For over 50 years, drug-induced mitochondrial toxicity has been investigated in academic studies (24). These drugs reduce the function of the electron transport chain (ETC), inhibit enzymes of the citric acid cycle, ATP generation, various mitochondrial transporters and the mitochondrial transcription and translational machinery (25). Therefore, despite the beneficial effects of drugs, due to the effect on mitochondrial activities, they can have deleterious side effects on the health of consumers (26). Unfortunately, most of these deleterious side effects were not detected in preclinical animal studies (27). These studies are usually done in young adult animals which have high robust mitochondrial reserves, lack of sufficient genetic diversity; insensitivity to tissue damage for revealing mitochondrial failure; absence of environmental factors and co-medication (27). Also, in these studies, the drugs would be studied separately without examining other effective parameters on toxicity such as genetic and environmental factors (28). Therefore, there are now several high-throughput preclinical organelle and in vitro cell models to address this issue (27). Furthermore, cell models cultured at high glucose levels for long periods are not suitable for assessing mitochondrial damage due to switching to glycolysis for energy production (29). Recently developed cell models grown in galactose or without glucose (such as HBSS buffer) and isolated mitochondria can more sensitively and accurately discriminate potential drug-induced mitochondrial toxicity (27, 30). Therefore, in this study, we focused on primary cells, RPTCs, obtained from kidney tissue with an incubation time of 4 h in HBSS buffer without glucose.
Mitochondrial toxicity caused by drugs has been recognized to induce organ toxicity to the kidney, skeletal muscle, heart, central nervous system and liver (24). Most of these observations have been obtained via studies performed in isolated mitochondria, cell lines and animal models (31). In current study, we searched for the first time the effect of colistin on RPTCs as a primary cell model to study nephrotoxic agents. Our results showed that colistin-induced cytotoxicity was associated with mitochondrial damage (MMP collapse and ATP depletion), ROS formation and oxidative stress (MDA content and GSH depletion). Consistent with our study, previous investigations have reported that mechanistically, colistin-induced nephrotoxicity is mainly on mitochondrial dysfunction triggered by oxidative stress (5, 18). Moreover, the beneficial effects of antioxidant and mitochondria protective agents such as panduratin A, melatonin, ascorbic acid and baicalein on colistin-induced nephrotoxicity, shows the importance of mitochondrial damage and oxidative stress in induction of kidney damage by this drug (14, 32–34). It has been reported that mitochondrial dysfunction and oxidative stress are important contributory factors to the lysosomal damages (35). Dysfunctional mitochondria can impact on the function of the lysosome by the formation of ROS as well as depriving the lysosome of ATP which is required by the V-ATPase proton pump to maintain the acidity of the lumen (35). Our results showed that colistin-induced lysosomal damages is associated with mitochondrial dysfunction, ROS formation and ATP depletion in RPTCs. In addition to, mitochondria regulate the activation of caspase and cell death via an event termed mitochondrial outer membrane permeabilization (MOMP); this leads to the release of various mitochondrial proteins that activate caspases, resulting in cell death (36). Our observations confirmed that colistin induces the loss of mitochondrial membrane potential and caspase 3 activation which is associated with cytotoxicity (measured with LDH) in RPTCs. The findings of our study and previous studies show that intervention in mitochondria can lead to reduction of colistin toxicity.
The beneficial effects of transplantation of mitochondria on drug toxicity, ischemic tissue damage and neurodegenerative disorder have been reported in previous studies (37). In this context, in the current study, we investigated whether the mitochondrial transplantation can prevent colistin-induced cytotoxicity in RPTCs. Our results showed that healthy isolated mitochondria treatment protect colistin-induced cytotoxicity in RPTCs. These findings confirmed that the positive effects of healthy isolated mitochondria treatment on colistin-induced cytotoxicity in RPTCs are associated with reduction of mitochondrial dysfunction, ROS formation, oxidative stress. A recent study has reported that mitochondrial transplantation isolated from mesenchymal stem cells reduces doxorubicin-mediated nephrotoxicity rats (38). In another study, mitochondrial transplantation caused histopathological stress, immunohistochemical stress (Bcl-2 and caspase-3), biochemical stress (urea, creatinine, blood urea nitrogen), and oxidative stress (lipid hyperlipidemia). oxidation, total superoxide dismutase, glutathione peroxidase) were reduced (38). They reported that mitochondrial transplantation improved the regeneration of tubular cells and the cellular antioxidant capacity (38). The findings of this research are largely close to our results in the current study. As presented in the results section, mitochondrial transplantation was able to reduce mitochondrial damage, ATP decline, ROS formation, GSH depletion, caspase 3 activation. Shi et al reported the positive effects of mitochondrial transplantation on acetaminophen-induced liver injury in mice (39). They observed the reduction of histopathological damage, serum transaminase, ROS production, mitochondrial swelling, and promotion of ATP levels and hepatic GSH after mitochondrial transplantation (39). Ulger et al. examined the effects of transplanted mitochondria on acetaminophen-induced toxic liver damage (40). They confirmed that mitochondrial transplantation can improve the liver histological structure to a similar level with healthy rats and decreases LDH levels, total oxidant levels, plasma ALT levels and apoptotic cells (40). It was also observed that mitochondrial transplantation increases GSH levels more effective than NAC treatment (40). The findings of the mentioned studies are in line with our observations in the current work. In addition, several studies using cell models, similar to our work have proven the beneficial effects of mitochondrial transplantation to reduce drug-induced cell damages (21, 41, 42).
In summary, colistin provoked damage in the RPTCs as evidenced by the increase of LDH levels, mitochondria membrane potential collapse, ROS formation, lysosomal dysfunction, depletion of GSH, ATP decline, oxidative stress and caspase 3 activation. The present study revealed for the first time that the healthy isolated mitochondria treatment could be helpful in reducing colistin-induced cytotoxicity in RPTCs via the maintenance of mitochondrial function, increase of antioxidant capacity, and inhibition of lysosomal damages and caspase 3 activation. The positive effect of mitochondrial transplantation against colistin-induced nephrotoxicity should be further searched by using animal and clinical trial studies. This study suggested that mitochondrial transplantation has the potential therapeutic against colistin-induced nephrotoxicity.