Indian Journal of Animal Research

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Indian Journal of Animal Research, volume 58 issue 3 (march 2024) : 459-469

Effects of Concomitant Use of N-acetylcysteine and Cyclosporine A on Acetaminophen-induced Acute Kidney Injury in Mice

Gonca Ozan Kocamüftüoğlu1,*, Nalan Kaya Tektemur2, Elif Erdem Güzel3, Ahmet Tektemur4, İbrahim Enver Ozan2
1Department of Biochemistry, Faculty of Veterinary Medicine, Burdur Mehmet Akif Ersoy University, Burdur, Turkey.
2Department of Histology and Embryology, Faculty of Medicine, Firat University, Elazig, Turkey.
3Department of Midwifery, Faculty of Health Sciences, Mardin Artuklu University, Mardin, Turkey.
4Department of Medical Biology, Faculty of Medicine, Firat University, Elazig, Turkey.
Cite article:- Kocamüftüoğlu Ozan Gonca, Tektemur Kaya Nalan, Güzel Erdem Elif, Tektemur Ahmet, Ozan Enver İbrahim (2024). Effects of Concomitant Use of N-acetylcysteine and Cyclosporine A on Acetaminophen-induced Acute Kidney Injury in Mice . Indian Journal of Animal Research. 58(3): 459-469. doi: 10.18805/IJAR.BF-1736.

Background: Acetaminophen (APAP), a commonly used analgesic, causes acute kidney injury (AKI) in overdose although it is rare. Mitochondrial dysfunction plays a major role in the pathophysiology of renal damage, although the exact molecular mechanism is unknown. This study aimed to evaluate the potential therapeutic effect of cyclosporin A (CsA), a mitochondrial membrane permeability transition pore (MPTP) inhibitor, with N-acetylcysteine (NAC) in APAP-induced AKI. 

Methods: Male BALB/c mice were divided into control, APAP, APAP+NAC, APAP+CsA and APAP+NAC+CsA groups (n=6). A single dose of APAP (400 mg/kg) was administered intraperitoneally. All other treatments (1200 mg/kg NAC, 50 mg/kg CsA) were performed intraperitoneally 3 h after APAP administration. All animals were decapitated and blood samples and kidney tissue samples were collected for evaluation. Serum creatinine (Cr) and blood urea nitrogen (BUN) levels were measured. The kidney tissue 8-hydroxy-deoxyguanosine (8-OHdG), cytochrome c (Cytc) and 3-nitrotyrosine (3-NT) levels and cytochrome c (Cytc) expressions were determined. 

Result: Increased Cr and BUN levels, histopathological examinations and expressions of 8-OHdG, 3-NT and Cytc were detected in the APAP group. Combined NAC+CsA treatment sufficiently reversed oxidative stress, serum Cr and BUN levels and histopathological alterations induced by APAP. Moreover, cytc levels and renal tubular injury were remarkably reduced by combined drug treatment compared to the APAP+NAC group. These data suggest that the therapeutic effect of combined NAC+CsA treatment in mice with APAP-induced nephrotoxicity can be related to the combination of the antioxidant effect of NAC and the mitochondrial MPTP inhibitor effect of CsA.

Acetaminophen (APAP), one of the most commonly used analgesics, is safe at therapeutic dosages. However, its overdose can lead to acute liver failure (ALF) and/or acute kidney injury (AKI). Renal tubular damage and acute renal failure may occur in nearly 2% of patients (Ko et al., 2017) and AKI can occur in patients with APAP overdose without liver failure. AKI is a syndrome associated with many etiologies and pathophysiologic processes leading to impaired kidney function (Akakpo et al., 2020). Regarding this clinical condition, there are many case reports in the literature. Such that the hemodialysis requirement of the patients has indicated the seriousness of these cases (Jeffery and Lafferty, 1981; Blakely and McDonald, 1995; Mazer and Perrone, 2008; Waring, 2009; Saleem and Iftikhar, 2019). Although the precise mechanism of APAP-induced AKI is obscure, a wide variety of processes from oxidative stress to mitochondrial dysfunction may have a crucial role in the primary mechanism of AKI (Ruan et al., 2019).

It is well known that oxidation of APAP in the liver led to a toxic metabolite, N-acetyl-p-benzoquinone imine (NAPQI) production, which is detoxified by hepatic glutathione (GSH). APAP overdose-mediated high NAPQI levels cause depletion of GSH, so reactive oxygen species (ROS) levels increase and oxidative stress occurs (Abdeen et al., 2019). Besides, it has been reported that APAP overdose-related excessive NAPQI product is excreted by the kidney, so it can conduce to renal injury (Chen et al., 2015). N-acetylcysteine (NAC), which is an amino acid that contains the thiol group, is a safe and cheap drug. NAC is a precursor of GSH, which is one of the most important antioxidants. Therefore, it is used for treating diseases induced by free oxygen radicals. Moreover, it has been used for the therapy of APAP-induced toxicity for a long time. It is universally effective in preventing hepatotoxicity if administered within 10 h of APAP overdose (Mokhtari et al., 2017). Although the efficacy of NAC against APAP-induced hepatoxicity has been reported many times in experimental studies, some studies have shown that it cannot effectively attenuate kidney damage caused by APAP overdose (Mazer and Perrone, 2008; Akakpo et al., 2020). Therefore, there is a need for additional drugs that can significantly improve kidney damage resulting from APAP overdose.

The kidney is a vital organ involved in the filtration of various substances and is particularly sensitive to drugs taken in toxic doses (Ansari et al., 2020). In kidneys, which require high ATP in terms of function, the density of mitochondria is high, especially in the renal tubules. Moreover, it is well known that mitochondria play important roles in many cellular processes, such as signal transduction, cell proliferation, cell growth and cell death. Therefore, mitochondrial dysfunction or damage could lead to impaired ATP generation and ROS balance. On the other hand, most therapeutic drugs may cause AKI because of their negative effects on the mitochondrion of the renal tubule (Hua et al., 2018). Moreover, mitochondrial damage through the opening of the mitochondrial membrane permeability transition pore (MPTP) has an important role in the mechanism of APAP-induced toxicity. Thus, mitochondria are a potential target for treating AKI. Cyclosporin A (CsA) is an immunosuppressive drug (Taylor et al., 2005) that prevents the opening of MPTP by binding to cyclophilin D, which penetrates the inner and outer membranes of mitochondria (Kim et al., 2003; Niimi et al., 2012). The opening of MPTP plays a crucial role in the mitochondrial dysfunction-induced cell death mechanism. Thus, the inactivation of cyclophilin D using CsA can block MPTP and suppress APAP-induced mitochondrial damage (Niimi et al., 2012).

In our previous study, it was revealed that hepatotoxicity occurred because of APAP overdose in mice and combined NAC+CsA treatment could be beneficial against hepatotoxicity (Kaya Tektemur et al., 2021). However, considering that hepatotoxicity caused by high-dose APAP is rarely seen together with renal damage and because NAC treatment alone is not sufficient to improve, especially, APAP overdose-related renal injury, we aimed to evaluate the potential therapeutic effects of NAC+CsA combined treatment on APAP-induced nephrotoxicity in mice.
Ethical statement

A total of 30 adult male BALB/c mice were used in this study. Mice were housed under optimal conditions (food, temperature, water, light) at the Firat University Animal Research Center (Elazig, Turkey). All experimental procedures were conducted according to the European Union guidelines (2010/63/EU) and ethical approval was obtained from the Firat University Animal Experiments Local Ethics Committee (Ethical number: 30.08.2021/3348).

Experimental procedure

Mice were randomly divided into five groups (n=6): Control, APAP, APAP+NAC, APAP+CsA and APAP+NAC+CsA. To induce the AKI model, APAP was administered in a single dose of 400 mg/kg (Salama et al., 2015). All other treatments were performed 3 h after APAP administration. NAC was administered in a single dose of 1200 mg/kg and/or CsA was administered in a single dose of 50 mg/kg. All drug administrations were performed intraperitoneally. However, no administration was made to the mice in the control group during the experimental period. Twelve hours after the administration of APAP, all animals were decapitated and blood samples were collected and stored -20°C for the evaluation of biochemical parameters. In addition, kidney tissue samples were rapidly removed and to evaluate the histological changes, one of the kidney tissues for each mouse was taken into 10% formalin and fixed. In addition, other kidney tissue samples were stored at -80°C after being frozen in liquid nitrogen to analyze the alterations in the gene expression levels.

Histological evaluations

To perform routine microscopic assessment, kidney samples fixed in 10% formalin solution were embedded in paraffin blocks. Subsequently, 5 μm of sections were taken from paraffin-embedded tissue samples and stained with hematoxylin and eosin (H&E). Histopathological alterations of kidney sections for each group were evaluated using a light microscope. On the other hand, the acute tubular injury score in kidney samples, stained with H&E, was conducted according to the scoring system used by Chen et al., (2013). In brief, 20 fields per kidney samples (×200 magnification) were evaluated in terms of tubular damage and scored from 0 to 5 (Score 0: no damage; Score 1: <10% damage; Score 2: 10-25% damage; Score 3: 25-50% damage; Score 4: 50-74% damage; Score 5: >75% damage).

Evaluation of renal function

The collected mouse blood samples were centrifuged at 4000 rpm for 10 min and their serumwas separated. Serum creatinine (Cr) and blood urea nitrogen (BUN) levels were measured as markers of renal function using an autoanalyzer (Gesan chem 200 Gesan Production srl, Campobello, Italy).

Immunohistochemistry (IHC)

The avidin-biotin-peroxidase complex method was used to evaluate the immunoreactivity levels of 3-nitrotyrosine (3-NT) (bs-8551R, Bioss, USA), cytosolic cytochrome c (Cytc) (bs-0013R, Bioss, USA) and 8-hydroxy-deoxyguanosine (8-OHdG) (ab48508, abcam) primary antibodies in kidney tissue samples. All sections were evaluated using a Leica DM500 microscope (DFC295; Leica, Wetzlar, Germany). Staining was evaluated by both its prevalence (0.1:<25%, 0.4: 26-50%, 0.6: 51-75%, 0.9: 76-100%) and severity (0: no, +0.5: very little, +1: little, +2: medium, +3: severe) (Histoscore=prevalence×severity).

Enzyme-linked immunosorbent assay (ELISA)

Enzyme-linked immunosorbent assay (ELISA) was performed to determine 3-NT and 8-OHdG levels in kidney tissue samples. Commercially available mice-specific 3-NT (E0019Ra, BT LAB, China) and 8-OHdG (YLA1580RA, YL Biont, China) ELISA kits were purchased. Briefly, kidney tissue samples were homogenized in phosphate-buffered saline (PBS) solution with a Bullet Blender Homogenizer (Next Advance, USA), centrifuged at 3000 rpm for 20 min and the obtained homogenates were evaluated in terms of protein concentration using a micro-spectrophotometer device (Allsheng, China). ELISA analyses were performed according to recommended protocols and a spectrophotometer (Multiskan FC, Thermo Scientific) was used to measure the optical density (OD) of kidney homogenate samples.

Quantitative real-time polymerase chain reaction (qRT-PCR)

Total RNA was extracted from mouse kidney samples using TRIzol Reagent (RiboEx, GeneAll, Korea) according to the recommended protocol. The quantity and purity of the obtained RNA samples were evaluated using a microspectrophotometer (Nano-400A, Allsheng, China). RNA samples were converted into complementary DNAs (cDNAs) using a reverse transcription kit (VitaScript, Procomcure, Austria). Quantitative real-time polymerase chain reaction (qRT-PCR) analysis was performed using an ABI 7500 Real-Time PCR device (Applied Biosystems, Singapore) and BrightGreen 2X qPCR MasterMix (abm, Canada). To analyze the Cytc mRNA expression level, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a reference gene (Table 1). At the end of the analysis, the 2-ΔΔCt method was used to calculate the variations in mRNA expression levels.

Table 1: Primer list of genes used for qPCR in the study.


 
Statistical analysis
 
Statistical analysis was performed using the IBM SPSS 22.0 package program. The Shapiro-Wilk test was used for normalizing all data. To compare median values among the groups, Kruskal-Wallis and Mann-Whitney U tests were used. On the other hand, for statistical analysis of qPCR data, the online Qiagen Gene Globe PCR data analysis method was used. All data are expressed as mean ± standard error (SE). p<0.05 was considered statistically significant. 
APAP-induced histopathological alterations and effects of NAC and/or CsA on kidney tissue samples of mice
 
We first histopathologically examined the effect of APAP overdose on the kidneys of mice (Fig 1A). Although normal kidney morphology was observed in the control group [Fig 1A (a)], mice subjected to 400 mg/kg APAP showed noticeable congestion and hemorrhage, intratubular cast formation, glomerular atrophy and excessive renal tubular injury [Fig 1A (b,c)]. NAC or CsA treatments reduced APAP-induced kidney injury indications, but marked congestion and hemorrhage and moderate renal tubular injury were observed [Fig 1A(d,e; respectively)]. On the other hand, combined NAC+CsA treatment improved almost all of the histological alterations caused by APAP in mouse kidney tissue samples [Fig 1A(f)]. In addition, when histological data were evaluated particularly in terms of renal tubular injury, statistically significant increases were seen in the APAP, APAP+NAC and APAP+CsA groups compared with the control group (p<0.001). Also, APAP+NAC+CsA group observed a significant decrease compared with the APAP group (p<0.001). Compared with the APAP+NAC and APAP+CsA groups, a significant decrease was detected in the APAP+NAC+CsA group (p<0.001) (Fig 1B).

Fig 1: Histopathological examination findings of the kidney tissues.


 
Effects of APAP, NAC and/or CsA treatment on serum creatinine (Cr) and blood urea nitrogen (BUN) levels
 
Cr and BUN levels, which are considered markers of renal function, were analyzed in serum samples of mice. Serum Cr and BUN levels were markedly increased in the APAP, APAP+NAC and APAP+CsA groups compared with the control group (p<0.001). On the other hand, serum Cr and BUN levels were remarkably decreased in the APAP+NAC, APAP+CsA and APAP+NAC+CsA groups compared with the APAP group (p<0.001). Moreover, significantly decreased serum Cr and BUN levels were detected in the APAP+NAC+CsA group compared with the APAP+NAC and APAP+CsA groups (p<0.001), however, no marked changes were observed against the control group (p>0.05) (Fig 2).

Fig 2: Biochemical analysis findings of the serum samples.


 
Effects of NAC and/or CsA treatment on 8-OHdG levels in APAP-induced acute nephrotoxicity
 
8-OHdG, a marker of oxidative DNA damage, was evaluated in terms of both immunoreactivity and protein concentration in kidney tissue samples of mice. In the immunoreactivity levels, a significant increase was observed in the APAP, APAP+NAC and APAP+CsA groups compared with the control group (p<0.001). Also, a remarkable decrease was detected in the APAP+NAC and APAP+NAC+CsA groups compared with the APAP group (p<0.001). Besides, a marked decrease was noticed in the APAP+NAC+CsA group compared with both APAP+NAC and APAP+CsA groups (p<0.001) (Fig 3A-B). On the other hand, in the protein concentration, a remarkable increase was observed in the APAP and APAP+CsA groups compared with the control group (p<0.001). Moreover, a significant decrease was detected in the APAP+NAC+CsA group compared with the APAP and APAP+CsA groups (p<0.001) (Fig 3C).

Fig 3: Alterations in 8-OHdG expression levels in kidney tissue samples.


 
Effects of NAC and/or CsA treatment on 3-nitrotyrosine levels in APAP-induced acute nephrotoxicity
 
3-NT, a molecule that is among the potential markers of oxidative stress, was evaluated in terms of both immunoreactivity and protein concentration in mouse kidney tissue samples. In the immunoreactivity levels, a remarkable increase was detected in the APAP, APAP+NAC and APAP+CsA groups compared with the control group (p<0.001). In addition, a marked decrease was detected in the APAP+NAC+CsA group compared with the APAP, APAP+NAC and APAP+CsA groups (p<0.001) (Fig 4A-B). On the other hand, in the protein concentration levels, a significant increase was observed in the APAP+NAC and APAP+CsA groups compared with the control group (p<0.001). Also, a significant decrease was detected in the APAP+CsA and APAP+NAC+CsA groups compared with the APAP group (p<0.001). Moreover, a remarkable decrease was detected in the APAP+NAC+CsA group compared with the APAP+CsA group (p<0.001) (Fig 4C).

Fig 4: Alterations in 3-NT expression levels in kidney tissue samples.


 
Effects of NAC and/or CsA treatment on cytochrome c levels in APAP-induced acute nephrotoxicity
 
In our study, Cytc, a potential biomarker for AKI, was assessed in terms of both immunoreactivity and mRNA levels in the kidney tissue samples of mice. When evaluated in terms of cytosolic immunoreactivity levels, a statistically significant increase was detected in the APAP, APAP+NAC and APAP+CsA groups compared with the control group (p<0.001). In addition, a remarkable decrease was detected in the APAP+NAC+CsA group compared with the APAP, APAP+NAC and APAP+CsA groups (p<0.001) (Fig 5A-B). Moreover, when evaluated in terms of mRNA levels, a statistically significant increase was detected only in the APAP group compared with the control group (p<0.05). In addition, a remarkable decrease was detected only in the APAP+NAC+CsA group compared with the APAP group (p<0.05) (Fig 5C).

Fig 5: Alterations in Cytc expression levels in kidney tissue samples.



There are previous experimental studies showing the safety effects of some plant extracts on kidney histology and blood parameters (Ammari et al., 2023; Ansari et al., 2020). On the other hand its known that kidney damage induced by drugs or chemicals has recently become a major clinical problem (Zhao  et al., 2011).  Several studies have indicated that an overdose of APAP induces nephrotoxicity, associated with AKI and death, both in humans and experimental animals (Karaali et al., 2019). Treatment of APAP-induced nephrotoxicity has life-saving significance because of the life-threatening outcomes of APAP overdose on kidneys (Demirbağ et al., 2010). However, a lack of effective treatment options for such diseases has drawn attention (Zhao et al., 2011). Thus, it is crucial to develop novel therapeutic approaches for APAP-induced nephrotoxicity (Ucar et al., 2013). To the best of our knowledge, for the first time, the potential therapeutic effect of NAC+CsA drug combination on APAP-induced nephrotoxicity in mice was evaluated in the current study. Our results demonstrated that combined NAC+CsA treatment provides remarkable ameliorative effects on APAP-induced nephrotoxicity. These were determined by histopathological, immunohistochemical, biochemical and molecular analysis. This approach may provide reinforcement to conventional NAC therapy, which indicates some restrictions for APAP poisoning.

To induce APAP-mediated toxicity, a single dose of APAP (400 mg/kg to 2000 mg/kg) was used in the majority of the experimental research (Chinnappan et al., 2019). The APAP-induced damage model used in our study was constituted according to the model in mice previously developed by Salama et al. (2015). It was shown histopathologically that overdose of APAP led to pathological changes in mouse kidney samples such as focal tubular cell detachment, vacuolar degeneration, necrosis and apoptosis (Zhao et al., 2011; Salama et al., 2015; Karaali et al., 2019). Similarly, it was determined histopathologically that APAP overdose caused critical negative results in mouse kidney tissues and in our study. Moreover, the increase in serum Cr and BUN levels, which are renal function markers, after APAP overdose also supports this finding.

In both the liver and kidney, APAP is oxidized by cytochrome P450 enzymes and the toxic electrophile N-acetyl-p-benzoquinone imine (NAPQI) is generated. At therapeutic doses of APAP, the produced NAPQI level is quite low and is conjugated with reduced GSH to produce nontoxic metabolites. However, toxic doses of APAP lead to the production of high NAPQI and only a part of it can be detoxified by GSH. Consequently, cellular GSH depletion occurs, which produces an imbalance between prooxidants and antioxidants (Zhao et al., 2011). In addition, the remaining free NAPQI binds to cellular proteins and other macromolecules. As a result, the production of superoxides (ROS and/or RNS)induces oxidative stress (Das et al., 2010; Ansari et al., 2020; Haidara et al., 2020; Alshahrani et al., 2021) and nitrosative stress (Xie et al., 2016; Yan-Zi et al., 2018). In addition, it can lead to DNA damage (McGill et al., 2012), mitochondrial MPT induction (Brown et al., 2012; McGill et al., 2012) and mitochondrial dysfunction (Xie et al., 2016; Haidara et al., 2020).

The precise molecular mechanism of kidney injury induced by APAP overdose remains obscure. However,  the increase in ROS produced by NAPQI and consequent oxidative stress plays a crucial role in APAP-caused nephrotoxicity. Research in experimental animals demonstrated that APAP overdose increases lipid peroxidation and inhibits antioxidant defense in kidney tissue (Ansari et al., 2020). Moreover, previous studies have shown that APAP overdose leads to DNA damage and impaired double-strand DNA break repair (Napirei et al., 2006; Borude et al., 2018). It is well known that this DNA damage occurs through oxidative stress-induced hydroxyl radicals that cause base modifications and breaks in DNA. 8-OHdG is located in both nuclear and mitochondrial DNA and is considered one of the most important markers of DNA damage (Benzer et al., 2018; Turk et al., 2019; Gelen et al., 2021). In addition, several studies have shown that ROS-mediated DNA damage can be prevented by inhibiting 8-OHdG (Cao et al., 2006; Kowluru and Kanwar, 2007; Turk et al., 2019; Gelen et al., 2021). Although DNA damage analysis was not performed in our study, in parallel with the literature, the remarkable increase in the 8-OHdG level due to APAP overdose suggested that DNA damage occurred in the kidney tissue.

The production of reactive nitrogen species (RNS) due to APAP-induced toxicity causes nitrotyrosine adduction to proteins after GSH depletion. Protein nitration is believed to occur by peroxynitrite (ONOO-), a highly reactive molecule produced by superoxide and nitric oxide (James et al., 2003; Kowluru et al., 2007; Brown et al., 2012; Xie et al., 2016; Yan-Zi et al., 2018). 3-NT, a diagnostic marker of peroxynitrite, is a stable end product frequently used to detect nitrosative stress (Ishii et al., 2006). Previous studies have reported the presence of 3-NT in APAP toxicity, particularly in the pathogenesis of APAP-induced hepatotoxicity (Banerjee et al., 2017; Yan-Zi et al., 2018). Similarly, the current research demonstrated that APAP overdose leads to increased 3-NT immunoreactivity and protein concentration in kidney tissue.

Mitochondrial dysfunction is a characteristic of APAP-induced toxicity and is related to increased oxidative stress in APAP overdose. The role of reactive metabolite NAPQI generation in mitochondrial dysfunction related to APAP overdose toxicity is well known. In addition, peroxynitrite formation due to APAP toxicity can lead to mitochondrial dysfunction (Brown et al., 2012). Moreover, it has been confirmed that numerous proteins related to antioxidant defense, energy supply, or fatty acid metabolism are nitrated by peroxynitrite in APAP hepatotoxicity. Mitochondrial dysfunction can also be caused by this condition (Xie et al., 2016). In addition, mitochondrial damage has been demonstrated in APAP overdose-induced hepatotoxicity in our previous study (Kaya Tektemur et al., 2021). Besides, Cytc release is one of the critical endpoints of cell death signaling and mitochondrial damage (Sepand et al., 2016). Similarly, the current research demonstrated that APAP overdose leads to increased Cyc immunoreactivity in the cytosol and mRNA levels in kidney tissue.

NAC, a well-known free radical scavenger, has universally confirmed beneficial effects on the prevention of APAP-induced hepatotoxicity (Mokhtari et al., 2017). This preventive effect of NAC in the liver occurs by replenishing depleted hepatic GSH (Mokhtari et al., 2017; Akakpo et al., 2020). Moreover, treatment with NAC decreases nitration of proteins in APAP-induced hepatotoxicity (Banerjee et al., 2017). Additionally, in our previous study, we showed that NAC treatment in APAP-mediated hepatotoxicity led to a certain level of improvement by reducing increased cytochrome c, AST and ALT levels and increasing decreased GSH levels, alleviating extremely massive and severe histopathological changes (Kaya Tektemur et al., 2021). On the other hand, it has been demonstrated that NAC cannot provide sufficient protection against APAP-induced nephrotoxicity (Akakpo et al., 2020). In addition, the current study clearly demonstrated that NAC therapy alone is not effective enough against APAP-induced nephrotoxicity, especially in terms of acute tubular damage. The reason for this may be that because the kidney is not a real producer of GSH like the liver, getting enough GSH from the circulation is critical for kidney tissue (Ucar et al., 2013). This reveals that searching for another protective molecule that would offer effective prevention against APAP-induced nephrotoxicity is required (Das et al., 2010). In kidneys, which require high ATP in terms of function, the density of mitochondria is high, especially in the renal tubules. Moreover, it is well known that mitochondria play important roles in many cellular processes such as signal transduction, cell proliferation, cell growth and cell death. Therefore, mitochondrial dysfunction or damage could lead to impaired ATP generation and ROS balance. On the other hand, most therapeutic drugs may cause AKI because of their negative effects on the mitochondrion of the renal tubule (Hua et al., 2018). It has been revealed that APAP-induced toxicity triggers the opening of mitochondrial MPTP. Thus, mitochondria are a potential target for treating AKI. Given the central role of mitochondria in the pathophysiology of AKI, in our study, it is not surprising that the therapeutic effects of CsA, an immunosuppressive drug known to inhibit the opening of MPTP (Taylor et al., 2005), together with NAC on APAP-induced renal damagewere evaluated. The opening of MPTP is characterized by the loss of membrane potential and cellular ATP depletion and has a crucial role in the mitochondrial dysfunction-induced cell death mechanism (Brown et al., 2012; Niimi et al., 2012). Additionally, the opening of MPTP is a prerequisite stage of Cytc release into the cytosol and consequently results in the swelling of mitochondria (Borutaite et al., 2003; Sepand et al., 2016). The observation of increased Cytc levels in mouse kidney tissue with APAP administration in our study may be related to this. On the other hand, Cytc levels and acute tubular injury were significantly reduced by combined NAC and CsA treatment compared to the APAP+NAC group indicating that mitochondrial MPTP-mediated mitochondrial damage may play a role in APAP-induced nephrotoxicity.
NAC or CsA treatments alone have not been sufficient to reduce APAP-mediated acute nephrotoxicity. However, the current study indicated that combined NAC and CsA treatment can effectively reduce APAP toxicity in mice kidney tissue. Based on the obtained data, we propose that CsA treatment contributes to NAC in providing protection against APAP-induced nephrotoxicity by inhibiting the opening of mitochondrial MPTPs in renal tubular cells.
This study was financially supported by the Firat University Scientific Research Projects Management Unit, Elazig, Turkey (Project No: TF.18.59).
All authors declared that there is no conflict of interest.

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