*3.4. The Induction of Mitophagy is Related to Protection by* α*M*

Dysfunctional mitochondria are usually eliminated through diverse processes, such as mitophagy [13], so we evaluated if mitophagy was related to CDDP-induced decrease in mitochondrial mass. Figure 8 shows that CDDP increased both PINK1 (Figure 8A) and Parkin (Figure 8B) protein levels and this increase was prevented by αM co-incubation. As the mitophagy process can be related to general macroautophagy, we pre-incubated LLC-PK1 cells with 30 μM CQ for 2 h before αM or CDDP-treatment, and with 10 nM of wortmannin 1 h before and during treatment, as autophagy inhibitors (Figure 9). Although autophagy inhibition has been related to the worsening of CDDP injury, Figure 9A shows that, apparently, CQ only has an effect on CDDP-treatment and does not have any effect over the protection by αM. CQ pre-treatment did not reduce cell viability in αM+CDDP-treatment. Wortmannin, as well as CQ, did not have any effect over the protection by αM (Figure 9B). Probably protection by αM has a mechanism independent of the induction of autophagy. Figure 9C shows micrographs representative of all treatments. CDDP treated cells showed less density at 24 h, as well as cell swelling and loss of structure compared to the control group. αM co-treatment prevented CDDP-alterations. In addition, in CQ and wortmannin + CDDP. we observed similar damage to the CDDP group. However, CQ or wortmannin did not alter the morphological characteristic compared with the αM+CDDP treatment.

**Figure 8.** Modulation of mitophagy induction in LLC-PK1 cells treated with αM, CDDP or both. After 24 h of αM-, CDDP-treatment or both, mitophagy proteins (**A**) phosphatase and tensin homologue (PTEN)-induced kinase 1 (PINK1) and (**B**) parkin RBR E3 ubiquitin protein ligase (Parkin) were evaluated. Representative blots are shown in (**C**). The data are presented as mean ± SD, *n* = 6–7. \* *p* < 0.05 vs. control. # *p* < 0.05 vs. CDDP. α-Tub = alpha-tubulin.

**Figure 9.** Effect of autophagy inhibition on the protection induced by αM vs. CDDP. LLC-PK1 cells were pretreated with (**A**) 30 μM chloroquine (CQ) for 2 h, as an inhibitor of degradation of cargo in the final autophagy phase, or (**B**) 10 nM wortmannin for 1 h and during the experiment, as an inhibitor of phosphoinositide 3-kinase (PI3K) in the initial autophagy phase induction. After pre-treatment, the cells were co-incubated with 4 μM αM and/or 30 μM CDDP for 24 h. Cell viability was determined, and (**C**) representative micrographs were taken. The data are presented as mean ± SD, *n* = 4–6. \*\*\* *p* < 0.001 vs. control. ### *p* < 0.001, ## *p* < 0.01 and # *p* < 0.05 vs. cisplatin. +++ *p* < 0.001 and ++*p* < 0.01 vs. (αM+CDDP). ddd *p* < 0.001 vs. (stressor+αM+CDDP). eee *p* < 0.001, ee *p*<0.01 and <sup>e</sup> *p* < 0.05 vs. (stressor+CDDP).

Figure 10C shows that CDDP-treatment induced an increase in p62 levels at 24 h, and a slight increase in microtubule-associated protein 1 light chain 3 alpha I (LC3-I) (Figure 10A) and the LC3-II/LC3-I ratio (Figure 10B). The increase in p62 is relevant because of the fact that p62 has been reported as one of receptors involved in PINK1/Parkin-mediated mitophagy balance [37] and aberrant p62 levels may be the result of mitochondrial dysfunction [38], which together with the autophagy results (Figure 9), suggest that mitophagy is involved in the observed mitochondrial mass decrease induced by CDDP.

**Figure 10.** Time-course effect of αM and CDDP on autophagy stress markers. LLC-PK1 cells were incubated with 4 μM αM, 30 μM CDDP or both during 0, 2, 4, 6, 8, 16, and 24 h. After each treatment, (**A**) microtubule-associated protein 1 light chain 3 alpha I (LC3-I), (**B**) lipidated form of LC3 (LC3-II), and (**C**) ubiquitin-binding protein p62 (p62) protein levels were evaluated. Panels A, B, and C show quantitative data, and panel D shows representative blots. Data are presented as mean ± SD, *n* = 3–5. \*\* *p* < 0.01 vs. control and ### *p* < 0.01 vs. CDDP. α-Tub = alpha-tubulin.
