NRF2 Activation by AR-20007 Preserves Renal Tubular Epithelial Cells from Antimycin A-Induced Cell Death via Glutathione Metabolism Regulation

Abstract

Oxidative stress plays a crucial role in the development and progression of various kidney diseases. Nuclear factor erythroid 2-related factor 2 (NRF2) is the primary transcription factor that protects cells from oxidative stress by regulating cytoprotective genes including those involved in the antioxidant glutathione (GSH) pathway. GSH maintains cellular redox status and affects redox signaling, cell proliferation, and cell death. Antimycin A, an inhibitor of complex III of the electron transport chain, causes oxidative stress and reduces GSH levels. In this study, we induced mitochondrial damage in rat renal proximal tubular cells using antimycin A and investigated cellular viability and levels of NRF2 and GSH. Treatment with antimycin A altered the expression of antioxidant genes, including reduction in the transcription of glutathione-cysteine ligase subunits (Gclc and Gclm) and glutathione reductase (Gsr1), followed by a reduction in total GSH content with a concomitant decrease in NRF2 protein expression. AR-20007, previously described as an NRF2 activator, stabilizes and increases NRF2 protein expression in cells. By stimulating NRF2, AR-20007 increased the expression of antioxidant and detoxifying enzymes, thereby enhancing protection against oxidative stress induced by antimycin A. These data suggest that NRF2 activation effectively inhibits antimycin A-induced oxidative stress and that NRF2 may be a promising therapeutic target for preventing cell death during acute kidney injury.

INTRODUCTION

Nuclear factor erythroid 2-related factor 2 (NRF2) is a pivotal regulator of cellular defense against oxidative stress.¹⁾ It operates by binding to the antioxidant response element (ARE) within the genome, thereby orchestrating the activation of genes involved in maintaining redox balance, fostering glutathione synthesis, managing heme metabolism, aiding detoxification, and facilitating drug excretion.¹⁾ The activity of NRF2 is intricately regulated by Kelch-like ECH-associated protein (Keap1). Keap1 acts as an adaptor molecule that associates with NRF2, promoting its ubiquitination via the E3 ubiquitin ligase complex, leading to NRF2’s subsequent degradation within the proteasome.²⁾ This mechanism maintains low basal expression level of NRF2 under normal physiological conditions. During periods of heightened oxidative stress, certain cysteine residues within Keap1 detect these stress signals and trigger conformational alteration in Keap1.³⁾ This structural change hampers ubiquitination, thus preventing the degradation of NRF2. Consequently, NRF2 is stabilized and activated, thereby fortifying the cellular defense against excessive oxidative stress. Based on its physiological role, pharmacological activation of NRF2 is a promising therapeutic approach for several acute and chronic diseases that are associated with oxidative stress and inflammation.⁴⁾ In various chronic disease models, NRF2 activators offer therapeutic benefits by restoring the therapeutic effects.⁵⁾ Most developed compounds activate NRF2 by specifically targeting its degradation via Keap1-dependent mechanisms.

Excessive oxidative stress can lead to the destruction of cellular components including lipids, proteins, and DNA, ultimately leading to cell death and tissue dysfunction.⁶⁾ Oxidative stress plays an important role in kidney disease development. Kidneys are particularly susceptible to oxidative stress for several reasons.^7,8) Renal proximal tubules have a high metabolic rate and oxygen consumption, resulting in an increased production of reactive oxygen species (ROS) as metabolic byproducts.^9,10) These cells contain a large number of mitochondria that produce large amounts of oxygen radicals, which, in turn, increase the susceptibility of the kidneys to oxidative stress-induced damage. In addition, the kidneys receive a high volume of blood flow and proximity to high oxygen concentrations in the blood can lead to increased oxidative stress.¹¹⁾ Exposure to toxins and waste products increases oxidative stress. Collectively, these factors make the kidneys particularly vulnerable to oxidative stress, which can contribute to the development and progression of various kidney diseases.

The kidneys are highly dependent on an adequate supply of glutathione (GSH) under both physiological and pathological conditions.¹²⁾ The tripeptide GSH is a major low-molecular-weight thiol compound that participates in cellular redox reactions. In addition to antioxidant defense, GSH is important for detoxification, immune function, and cellular repair and maintenance.¹³⁾ Under oxidative stress, GSH is oxidized to glutathione disulfide and further to other products such as sulfonates. Maintenance of GSH metabolism is critical for cellular redox homeostasis and for determining susceptibility to ROS. Decreased GSH levels are often observed in kidneys under diseased conditions.¹⁴⁾ This decline may contribute to increased oxidative stress and damage to renal cells, thereby exacerbating disease progression. Some studies have explored the potential benefits of GSH supplementation in protecting the kidneys, particularly under conditions associated with oxidative stress.¹⁵⁾ Understanding the role of GSH in kidney physiology and pathology may lead to the development of new therapeutic approaches for the management of kidney diseases.

Previous studies have identified an endogenous NRF2 activator that integrates the glycolysis and Keap1–NRF2 antioxidant pathways.^16–18) Based on this activation mechanism, they identified a small-molecule inhibitor of the glycolytic enzyme PGK1, which revealed a direct link between glycolysis and NRF2 signaling. Based on previous results, we aimed to evaluate the activity of a PGK1 inhibitor in kidney cells. To evaluate whether the PGK1 inhibitor activates NRF2 in kidney cells, we used NRK52E renal tubule epithelial cells. Using an antimycin A-induced oxidative stress model, we evaluated the protective effects of a PGK1 inhibitor by modulating GSH metabolism.

MATERIALS AND METHODS

Cell Culture and Treatments

NRK52E rat renal tubular epithelial cells from ATCC (CRL-1571, Manassas, VA, U.S.A.) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) medium supplemented with 5% fetal bovine serum and incubated at 37 °C in 5% CO₂. In experiments, NRK52E cells were treated with the NRF2-specific activator AR-20007 (20 µM). To assess the effect of AR-20007 on antimycin-A induced mitochondrial damage and GSH depletion, cells were pre-treated with AR-20007 (20 µM) 1 h 30 min before antimycin A (20 µM) treatment. Protein and RNA samples were collected 24 h after antimycin A treatment to determine the effects of AR-20007.

Cell Viability Assay

Cell viability was measured to determine the cytotoxicity of AR-20007 on NRK52E cells. EZ-Cytox Cell Viability Assay Kit (EZ-500; Dogen Bio, Seoul, Republic of Korea) was used according to the manufacturer’s instructions. Briefly, NRK52E cells were incubated to 80% confluence in a serum-free medium and treated with AR-20007 at the indicated concentrations for 24 h. The cells were further cultured for 30 min by adding a cell viability solution (10 µM) to the incubator. After shaking for 1 min, absorbance was measured at 450 nm using a spectrophotometer. The percentage of viable cells was calculated using the following formula: ([chemically treated group/control group] × 100).

ROS Scavenging Assays

The direct ROS scavenging activity of the AR-20007 on cell free system were measured using an oxidant-sensitive fluorescent probe. AR-20007 dissolved in 10 µL of solvent were prepared in different concentration on a black 96-well plate. Then, 10 µL of SIN-1 (10 µM, dissolved in distilled water, 657028, Sigma-Aldrich, St. Louis, MO, U.S.A.) was added to induce ROS generation and mixed with 180 µL of sodium phosphate buffer (pH 7.4) containing 12.5 µM DCF-DA and 600 units of esterase. Fluorescence intensity was measured at emission and excitation wavelengths of 535 and 485 nm, respectively, using a fluorescence microplate reader (Berthold Technologies GmbH & Co., Wien, Austria).

Western Immunoblotting

Proteins from cultured NRK52E cells were extracted using radio immunoprecipitation assay (RIPA) buffer (#9806, Cell Signaling Technology, Danvers, MA, U.S.A.) containing a protease inhibitor cocktail (GenDEPOT, Katy, TX, U.S.A.). Protein quantification in cell lysates was performed using a bicinchoninic acid (BCA) assay kit (Thermo Fisher Scientific, Waltham, MA, U.S.A.), and 15 µg of protein were loaded onto 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) gels for separation. Immunoblotting was conducted overnight at 4 °C using an anti-Nrf2 antibody (rabbit, 1 : 1000, sc-722; Santa Cruz Biotechnology, Santa Cruz, CA, U.S.A.). Then, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody, anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (mouse, 1 : 500, sc-365062; Santa Cruz Biotechnology) at 25 °C for 1 h. The resulting immunoblots were visualized using Western Bright Peroxide solution (Advansta, San Jose, CA, U.S.A.) and a ChemiDoc imaging system (Bio-Rad, Hercules, CA, U.S.A.), according to the manufacturer’s instructions.

Quantitative Real-Time PCR Analysis

Total RNA was extracted from cultured cells using RiboEx (Geneall, 301-001, Seoul, Korea), following the manufacturer’s protocol. Total RNA was reverse-transcribed into cDNA using SuPrimeScript RT-PCR Premix (SR-4100; GenDEPOT). Quantitative real-time PCR was performed using SYBR Green Master Mix (Bioline, Taunton, MA, U.S.A.) on a CFX Connect System (Bio-Rad). The primer sequences are listed in Table 1. The 2-ΔΔCt method was used for the calculation of fold changes. 18s gene was served as the reference gene.

Table 1. Primer Sequences of Quantitative (q)RT-PCR

Gene	Forward (5′–3′)	Reverse (3′–5′)
18s	CGC GGT TCT ATT TTG TTG GT	AGT CGG CAT CGT TTA TGG TC
Gclm	AGA CCG GGA ACC TGC TCA AC	GAT TTG GGA GCT CCA TTC ATT CA
Gclc	CTG CAC ATC TAC CAC GCA GTC A	ATC GCC GCC ATT CAG TAA CAA
Gsr1	GTG GAA GTC AAC GGG AAA AA	ATC TCC ACG GCA ATG TAA CC
Gstα1	GGA CAA AGC AAG GAA CCG TT	CTT CTT CAC TGT GGG GAG GT
Slc7a11	CCT CTG TTC ATC CCA GCA TT	CGT CTG AAC CAC TTG GGT TT
Hmox1	CAC GCA TAT ACC CGC TAC CT	CCA GAG TGT TCA TTC GAC CA
Nqo1	TGG AAG CTG CAG ACC TGG TG	CCC TTG TCA TAC ATG GTG GCA TAC
Nqo1	TGC TGG TGC TGA GTA TGT CG	TTG AGA GCA ATG CCA GCC

Determination of GSH Levels in the Cells

Cells were homogenized as described above, and the supernatant was collected after centrifugation. Briefly, a 25% meta-phosphoric acid-added homogenate was centrifuged at 12000 rpm for 10 min and the supernatant was collected for the assay. Fifty nanomoles phosphate buffer containing 1 mM ethylenediaminetetraacetic acid (EDTA) (pH 7.4) was added to supernatant, followed by the addition of 10 µL phthaldialdehyde (P-0657, Sigma-Aldrich Quimica SA, Madrid, Spain). After 25 min of shaking at room temperature, fluorescence was measured at excitation and emission wavelengths of 350 and 420 nm, respectively.

Quantification and Statistical Analysis

The difference between the two groups was analyzed using Student’s t-test, while ANOVA was utilized to analyze differences among multiple groups. Data analysis was conducted using GraphPad Prism version 5 (GraphPad Software Inc., San Diego, CA, U.S.A.). All data are presented as the mean values (± standard error of the mean (S.E.M.)). Statistical significance was set at p < 0.05. significant.

RESULTS

PGK1 Inhibitor, AR-20007 Activates NRF2 in NRK52E Renal Tubule Epithelial Cells

Previous studies have demonstrated that compounds containing the 3-aminotetrahydrothiophene 1,1-dioxides moiety act as inhibitors of PGK1 while possessing NRF2-activating properties. Based on these results, we evaluated AR-20007 as an NRF2 activator in renal tubule epithelial cells (Fig. 1A). Prior to assessing if AR-20007 induces NRF2 activation in renal cells, we initially examined its potential for directly scavenging ROS species. However, AR-20007 showed no direct scavenging effect on ROS in a cell-free system (Fig. 1B). Treatment with 20 µM of AR-20007 significantly increased NRF2 expression in the cells (Figs. 1C, 1D). We further examined whether the increase in NRF2 protein expression was due to an increase in its transcription level. However, AR-20007 did not increase NRF2 gene expression, implying that the compound stabilized NRF2 protein expression (Fig. 1E). We also confirmed that AR-20007 was not significantly toxic to NRK52E cells (Fig. 1F). These results indicated that AR-20007 is a potent NRF2 activator in renal tubule epithelial cells.

Fig. 1. AR-20007 Activates NRF2 in NRK52E Renal Tubule Epithelial Cells

(A) Chemical structure of AR-20007. (B) The ROS scavenging effect of AR-20007 was evaluated in a cell-free system using the DCFDA assay. AR-20007 did not alter SIN1-induced ROS production in the test tube. *** p < 0.001 versus blank group. (C) NRF2 protein level was detected by Western blot analysis after AR-20007 treatment in NRK52E cells. GAPDH was used as internal control. (D) Relative NRF2 protein expressions were quantified using densitometry. * p < 0.05 versus control group. (E) Relative mRNA expression of Nfe2l2. (F) The cytotoxicity of AR-20007 was evaluated using the MTT assay to assess NRK52E cell viability at different treatment durations (2, 4, 8, and 24 h). * p < 0.05 vs. control group. *** p < 0.001 versus blank group.

AR-20007 Increases Antioxidant and GSH Metabolism Genes, Elevating GSH Levels in NRK52E Cells

The role of NRF2 activation in kidney cells has been previously reported. We further evaluated whether NRF2 activation induced the expression of genes associated with antioxidant activity and GSH metabolism. AR-20007 significantly increased the expression of GSH metabolism-associated genes (Gclm, Gclc, Gcr1, Gsta1, and Slc7a11) in a time-dependent manner (Fig. 2A). Other NRF2 target genes, Hmox1 and Nqo, which play important roles as antioxidants, also increased after AR-20007 treatment (Fig. 2B). We further examined whether changes in GSH metabolism-associated genes were related to changes in GSH levels. AR-20007 treatment significantly increased the cellular GSH levels in NRK52E cells. Collectively, these results indicate that NRF2 activation by AR-20007 significantly induces the expression of antioxidant and GSH metabolism genes, thus elevating GSH levels in kidney cells.

Fig. 2. AR-20007 Induces NRF2 Activation, Leading to an Enhancement in Antioxidant Capacity and Elevated Levels of GSH in NRK52E Cells

(A) The mRNA expression levels of GSH metabolism-related genes, including Gclm, Gclc, Gsr1, Gsta1, and Slc7a11, were assessed following a 20 µM AR-20007 treatment for 2 and 4 h. * p < 0.05 versus control groups. (B) The relative mRNA expression of antioxidant genes, including Hmox1 and Nqo, was examined following a 20 µM AR-20007 treatment for 2 and 4 h. * p < 0.05 versus control groups. (C) Cellular GSH levels were measured after AR-20007 treatment. * p < 0.05 versus control groups.

Induction of Oxidative Stress in NRK52E Cells through Antimycin A Treatment

Next, we investigated whether mitochondrial damage induces oxidative stress by regulating NRF2 expression in renal cells. To induce mitochondrial damage, the renal cells were treated with antimycin A. Antimycin A treatment significantly increased cell death in a dose-dependent manner (Fig. 3A). We further investigated whether NRF2 expression was regulated by antimycin A treatment. Antimycin A treatment significantly reduced NRF2 protein expression in these cells (Figs. 3B, 3C). Reduced NRF2 protein expression was associated with changes in GSH levels in the cells (Fig. 3D). Additionally, antimycin A treatment altered gene expression associated with GSH metabolism. Antimycin A treatment decreased Gclm, Gclc, and Gsr1 gene expression and increased Gsta1 expression (Fig. 3E). Antimycin A treatment decreased Hmox1 gene expression and increased Nqo1 gene expression (Fig. 3E). These results suggest that mitochondrial damage induced by antimycin A treatment significantly alters NRF2 expression and oxidative stress in the cells, leading to cell death.

Fig. 3. Antimycin A Treatment Induces Oxidative Stress and GSH Depletion Leading to Cell Cytotoxicity in Renal Tubular Epithelial Cells

(A) The assessment of NRK52E cells viability with various concentration of antimycin A (10 and 20 μM). * p < 0.05 versus control groups. (B) NRF2 protein level was detected by Western blot analysis after antimycin A treatment in NRK52E cells. (C) Relative NRF2 protein expressions were quantified using densitometry. * p < 0.05 versus control group. (D) GSH levels were measured after antimycin A treatment. * p < 0.05 versus control groups. (E) Relative mRNA expression of GSH metabolism-related and antioxidant genes, such as Gclm, Gclc, Gsr1, Gsta1, Slc7a11, Hmox1, and Nqo1 after antimycin A treatment. * p < 0.05 versus control groups.

AR-20007 Treatment Relieves Oxidative Stress and Prevents Cell Death Induced by Antimycin A Treatment in Renal Cells

Next, we evaluated whether NRF2 activation by AR-20007 can relieve oxidative stress and prevent antimycin A-induced cell death in renal cells. AR-20007 pretreatment significantly increased Nrf2 protein expression, which was decreased by Antimycin A treatment (Fig. 4A). Pretreatment with AR-20007 significantly inhibited antimycin A-induced renal cell death (Fig. 4B). We further examined whether AR-20007 could restore GSH levels in these cells. AR-20007 pre-treatment significantly blocked the antimycin A-induced decrease in GSH levels (Fig. 4C). Finally, we checked whether AR-20007 increased GSH metabolism and the expression of antioxidant-related genes under antimycin A-treated conditions. AR-20007 pretreatment significantly increased the expression of GSH metabolism genes (Gclc, Fsr1, Gsta1, and Slc7a11) and antioxidant genes (Hmox1 and Nqo1) in the cells (Fig. 4D). Collectively, these results suggest that NRF2 activation by AR-20007 protects renal tubular epithelial cells from antimycin A-induced cell death by regulating GSH metabolism.

Fig. 4. NRF2 Activation by AR-20007 Attenuates Antimycin A-Induced Oxidative Stress in NRK52E Cells

(A) NRF2 protein level was detected by Western blot analysis after AR-20007 and/or Antimycin A treatment in NRK52E cells. (B) The assessment of NRK52E cells viability was conducted by pre-treating the cells with AR-20007, followed by treatment with antimycin A for 24 h. * p < 0.05 versus control groups. ^# p < 0.05 versus antimycin A groups. (C) GSH activity was measured after AR-20007 and antimycin A treatment in NRK52E cells. * p < 0.05 versus control groups. ^# p < 0.05 versus antimycin A groups. (D) Relative mRNA expression of antioxidant genes, such as Gclm, Gclc, Gsr1, Gsta1, and Slc7a11, and Hmox1 and Nqo1. * p < 0.05 versus control groups. ^# p < 0.05 versus antimycin A groups.

AR-20007 Treatment Alleviates Cisplatin-Induced Oxidative Stress without Inhibiting Cell Death

Lastly, we further implemented experiments to check whether NRF2 activation blocks cisplatin-induced cell death in renal cells. Cisplatin treatment significantly decreased NRF2 protein expression levels and induced renal cell death (Figs. 5A, 5B). NRF2 activation by AR-20007 treatment increased the expression of GSH metabolism genes (Gclm, Gclc, Gsr1, Gsta1, and Slc7a11) and antioxidant genes (Hmox1 and Nqo1) in the cells and restored cisplatin-induced decrease in GSH levels (Figs. 5C, 5D). However, NRF2 activation did not inhibit cisplatin-induced cell death (Fig. 5E). These findings indicate that while AR-20007-mediated NRF2 activation alleviates cisplatin-induced oxidative stress, it is insufficient to prevent cisplatin-induced cell death.

Fig. 5. NRF2 Activation by AR-20007 Attenuates Cisplatin-Induced Oxidative Stress in NRK52E Cells

(A) NRF2 protein level was detected by Western blot analysis after cisplatin treatment in NRK52E cells. (B) The assessment of NRK52E cells viability was conducted by pre-treating the cells with cisplatin for 24 h. *** p < 0.001 versus control groups. (C) Relative mRNA expression of antioxidant genes, such as Gclm, Gclc, Gsr1, Gsta1, and Slc7a11, and Hmox1 and Nqo1. * p < 0.05 versus control groups. ^# p < 0.05 versus cisplatin groups. (D) GSH activity was measured after AR-20007 and/or cisplatin treatment in NRK52E cells. * p < 0.05 versus control groups. ^# p < 0.05 versus cisplatin groups. (E) The assessment of NRK52E cells viability was conducted by pre-treating the cells with AR-20007, followed by treatment with cisplatin for 24 h. ^# p < 0.05 versus control groups.

DISCUSSION

Maintaining an effective antioxidant system is essential for the preservation of cellular physiology. Owing to the susceptibility of the kidney to oxidative stress, it is crucial to maintain an effective antioxidant system. Elevated oxidative stress is associated with diverse pathophysiological conditions in kidneys. Oxidative stress plays a significant role in renal cell death.^19,20) Elevated oxidative stress is associated with the dysfunction of kidney cell mitochondria, inflammation, disruptions in signaling pathways, and DNA damage, collectively contributing to alterations in the cell fate within the kidney.²¹⁾ In the present study, we assessed whether NRF2 activation mitigates oxidative stress, thereby preventing cell death in renal tubular epithelial cells. AR-20007, a new class of NRF2 activators, markedly influences GSH metabolism in renal tubule cells and directly elevates cellular GSH levels. We also investigated the potential of AR-20007 in preventing oxidative stress and cell death induced by antimycin A. Treatment with antimycin A significantly lowered NRF2 expression and GSH levels, ultimately resulting in cell death. Ultimately, our findings demonstrate that AR-20007 effectively prevents antimycin A-induced cell death by alleviating oxidative stress. Collectively, our findings demonstrate that NRF2 activation protects renal tubular epithelial cells from antimycin A-induced cell death by regulating GSH metabolism (Fig. 6).

Fig. 6. The Mechanism of AR-20007’s Action on Antimycin A-Induced Cell Death and Oxidative Stress in Renal Tubular Epithelial Cells

NRF2 plays a crucial role in the cellular defense against oxidative stress.²²⁾ When cells experience oxidative stress due to an imbalance in ROS, NRF2 is activated. Under conditions of excessive oxidative stress, cistein residues in Keap1 undergo oxidation or alkylation. This modification facilitates the accumulation of NRF2 in the nucleus. Activated NRF2 promotes gene upregulation by binding to ARE sites in genomic loci.²³⁾ Based on this activation mechanism, numerous small molecules with the capacity to induce NRF2 activation have been identified.²⁴⁾ Most previously identified molecules are either electrophiles or undergo cellular transformations to generate electrophiles, leading to direct covalent modifications of Keap1.²⁵⁾ Although these molecules effectively activate NRF2, small electrophilic molecules are generally not considered promising drug candidates.¹⁶⁾ They react broadly with cellular nucleophiles, resulting in cytotoxicity. In our study, we employed a PGK1 inhibitor known for its ability to elevate methylglyoxal levels within cells, consequently triggering the activation of NRF2. In kidney cells, AR-20007 significantly increased NRF2 protein expression without causing cytotoxicity. In addition, it increased the expression of NRF2 target genes, including antioxidant genes and those associated with GSH metabolism. These findings indicate that the inhibition of PGK1 serves as a promising therapeutic approach for NRF2 activation in kidney cells.

NRF2 enhances the expression of a wide range of antioxidant and detoxification genes, many of which are directly involved in GSH metabolism.²⁶⁾ These genes are critical for GSH synthesis, utilization, and protection against oxidative stress. Gclc and Gclm form the rate-limiting enzyme in GSH synthesis, where Gclc catalyzes the initial step and Gclm modulates its activity. Slc711a encodes a component of the cystine/glutamate antiporter, which imports cystine into the cell, subsequently reduced to cysteine, a precursor for GSH synthesis. Gsta1 catalyze the conjugation of GSH to various harmful compounds, facilitating their detoxification and excretion. Additionally, Gsr is responsible for reducing GSSG back to its reduced form GSH, maintaining the cellular GSH pool. In our study, the expression of these genes was differentially regulated under stressed conditions. However, AR-20007 treatment increased their expression under both normal and Antimycin A-induced stressed conditions. Alongside the direct antioxidant genes Hmox1 and Nqo1, the regulation of GSH metabolism appears to be an important defense mechanism facilitated by NRF2 activation under oxidative stress conditions.

In kidney tubule epithelial cells, mitochondria are particularly important for meeting the high energy demands associated with the active transport processes involved in reabsorption and secretion.^27,28) These cells rely on ATP generated by the mitochondria to fuel the pumps and transporters responsible for moving substances across the renal tubular epithelium. Additionally, mitochondria play a role in maintaining cellular redox balance and participate in signaling pathways related to cell survival, regulation of apoptosis (programmed cell death), and maintenance of cellular homeostasis. Mitochondrial dysfunction in kidney tubule epithelial cells has been implicated in various kidney disorders, including acute kidney injury and chronic kidney disease.^29,30) In our study, exposure to antimycin A, a potent inhibitor of the mitochondrial electron transport chain, reduced NRF2 expression and elevated oxidative stress. These alterations ultimately contribute to cell death in kidney cells. Using an antimycin A-induced oxidative stress model, we evaluated the protective effects of AR-20007 on kidney cells. Administration of AR-20007 notably counteracted the alterations induced by antimycin A in GSH metabolism, demonstrating a cytoprotective effect in kidney cells.

Although activation of NRF2 by AR-20007 has been demonstrated to exert cytoprotective effects through the regulation of GSH metabolism and antioxidant systems, several questions remain unanswered and warrant further investigation. Firstly, AR-20007 did not exhibit a significant protective effect in a cisplatin-induced renal cell toxicity model. Cisplatin is known to induce significant cytotoxicity in renal cells by triggering oxidative stress. Our data indicate that AR-20007 significantly upregulated genes associated with GSH metabolism and restored GSH levels depleted by cisplatin treatment. While both antimycin A and cisplatin treatment reduced NRF2 expression and induced substantial oxidative stress in the cells, AR-20007 only protected against antimycin A-induced renal cell death. Further research is needed to elucidate the precise mechanism and potential benefits of NRF2 activation under various cytotoxic conditions. Secondly, additional in vivo investigations are required to validate the role of NRF2 activation in kidney injury. Previous studies have demonstrated the protective effects of NRF2 activators in kidney injury by mitigating oxidative stress. Given that AR-20007 activates NRF2 through a distinct mechanism, assessing its efficacy in vivo is imperative. In summary, our findings suggest that NRF2 activation effectively mitigates antimycin A-induced oxidative stress, underscoring NRF2 as a promising therapeutic target for preventing cell death in acute kidney injury.

Funding

This work was supported by a 2-year Research Grant of Pusan National University.

Author Contributions

JY: Performed experiments, Writing; JJ: Performed experiments, Materials provided; SH: Performed experiments; MK: Performed experiments; JK: Materials provided, Performed experiments; HL: Performed experiments; DK: Performed experiments; BMK: Performed experiments; JK: Performed experiments; MK: Materials provided, Conceptualization, HY: Materials provided, Conceptualization; KWC: Writing, Conceptualization, Funding acquisition.

Conflict of Interest

The authors declare no conflict of interest.

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