as mitochondrial mass markers were quantified and (**E**,**G**) representative protein expression is shown. Densitometry values were normalized by Ponceau red staining. The data are presented as mean <sup>±</sup> SD, *<sup>n</sup>* <sup>=</sup> 4–8. \*\*\* *<sup>p</sup>* <sup>&</sup>lt; 0.001, \*\* *<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 vs. (α<sup>M</sup> <sup>+</sup> CDDP). NDUFB8 = NADH: ubiquinone oxidoreductase subunit B8, SDHB = succinate dehydrogenase complex iron–sulfur subunit B, MTCO1 = mitochondrial cytochrome c oxidase I catalytic subunit, ATP5A = ATP synthase F subunit alpha, α-Tub = alpha-tubulin.

**Figure 6.** CDDP-induced toxicity is related to a decrease of mitochondrial fusion. LLC-PK1 cells were treated with αM, CDDP or both for 24 h. After treatment, (**A**) representative micrographs of MitoTracker green, mitochondrial mass marker, at 20x and posterior zoom 1:1 and western blotting of (**B**) Fusion marker, mitofusin 2 (MNF2) level and representative blot in (**C**). The data are presented as mean <sup>±</sup> SD, *n* = 6–8. \*\*\* *p* < 0.001 vs. control. ### *p* < 0.001, ## *p* < 0.01 vs. CDDP. <sup>+</sup> *p* < 0.05 vs. (αM+CDDP). α-Tub = alpha-tubulin.

**Figure 7.** Modulation of mitochondrial biogenesis in LLC-PK1 cells treated with αM, CDDP or both. After 24 h of treatment, protein levels of (**A**) peroxisome proliferator-activated receptor gamma (PPARγ) coactivator 1-alpha (PGC1α), (**B**) nuclear respiratory factor 1 (NRF1), (**C**) mitochondrial transcription factor A (TFAM) were quantified. Representative blots are shown in (**D**). The data are presented as mean <sup>±</sup> SD, *<sup>n</sup>* <sup>=</sup> 5–7. \*\*\* *<sup>p</sup>* <sup>&</sup>lt; 0.001. ### *<sup>p</sup>* <sup>&</sup>lt; 0.001 and ## *<sup>p</sup>* <sup>&</sup>lt; 0.01 vs. CDDP. <sup>α</sup>-Tub <sup>=</sup> alpha-tubulin.
