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Renoprotective Effects of Carbon Monoxide–Releasing Molecule 3 in Ischemia-Reperfusion Injury and Cisplatin-Induced Toxicity
Renoprotective Effects of Carbon MonoxideeReleasing Molecule 3 in Ischemia-Reperfusion Injury and Cisplatin-Induced Toxicity Y.E. Yoona, K.S. Leeb, Y.J. Leea, H.H. Leea, and W.K. Hana,* a Department of Urology, Urological Science Institute, Yonsei University College of Medicine, Seoul, Korea; and bDepartment of Urology, Gangnam Severance Hospital, Yonsei University College of Medicine, Seoul, Korea
ABSTRACT Background. We investigated the effects of a soluble carbon monoxideereleasing molecule (CORM) in cisplatin-induced cytotoxicity and ischemia-reperfusion injury (IRI) in vitro. Methods. The effects of CORM-3 (12.5e200 mM) were assessed in normal kidney epithelial cells (HK-2, LLC-PK1) and renal cancer cells (Caki-1, Caki-2) subjected to cisplatin (50e200 mM) or IRI. To induce IRI, cells were placed in an anaerobic chamber (37 C, 95% nitrogen, 5% carbon dioxide) for 48 hours. Cells were transferred to complete medium and incubated at 37 C, 5% carbon dioxide for 6 hours. Cell viability (CCK assays), tumor necrosis factor (TNF)-a messenger RNA (mRNA) levels (quantitative reverse-transcriptase polymerase chain reaction), and protein expression of cleaved-caspase 3 and oxidative stress markers (including Erk1/2, JNK, and P38; Western blot) were assessed. Results. Viability after IRI was approximately 40% of control. Protective effects of CORM-3 in the IRI model were dose-dependent. Cell viability was 40% recovered in 200-mM CORM-3-pretreated cells compared with control. The protective effects of CORM-3 in cells exposed to cisplatin for 24 hours were weaker than in the IRI model. TNF-a mRNA was induced by stimulated IRI or cisplatin exposure; CORM-3 pretreatment attenuated the rise in TNF-a mRNA. IRI or cisplatin-induced activated oxidative stress markers decreased in CORM-3-pretreated cells. CORM-3 reduced expression of the apoptotic marker cleaved-caspase 3. Conclusion. Our data demonstrate the protective effects of CORM-3 in cisplatin cytotoxicity and IRI in both normal kidney cells and renal cancer cells in vitro. CORM-3 exerts these effects by ameliorating inflammatory and oxidative stress pathways.
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ARBON monoxide (CO) is a molecule that has protective effects in tissues subjected to ischemiareperfusion injury (IRI) and inflammation [1,2]. However, the use of CO in humans is very challenging because of its toxicity characterized by inhibition of oxygen delivery at high concentrations [1]. Thus, clinical application of CO is very limited at present. CO-releasing molecules (CORMs) that can deliver CO to tissues were initially developed and evaluated over a decade ago [3]. Among the various CORMs, CORM-3 [tricarbonylchloro(glycinato)ruthenium (II)] is water soluble and thus easy to handle and apply to cells and organs [3]. CORMs exhibit organ protective effects ª 2017 Elsevier Inc. All rights reserved. 230 Park Avenue, New York, NY 10169
Transplantation Proceedings, 49, 1175e1182 (2017)
in transplantation, inflammation, liver failure, and exposure to cytotoxic agents [4e7]. This study was financially supported by the “Dongwha” Faculty Research Assistance Program of Yonsei University College of Medicine (6-2015-0170) and the National Research Foundation grant (NRF- 2012R1A1A1042968) funded by the Korean government (MEST). The funding sources had no involvement in this study. *Address correspondence to Woong Kyu Han, MD, PhD, Department of Urology, Urological Science Institute, Yonsei University College of Medicine, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-752, Korea. E-mail: [email protected] 0041-1345/17 http://dx.doi.org/10.1016/j.transproceed.2017.03.067
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Previous research regarding the effects of CORM-3 on kidneys or kidney cells has primarily examined its role in kidney transplantation and IRI [7e11]. These studies unanimously concluded that CORM-3 has a renoprotective effect in kidney transplantation because of its protective effects against IRI. However, most of these studies did not use human kidney cell lines. Furthermore, renal cell carcinoma (RCC) cells were used in none of these experiments. Because RCC cells may require more oxygen to survive than normal cells, the effect of CORM-3 on RCC cells should be evaluated [12]. The effect of CORM-3 during cytotoxic exposure also requires evaluation [4]. Therefore, we conducted this study to evaluate the effects of CORM-3 in normal kidney cells and RCC cells in the settings of IRI and cisplatin-induced cytotoxicity. MATERIALS AND METHODS Cell Cultures LLC-PK1 cells (porcine renal tubule epithelial cells), HK-2 cells (human renal tubular epithelial cells), and 2 types of renal clear cell cancer cells, Caki-1 (originated from clear cell RCC) and Caki-2 (originated from high-grade papillary clear cell RCC), were obtained from American Type Culture Collection (ATCC; Manassas, Va, USA). The LLC-PK1 cells were cultured on growth media consisting of M199 medium (Welgene, Gyeongsan, Korea) with 3% fetal bovine serum (FBS; Gibco, Grand Island, NY, USA) and 1% antibiotics (Gibco). HK-2 cells were cultured in RPMI1640 medium (Welgene) with 10% fetal bovine serum (FBS) and 1% antibiotics. Caki-1 and Caki-2 cells were cultured in McCoy’s medium (ATCC) supplemented with 10% FBS and 1% antibiotics. Cell lines were cultured according to ATCC protocols.
Reagents CORM-3 was purchased from Sigma (Sigma-Aldrich, Poole, UK). CORM-3 was freshly prepared by dissolution in distilled water. An inactive CORM-3 compound (iCORM-3) was prepared by adding CORM-3 (2.5 mM) to phosphate-buffered saline (pH 7.4) and leaving this solution at room temperature for 1 day [13]. Cisplatininjection solution was purchased from Il-dong Pharmaceutical Company (Seoul, Korea) and diluted with normal saline.
Stimulation of IRI in Vitro The cells were grown until confluent. Ischemia was induced by placing cells for 48 hours within an anaerobic chamber (37 C, 95% nitrogen, 5% carbon dioxide [CO2]) in RPMI1640 medium without glucose, but supplemented with 0.5% FBS. Cell culture medium was then changed to complete medium, and the cells were maintained in a humidified 5% CO2 incubator for another 6 hours.
Cell Viability Assay Normal and RCC cell lines (5000 cells/well) were plated onto a 96-well plate with 200 mL of culture medium. The cells were pretreated for 3 hours with various doses (0, 12.5, 25, 50, 100, and 200 mM) of CORM-3 or 200 mM of iCORM-3, and then the cells were subjected to IRI or cisplatin-induced cell damage. The cells were exposed to the Cell Counting Kit assay reagent (EZ-CYTOX, DoGenBio, Seoul, Korea) for 2 hours in the 5% CO2 incubator. The optical density was read at 450 nm using a VersaMax ELISA Microplate Reader (Molecular Devices, Sunnyvale, Calif, USA).
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Tumor Necrosis Factor-a Quantitative Reverse Transcriptase Polymerase Chain Reaction Expression of the tumor necrosis factor (TNF)-a gene was quantified by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) using total RNA obtained from the cell lines. Total RNA was isolated from the cell lines using the TRIzol Reagent (Ambion, Grand Island, NY, USA). Reverse transcription was performed using a high-capacity RNA to cDNA kit (Applied Biosystems, Grand Island, NY, USA). qRT-PCR was performed using SYBR Green PCR master mix (Applied Biosystems, Odessa, Tex, USA) and StepOnePlus Real-Time PCR System (Applied Biosystems). The qRT-PCR primer sets were as follows: porcine TNF-a sense primer, 50 -GGCCCAAGGACTCAGATCAT-30 , and antisense primer, 50 -GCATACCCACTCTGCCATTG-30 ; porcine actin sense primer, 50 -CACCTTCTACAACGAGCTGC-30 , and antisense primer, 50 -TCATCTTCTCACGGTTGGCT-30 ; human TNF-a sense primer, 50 -TCAACCTCCTCTCTGCCATC-30 , and antisense primer, 50 -CCAAAGTAGACCTGCCCAGA-0 ; and human glyceraldehyde 3-phosphate dehydrogenase (GAPDH) sense primer, 50 -GGGTCATTGATGGCAACAATATC-30 , and antisense primer, 50 -ATGGGGAAGGTGAAGGTCG-30 . The primers were purchased from Bioneer (Daejeon, Korea). The relative amounts of TNF-a were normalized versus actin or GAPDH.
Western Blot Analysis Cells were collected and lysed with Radioimmunoprecipitation Assay Lysis Buffer (ELPIS, Daejeon, Korea), supplemented with 1 protease inhibitor cocktail (Sigma-Aldrich) and 1 mM phenylmethylsulfonyl fluoride (Sigma-Aldrich). Protein concentrations were determined using a Pierce BCA protein assay kit (Thermo Fisher Scientific Ind, Waltham, Mass, USA). Equal quantities of protein isolates were separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis and later transferred to a polyvinylidene difluoride membrane (Millipore, Billerica, Mass, USA). After blocking for 1 hour with Tris-buffered saline plus 0.05% Tween20 (TBST; ELPIS) containing 5% skim milk, the membranes were incubated at 4 C overnight with the appropriate primary antibody in TBST containing 3% bovine serum albumin (Bovogen, Victoria, Australia). For protein expression of Erk1/2/p-Erk1/2, JNK/p-JNK, P38/p-P38, and cleaved-caspase-3, we used primary antibodies (diluted 1:1000, Cell Signaling, Boston, Mass, USA). For visualization, we used horseradish peroxidase-coupled secondary antibody (Goat Anti-Rabbit IgG; Thermo Fisher Scientific Ind) and the ECL chemiluminescence substrate solution (Bio-Rad, Calif, USA).
Statistical Analysis All data are expressed as means standard deviation. Statistical analyses were performed using SPSS 21 (SPSS Inc, Chicago, Ill, USA) and GraphPad Prism 5 (GraphPad Software, San Diego, Calif, USA). A P value of <.05 was considered statistically significant.
RESULTS Establishment of IRI in Vitro and CORM-3 Recovery of IRIStimulated Inhibition of Proliferation in Normal Renal Cells and RCC Cells
To establish the in vitro models of IRI, we analyzed cell viability of normal renal cells (LLC-PK1, HK-2) and RCC cells (Caki-1, Caki-2) under various conditions: 24 and 48
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hours of stimulated ischemia followed by 0, 6, and 24 hours of reperfusion. Cell viability was approximately 50% decreased following 24 hours of ischemia in LLC-PK1 cells compared with the control. However, cell viability of the other cell lines decreased less than 50% after 24 hours of ischemia under most reperfusion conditions (Fig 1A). Next, the different cell lines were subjected to 48 hours of ischemia followed by 0, 6, and 24 hours of reperfusion; under these conditions, the cell viability of the normal renal cells and RCC cells was decreased over 60% compared with the control (Fig 1B). Based on these results, we established the IRI conditions as 48 hours of ischemia and followed by 6 hours of reperfusion. To study the effects of CORM-3 on the IRI in vitro models, the different confluent cell lines were pretreated with 0, 12.5, 25, 50, 100, and 200 mM CORM-3 for 3 hours and then stimulated with 48 hours of ischemia and 6 hours
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of reperfusion. CORM-3 significantly protected cell viability of the different cell lines in a dose-dependent manner (Fig 1C). At 200 mM, CORM-3 protected the cell viability of LLC-PK1, HK-2, Caki-1, and Caki-2 cells by 38%, 32%, 43%, and 39%, respectively, compared with cells not treated with CORM-3 (ie, 0 mM CORM-3; Fig 1C). Furthermore, iCORM-3, which is incapable of liberating CO, did not any exhibit any effects on the inhibition of cell proliferation by IRI. Cell morphology was observed with an inverted microscope (Fig 1D) and confirmed cell viability. Protective Effects of CORM-3 During Cisplatin Treatment in Normal Renal Cells and Renal Cell Cancer Cells
To establish the in vitro models of cisplatin cytotoxicity, confluent normal renal cells and RCC cells were exposed to cisplatin doses of 0, 6.25, 12.5, 25, 50, 100, and 200 mM for 24 hours. The results showed that normal renal cells
Fig 1. Cell viability assays of LLC-PK1, HK-2, Caki-1, and Caki-2 cells after ischemia reperfusion injury (IRI) and the effects of carbon monoxideereleasing molecule 3 (CORM-3) on these IRI in vitro models. Confluent LLC-PK-1, HK-2, Caki-1, and Caki-2 cells were placed in an anaerobic chamber (37 C, 95% nitrogen, 5% carbon dioxide) for 24 hours (A) or 48 hours (B) and cells underwent reoxygenation (reoxy) for 0, 6, and 24 hours. After pretreatment with CORM-3 (0, 12.5, 25, 50, 100, and 200 mM) or 200 mM of inactive CORM-3 (iCORM-3) for 3 hours, the cells were subjected to ischemia for 48 hours and reoxygenation for 6 hours (C). Cell morphology was observed with an inverted microscope (D). Cell viability was analyzed using the Cell Counting Kit assay (with the control [Cont] arbitrarily set as 100% viable cells). Error bars represent standard deviation of the independent experiments. *P < .05 vs IRIinduced cells (0 mM of CORM-3). yP < .05 vs control (no treatment).
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(LLC-PK1, HK-2) exposed to 12.5 to 200 mM cisplatin had significantly decreased cell viability, which was proportional to the cisplatin concentration. RCC cells were more likely to be resistant to cisplatin, but cell viability significantly decreased with 200 mM cisplatin treatment in RCC cells (Caki-1, Caki-2; Fig 2A). Based on the results of these
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experiments, we established the cytotoxic concentrations of cisplatin as 50 mM in normal renal cells and 200 mM in RCC cells. To study the effects of CORM-3 on cisplatin-treated cells, the different confluent cell lines were pretreated with 0, 12.5, 25, 50, 100, and 200 mM CORM-3 for 3 hours and then
Fig 2. Cell viability assays of normal renal cells and renal cell cancer (RCC) cells after cisplatin (CP) treatment and inhibition of CP-induced cytotoxicity in normal renal cells and RCC cells by carbon monoxideereleasing molecule 3 (CORM-3). Confluent cells were treated with CP (0, 6.25, 12.5, 25, 50, 100, and 200 mM) for 24 hours and the Cell Counting Kit (CCK) assay was performed (A). Normal renal cells and RCC cells were incubated with different concentrations of CORM-3 (0e200 mM) for 3 hours, after which the LLC-PK1 cells and HK-2 cells were exposed to 50 mM CP and the Caki-1 and Caki-2 cells were exposed to 200 mM CP (B). Inactive CORM-3 (iCORM-3) was used as a negative control (Cont). After 24-hour incubation, the CCK assay was performed. Error bars represent standard deviation of the independent experiments. *P < .05 vs CP-exposed cells (0 mM of CORM-3). yP < .05 vs control (no treatment).
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Fig 3. Carbon monoxideereleasing molecule 3 (CORM-3; abbreviated “C” in the figure) inhibition of ischemia reperfusion injury (IRI)induced and cisplatin (CP)-induced tumor necrosis factor (TNF)-a mRNA expression in normal renal cells and RCC cells. Confluent LLC-PK1 (A, E), HK-2 (B, F), Caki-1 (C, G), and Caki-2 (D, H) cells were exposed to CORM-3 (0e200 mM) for 3 hours. Inactive iCORM-3 (iC) was used as a negative control (Cont). For the IRI models, the cells were placed in an anaerobic chamber for 24 hours and the cell culture medium was changed to a no-glucose RPMI medium containing 0.5% fetal bovine serum. This was followed by 6-hour reoxygenation, during which the cell medium was changed to the complete medium. For the CP-cytotoxicity models, LLC-PK1 cell and HK-2 cells were exposed to 50 mM CP and Caki-1 cells and Caki-2 cells were exposed to 200 mM CP for 24 hours. Relative expression of TNF-a was assessed by quantitative reverse transcriptase polymerase chain reaction (x-fold expression, DDCt-method). *P < .05 vs IRI-induced cells or CP-exposed cells (no treatment with CORM-3). yP < .05 vs control (no treatment).
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Fig 4. Ischemia reperfusion injury (IRI) stimulation of oxidative stress signaling and apoptosis, as well as carbon monoxideereleasing molecule 3 (CORM-3; abbreviated “C” in the figure) inhibition of IRI-induced oxidative stress markers and cleaved caspase-3 (C-caspase 3) protein expression, in normal renal cells and RCC cells. Normal renal cells and RCC cells were treated with CORM-3 (200 mM) for 3 hours. Inactive iCORM-3 (iC) was used as a negative control (con). The cell plates were placed in an anaerobic chamber (37 C, 95% nitrogen, 5% carbon dioxide [CO2]) for 48 hours, then the cell medium was changed back to the complete medium and the cells were incubated in a 37 C humidified CO2 incubator for 6 hours. Cell lysates were resolved by sodium dodecyl sulfatepolyacrylamide gel electrophoresis. Immunoblot analysis was performed using antibodies against Erk1/2, p-Erk1/2, P38, p-P38, JNK, p-JNK, cleaved-caspase 3 (C-caspase 3), and b-actin.
exposed to cisplatin for 24 hours. The results showed that 200 mM CORM-3 protected cell viability of LLC-PK1, HK-2, Caki-1, and Caki-2 cells by 15%, 25%, 8%, and 10%, respectively, compared with cells not treated with CORM-3 (Fig 2B). Inhibition of Elevated TNF-a Messenger RNA Levels by CORM-3 Following IRI or Cisplatin Treatment
We assessed the induction of the inflammation marker TNF-a, which is important in IRI-induced or cisplatinexposed normal renal cells and RCC cells, and used TNFa messenger RNA (mRNA) levels to investigate the effects of CORM-3 on cell inflammation. As shown in Fig 3, IRIinduced or cisplatin-exposed cells exhibited significantly upregulated TNF-a mRNA levels in the different cell lines, whereas CORM-3-pretreated cells showed reduced TNF-a mRNA levels compared with cells not pretreated with CORM-3. Expression of Oxidative Stress-Related Proteins and Cleaved-Caspase 3
The expression of proteins involved in the oxidative stress pathway was evaluated following IRI or cisplatin treatment. Western blot results showed that IRI induced significant upregulation of oxidative stress-related proteins (p-JNK, p-P38) and downregulation of p-Erk1/2 protein expression in renal cells (LLC-PK1, HK-2). These effects were significantly decreased when the cells were pretreated with
CORM-3 (Fig 4). In contrast to the effects in normal kidney cell lines, pretreatment with CORM-3 did not affect oxidative stress markers in the RCC cell lines (Caki-1, Caki-2). However, all cell lines showed decreased cleaved-caspase 3 after CORM-3 treatment. In the cisplatin-induced toxicity experiments, all cell lines showed little difference in expression patterns between groups treated with CORM-3 or iCORM-3 or not treated with CORM-3 (Fig 5). LLCPK1 cells showed slightly decreased p-JNK and p-P38 expression when pretreated with CORM-3.
DISCUSSION
The aim of this study was to evaluate the effects of CORM-3 in renal cells in the settings of IRI and cisplatin-induced cytotoxicity. CORM-3 showed protective effects in not only normal kidney epithelial cells, but also in RCC cell lines in IRI. These findings are consistent with the results of previous studies, which demonstrated that CORM-3 has renoprotective effects in IRI. Sener et al demonstrated that CORM-3 could prevent transplantation-related apoptosis through the upregulation of mitochondrial Bcl-2 survival factors [7]. However, in their in vitro experiments, the authors used human umbilical vein endothelial cells to assess the effects of CORM-3 in transplant-relevant conditions of cold storage [7]. To our knowledge, the current study is the first to use kidney cell lines to assess the effects of CORM-3 in IRI.
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Fig 5. Cisplatin (CP) stimulation of oxidative stress signaling and apoptosis, as well as carbon monoxideereleasing molecule 3 (CORM-3; abbreviated “C” in the figure) inhibition of CP-induced oxidative stress markers and cleaved caspase-3 protein expression, in normal renal cells and renal cell carcinoma (RCC) cells. Normal renal cells and RCC cells were pretreated with CORM-3 (200 mM) for 3 hours, then LLC-PK1 and HK-2 cells were exposed to 50 mM CP and Caki-1 and Caki-2 cells were exposed to 200 mM CP. Inactive iCORM-3 (iC) was used as a negative control (Con). After 24-hour incubation, cell lysates were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Immunoblot analysis was performed using antibodies against Erk1/2, p-Erk1/2, P38, p-P38, JNK, p-JNK, cleavedcaspase 3 (C-caspase 3), and b-actin.
TNF-a is a proinflammatory cytokine that is an important mediator of renal cell injury in IRI and an inducer of apoptosis [14,15]. Normal renal cells and renal cancer cells pretreated with CORM-3 showed lower levels of TNF-a than untreated cells, demonstrating the effects of CORM-3 in protecting against inflammation and subsequent cell death. Our results also indicate that these protective effects could be acting through a reduction of oxidative stress, suggesting that the MAPKs-p53-caspase-3 signaling pathway is involved in the protective effects of CORM-3 [16]. Activation of ERK1/2 improves cell survival through inhibition of apoptosis, and activation of JNK and P38 is linked to cell death [17]. In our results, ERK1/2 was increased and JNK and P38 were decreased, suggesting that CORM-3 acted by preventing cell death. We also performed experiments with cisplatin to evaluate the effects of CORM-3 during cytotoxic injury. In general, the effects of CORM-3 in cisplatin-induced cytotoxicity were less protective than in IRI. Tayem et al demonstrated that CORM-3 could be used as a potent therapeutic adjuvant in the treatment of cisplatin-induced nephrotoxicity [4]. These authors used LLC-PK1, which is renal tubule epithelial cell, and noted that treatment with CORM-3 (1e50 mM) resulted in a remarkable and concentrationdependent decrease in cisplatin-induced caspase-3 activity and cell detachment [4]. In the current study, we demonstrated similar results regarding cell viability and the inflammatory pathway marker TNF-a. We also evaluated the effects of CORM-3 in RCC cells because these cells are
resistant to chemotherapeutic agents such as cisplatin [18]. Although slightly improved cell viability and reduction of TNF-a levels were seen in RCC cells pretreated with CORM-3, CORM-3 did not produce discernible effects during Western blot analysis, including cleaved-caspase 3 levels. Perhaps our model of cisplatin-induced cytotoxicity was insufficient to show the effects of CORM-3; alternatively, CORM-3 may indeed have no effect on RCCs exposed to this type of injury. Additional detailed experiments are required to determine the interaction between RCC cells and CORM-3. We conclude that CORM-3 has protective effects in IRI and cisplatin-induced cytotoxicity models in vitro. Our results suggest that CORM-3 attenuates IRI and cisplatininduced cytotoxicity by amelioration of inflammatory and oxidative stress pathways. These effects were observed in not only normal kidney cells but also in RCC cells.
REFERENCES [1] Ozaki KS, Kimura S, Murase N. Use of carbon monoxide in minimizing ischemia/reperfusion injury in transplantation. Transplant Rev (Orlando) 2012;26:125e39. [2] Baumgart K, Radermacher P, Wagner F. Applying gases for microcirculatory and cellular oxygenation in sepsis: effects of nitric oxide, carbon monoxide, and hydrogen sulfide. Curr Opin Anaesthesiol 2009;22:168e76. [3] Clark JE, Naughton P, Shurey S, Green CJ, Johnson TR, Mann BE, et al. Cardioprotective actions by a water-soluble carbon monoxide-releasing molecule. Circ Res 2003;93:e2e8.
1182 [4] Tayem Y, Johnson TR, Mann BE, Green CJ, Motterlini R. Protection against cisplatin-induced nephrotoxicity by a carbon monoxidereleasing molecule. Am J Physiol Renal Physiol 2006;290:F789e94. [5] Yan BZ, Yang BS, Li H, Zhang YF, Pei FH, Zhu AC, et al. The therapeutic effect of CORM-3 on acute liver failure induced by lipopolysaccharide/D-galactosamine in mice. Hepatobiliary Pancreat Dis Int 2016;15:73e80. [6] Uddin MJ, Li CS, Joe Y, Chen Y, Zhang Q, Ryter SW, et al. Carbon monoxide inhibits tenascin-C mediated inflammation via IL-10 expression in a septic mouse model. Mediators Inflamm 2015;2015:613249. [7] Sener A, Tran KC, Deng JP, Garcia B, Lan Z, Liu W, et al. Carbon monoxide releasing molecules inhibit cell death resulting from renal transplantation related stress. J Urol 2013;190:772e8. [8] Caumartin Y, Stephen J, Deng JP, Lian D, Lan Z, Liu W, et al. Carbon monoxide-releasing molecules protect against ischemia-reperfusion injury during kidney transplantation. Kidney Int 2011;79:1080e9. [9] Bagul A, Hosgood SA, Kaushik M, Nicholson ML. Carbon monoxide protects against ischemia-reperfusion injury in an experimental model of controlled nonheartbeating donor kidney. Transplantation 2008;85:576e81. [10] Sandouka A, Fuller BJ, Mann BE, Green CJ, Foresti R, Motterlini R. Treatment with CO-RMs during cold storage improves renal function at reperfusion. Kidney Int 2006;69:239e47. [11] Vera T, Henegar JR, Drummond HA, Rimoldi JM, Stec DE. Protective effect of carbon monoxide-releasing
YOON, LEE, LEE ET AL compounds in ischemia-induced acute renal failure. J Am Soc Nephrol 2005;16:950e8. [12] Haase VH. Renal cancer: oxygen meets metabolism. Exp Cell Res 2012;318:1057e67. [13] Winburn IC, Gunatunga K, McKernan RD, Walker RJ, Sammut IA, Harrison JC. Cell damage following carbon monoxide releasing molecule exposure: implications for therapeutic applications. Basic Clin Pharmacol Toxicol 2012;111:31e41. [14] Donnahoo KK, Meng X, Ayala A, Cain MP, Harken AH, Meldrum DR. Early kidney TNF-alpha expression mediates neutrophil infiltration and injury after renal ischemia-reperfusion. Am J Physiol 1999;277:R922e9. [15] Hsu H, Xiong J, Goeddel DV. The TNF receptor 1-associated protein TRADD signals cell death and NF-kappa B activation. Cell 1995;81:495e504. [16] Lee D, Kim KH, Moon SW, Lee H, Kang KS, Lee JW. Synthesis and biological evaluation of chalcone analogues as protective agents against cisplatin-induced cytotoxicity in kidney cells. Bioorg Med Chem Lett 2015;25:1929e32. [17] Song H, Han IY, Kim Y, Kim YH, Choi IW, Seo SK, et al. The NADPH oxidase inhibitor DPI can abolish hypoxia-induced apoptosis of human kidney proximal tubular epithelial cells through Bcl2 up-regulation via ERK activation without ROS reduction. Life Sci 2015;126:69e75. [18] Hueber PA, Waters P, Clark P, Eccles M, Goodyer P. PAX2 inactivation enhances cisplatin-induced apoptosis in renal carcinoma cells. Kidney Int 2006;69:1139e45.
ABSTRACT Background. We investigated the effects of a soluble carbon monoxideereleasing molecule (CORM) in cisplatin-induced cytotoxicity and ischemia-reperfusion injury (IRI) in vitro. Methods. The effects of CORM-3 (12.5e200 mM) were assessed in normal kidney epithelial cells (HK-2, LLC-PK1) and renal cancer cells (Caki-1, Caki-2) subjected to cisplatin (50e200 mM) or IRI. To induce IRI, cells were placed in an anaerobic chamber (37 C, 95% nitrogen, 5% carbon dioxide) for 48 hours. Cells were transferred to complete medium and incubated at 37 C, 5% carbon dioxide for 6 hours. Cell viability (CCK assays), tumor necrosis factor (TNF)-a messenger RNA (mRNA) levels (quantitative reverse-transcriptase polymerase chain reaction), and protein expression of cleaved-caspase 3 and oxidative stress markers (including Erk1/2, JNK, and P38; Western blot) were assessed. Results. Viability after IRI was approximately 40% of control. Protective effects of CORM-3 in the IRI model were dose-dependent. Cell viability was 40% recovered in 200-mM CORM-3-pretreated cells compared with control. The protective effects of CORM-3 in cells exposed to cisplatin for 24 hours were weaker than in the IRI model. TNF-a mRNA was induced by stimulated IRI or cisplatin exposure; CORM-3 pretreatment attenuated the rise in TNF-a mRNA. IRI or cisplatin-induced activated oxidative stress markers decreased in CORM-3-pretreated cells. CORM-3 reduced expression of the apoptotic marker cleaved-caspase 3. Conclusion. Our data demonstrate the protective effects of CORM-3 in cisplatin cytotoxicity and IRI in both normal kidney cells and renal cancer cells in vitro. CORM-3 exerts these effects by ameliorating inflammatory and oxidative stress pathways.
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ARBON monoxide (CO) is a molecule that has protective effects in tissues subjected to ischemiareperfusion injury (IRI) and inflammation [1,2]. However, the use of CO in humans is very challenging because of its toxicity characterized by inhibition of oxygen delivery at high concentrations [1]. Thus, clinical application of CO is very limited at present. CO-releasing molecules (CORMs) that can deliver CO to tissues were initially developed and evaluated over a decade ago [3]. Among the various CORMs, CORM-3 [tricarbonylchloro(glycinato)ruthenium (II)] is water soluble and thus easy to handle and apply to cells and organs [3]. CORMs exhibit organ protective effects ª 2017 Elsevier Inc. All rights reserved. 230 Park Avenue, New York, NY 10169
Transplantation Proceedings, 49, 1175e1182 (2017)
in transplantation, inflammation, liver failure, and exposure to cytotoxic agents [4e7]. This study was financially supported by the “Dongwha” Faculty Research Assistance Program of Yonsei University College of Medicine (6-2015-0170) and the National Research Foundation grant (NRF- 2012R1A1A1042968) funded by the Korean government (MEST). The funding sources had no involvement in this study. *Address correspondence to Woong Kyu Han, MD, PhD, Department of Urology, Urological Science Institute, Yonsei University College of Medicine, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-752, Korea. E-mail: [email protected] 0041-1345/17 http://dx.doi.org/10.1016/j.transproceed.2017.03.067
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Previous research regarding the effects of CORM-3 on kidneys or kidney cells has primarily examined its role in kidney transplantation and IRI [7e11]. These studies unanimously concluded that CORM-3 has a renoprotective effect in kidney transplantation because of its protective effects against IRI. However, most of these studies did not use human kidney cell lines. Furthermore, renal cell carcinoma (RCC) cells were used in none of these experiments. Because RCC cells may require more oxygen to survive than normal cells, the effect of CORM-3 on RCC cells should be evaluated [12]. The effect of CORM-3 during cytotoxic exposure also requires evaluation [4]. Therefore, we conducted this study to evaluate the effects of CORM-3 in normal kidney cells and RCC cells in the settings of IRI and cisplatin-induced cytotoxicity. MATERIALS AND METHODS Cell Cultures LLC-PK1 cells (porcine renal tubule epithelial cells), HK-2 cells (human renal tubular epithelial cells), and 2 types of renal clear cell cancer cells, Caki-1 (originated from clear cell RCC) and Caki-2 (originated from high-grade papillary clear cell RCC), were obtained from American Type Culture Collection (ATCC; Manassas, Va, USA). The LLC-PK1 cells were cultured on growth media consisting of M199 medium (Welgene, Gyeongsan, Korea) with 3% fetal bovine serum (FBS; Gibco, Grand Island, NY, USA) and 1% antibiotics (Gibco). HK-2 cells were cultured in RPMI1640 medium (Welgene) with 10% fetal bovine serum (FBS) and 1% antibiotics. Caki-1 and Caki-2 cells were cultured in McCoy’s medium (ATCC) supplemented with 10% FBS and 1% antibiotics. Cell lines were cultured according to ATCC protocols.
Reagents CORM-3 was purchased from Sigma (Sigma-Aldrich, Poole, UK). CORM-3 was freshly prepared by dissolution in distilled water. An inactive CORM-3 compound (iCORM-3) was prepared by adding CORM-3 (2.5 mM) to phosphate-buffered saline (pH 7.4) and leaving this solution at room temperature for 1 day [13]. Cisplatininjection solution was purchased from Il-dong Pharmaceutical Company (Seoul, Korea) and diluted with normal saline.
Stimulation of IRI in Vitro The cells were grown until confluent. Ischemia was induced by placing cells for 48 hours within an anaerobic chamber (37 C, 95% nitrogen, 5% carbon dioxide [CO2]) in RPMI1640 medium without glucose, but supplemented with 0.5% FBS. Cell culture medium was then changed to complete medium, and the cells were maintained in a humidified 5% CO2 incubator for another 6 hours.
Cell Viability Assay Normal and RCC cell lines (5000 cells/well) were plated onto a 96-well plate with 200 mL of culture medium. The cells were pretreated for 3 hours with various doses (0, 12.5, 25, 50, 100, and 200 mM) of CORM-3 or 200 mM of iCORM-3, and then the cells were subjected to IRI or cisplatin-induced cell damage. The cells were exposed to the Cell Counting Kit assay reagent (EZ-CYTOX, DoGenBio, Seoul, Korea) for 2 hours in the 5% CO2 incubator. The optical density was read at 450 nm using a VersaMax ELISA Microplate Reader (Molecular Devices, Sunnyvale, Calif, USA).
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Tumor Necrosis Factor-a Quantitative Reverse Transcriptase Polymerase Chain Reaction Expression of the tumor necrosis factor (TNF)-a gene was quantified by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) using total RNA obtained from the cell lines. Total RNA was isolated from the cell lines using the TRIzol Reagent (Ambion, Grand Island, NY, USA). Reverse transcription was performed using a high-capacity RNA to cDNA kit (Applied Biosystems, Grand Island, NY, USA). qRT-PCR was performed using SYBR Green PCR master mix (Applied Biosystems, Odessa, Tex, USA) and StepOnePlus Real-Time PCR System (Applied Biosystems). The qRT-PCR primer sets were as follows: porcine TNF-a sense primer, 50 -GGCCCAAGGACTCAGATCAT-30 , and antisense primer, 50 -GCATACCCACTCTGCCATTG-30 ; porcine actin sense primer, 50 -CACCTTCTACAACGAGCTGC-30 , and antisense primer, 50 -TCATCTTCTCACGGTTGGCT-30 ; human TNF-a sense primer, 50 -TCAACCTCCTCTCTGCCATC-30 , and antisense primer, 50 -CCAAAGTAGACCTGCCCAGA-0 ; and human glyceraldehyde 3-phosphate dehydrogenase (GAPDH) sense primer, 50 -GGGTCATTGATGGCAACAATATC-30 , and antisense primer, 50 -ATGGGGAAGGTGAAGGTCG-30 . The primers were purchased from Bioneer (Daejeon, Korea). The relative amounts of TNF-a were normalized versus actin or GAPDH.
Western Blot Analysis Cells were collected and lysed with Radioimmunoprecipitation Assay Lysis Buffer (ELPIS, Daejeon, Korea), supplemented with 1 protease inhibitor cocktail (Sigma-Aldrich) and 1 mM phenylmethylsulfonyl fluoride (Sigma-Aldrich). Protein concentrations were determined using a Pierce BCA protein assay kit (Thermo Fisher Scientific Ind, Waltham, Mass, USA). Equal quantities of protein isolates were separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis and later transferred to a polyvinylidene difluoride membrane (Millipore, Billerica, Mass, USA). After blocking for 1 hour with Tris-buffered saline plus 0.05% Tween20 (TBST; ELPIS) containing 5% skim milk, the membranes were incubated at 4 C overnight with the appropriate primary antibody in TBST containing 3% bovine serum albumin (Bovogen, Victoria, Australia). For protein expression of Erk1/2/p-Erk1/2, JNK/p-JNK, P38/p-P38, and cleaved-caspase-3, we used primary antibodies (diluted 1:1000, Cell Signaling, Boston, Mass, USA). For visualization, we used horseradish peroxidase-coupled secondary antibody (Goat Anti-Rabbit IgG; Thermo Fisher Scientific Ind) and the ECL chemiluminescence substrate solution (Bio-Rad, Calif, USA).
Statistical Analysis All data are expressed as means standard deviation. Statistical analyses were performed using SPSS 21 (SPSS Inc, Chicago, Ill, USA) and GraphPad Prism 5 (GraphPad Software, San Diego, Calif, USA). A P value of <.05 was considered statistically significant.
RESULTS Establishment of IRI in Vitro and CORM-3 Recovery of IRIStimulated Inhibition of Proliferation in Normal Renal Cells and RCC Cells
To establish the in vitro models of IRI, we analyzed cell viability of normal renal cells (LLC-PK1, HK-2) and RCC cells (Caki-1, Caki-2) under various conditions: 24 and 48
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hours of stimulated ischemia followed by 0, 6, and 24 hours of reperfusion. Cell viability was approximately 50% decreased following 24 hours of ischemia in LLC-PK1 cells compared with the control. However, cell viability of the other cell lines decreased less than 50% after 24 hours of ischemia under most reperfusion conditions (Fig 1A). Next, the different cell lines were subjected to 48 hours of ischemia followed by 0, 6, and 24 hours of reperfusion; under these conditions, the cell viability of the normal renal cells and RCC cells was decreased over 60% compared with the control (Fig 1B). Based on these results, we established the IRI conditions as 48 hours of ischemia and followed by 6 hours of reperfusion. To study the effects of CORM-3 on the IRI in vitro models, the different confluent cell lines were pretreated with 0, 12.5, 25, 50, 100, and 200 mM CORM-3 for 3 hours and then stimulated with 48 hours of ischemia and 6 hours
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of reperfusion. CORM-3 significantly protected cell viability of the different cell lines in a dose-dependent manner (Fig 1C). At 200 mM, CORM-3 protected the cell viability of LLC-PK1, HK-2, Caki-1, and Caki-2 cells by 38%, 32%, 43%, and 39%, respectively, compared with cells not treated with CORM-3 (ie, 0 mM CORM-3; Fig 1C). Furthermore, iCORM-3, which is incapable of liberating CO, did not any exhibit any effects on the inhibition of cell proliferation by IRI. Cell morphology was observed with an inverted microscope (Fig 1D) and confirmed cell viability. Protective Effects of CORM-3 During Cisplatin Treatment in Normal Renal Cells and Renal Cell Cancer Cells
To establish the in vitro models of cisplatin cytotoxicity, confluent normal renal cells and RCC cells were exposed to cisplatin doses of 0, 6.25, 12.5, 25, 50, 100, and 200 mM for 24 hours. The results showed that normal renal cells
Fig 1. Cell viability assays of LLC-PK1, HK-2, Caki-1, and Caki-2 cells after ischemia reperfusion injury (IRI) and the effects of carbon monoxideereleasing molecule 3 (CORM-3) on these IRI in vitro models. Confluent LLC-PK-1, HK-2, Caki-1, and Caki-2 cells were placed in an anaerobic chamber (37 C, 95% nitrogen, 5% carbon dioxide) for 24 hours (A) or 48 hours (B) and cells underwent reoxygenation (reoxy) for 0, 6, and 24 hours. After pretreatment with CORM-3 (0, 12.5, 25, 50, 100, and 200 mM) or 200 mM of inactive CORM-3 (iCORM-3) for 3 hours, the cells were subjected to ischemia for 48 hours and reoxygenation for 6 hours (C). Cell morphology was observed with an inverted microscope (D). Cell viability was analyzed using the Cell Counting Kit assay (with the control [Cont] arbitrarily set as 100% viable cells). Error bars represent standard deviation of the independent experiments. *P < .05 vs IRIinduced cells (0 mM of CORM-3). yP < .05 vs control (no treatment).
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(LLC-PK1, HK-2) exposed to 12.5 to 200 mM cisplatin had significantly decreased cell viability, which was proportional to the cisplatin concentration. RCC cells were more likely to be resistant to cisplatin, but cell viability significantly decreased with 200 mM cisplatin treatment in RCC cells (Caki-1, Caki-2; Fig 2A). Based on the results of these
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experiments, we established the cytotoxic concentrations of cisplatin as 50 mM in normal renal cells and 200 mM in RCC cells. To study the effects of CORM-3 on cisplatin-treated cells, the different confluent cell lines were pretreated with 0, 12.5, 25, 50, 100, and 200 mM CORM-3 for 3 hours and then
Fig 2. Cell viability assays of normal renal cells and renal cell cancer (RCC) cells after cisplatin (CP) treatment and inhibition of CP-induced cytotoxicity in normal renal cells and RCC cells by carbon monoxideereleasing molecule 3 (CORM-3). Confluent cells were treated with CP (0, 6.25, 12.5, 25, 50, 100, and 200 mM) for 24 hours and the Cell Counting Kit (CCK) assay was performed (A). Normal renal cells and RCC cells were incubated with different concentrations of CORM-3 (0e200 mM) for 3 hours, after which the LLC-PK1 cells and HK-2 cells were exposed to 50 mM CP and the Caki-1 and Caki-2 cells were exposed to 200 mM CP (B). Inactive CORM-3 (iCORM-3) was used as a negative control (Cont). After 24-hour incubation, the CCK assay was performed. Error bars represent standard deviation of the independent experiments. *P < .05 vs CP-exposed cells (0 mM of CORM-3). yP < .05 vs control (no treatment).
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Fig 3. Carbon monoxideereleasing molecule 3 (CORM-3; abbreviated “C” in the figure) inhibition of ischemia reperfusion injury (IRI)induced and cisplatin (CP)-induced tumor necrosis factor (TNF)-a mRNA expression in normal renal cells and RCC cells. Confluent LLC-PK1 (A, E), HK-2 (B, F), Caki-1 (C, G), and Caki-2 (D, H) cells were exposed to CORM-3 (0e200 mM) for 3 hours. Inactive iCORM-3 (iC) was used as a negative control (Cont). For the IRI models, the cells were placed in an anaerobic chamber for 24 hours and the cell culture medium was changed to a no-glucose RPMI medium containing 0.5% fetal bovine serum. This was followed by 6-hour reoxygenation, during which the cell medium was changed to the complete medium. For the CP-cytotoxicity models, LLC-PK1 cell and HK-2 cells were exposed to 50 mM CP and Caki-1 cells and Caki-2 cells were exposed to 200 mM CP for 24 hours. Relative expression of TNF-a was assessed by quantitative reverse transcriptase polymerase chain reaction (x-fold expression, DDCt-method). *P < .05 vs IRI-induced cells or CP-exposed cells (no treatment with CORM-3). yP < .05 vs control (no treatment).
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Fig 4. Ischemia reperfusion injury (IRI) stimulation of oxidative stress signaling and apoptosis, as well as carbon monoxideereleasing molecule 3 (CORM-3; abbreviated “C” in the figure) inhibition of IRI-induced oxidative stress markers and cleaved caspase-3 (C-caspase 3) protein expression, in normal renal cells and RCC cells. Normal renal cells and RCC cells were treated with CORM-3 (200 mM) for 3 hours. Inactive iCORM-3 (iC) was used as a negative control (con). The cell plates were placed in an anaerobic chamber (37 C, 95% nitrogen, 5% carbon dioxide [CO2]) for 48 hours, then the cell medium was changed back to the complete medium and the cells were incubated in a 37 C humidified CO2 incubator for 6 hours. Cell lysates were resolved by sodium dodecyl sulfatepolyacrylamide gel electrophoresis. Immunoblot analysis was performed using antibodies against Erk1/2, p-Erk1/2, P38, p-P38, JNK, p-JNK, cleaved-caspase 3 (C-caspase 3), and b-actin.
exposed to cisplatin for 24 hours. The results showed that 200 mM CORM-3 protected cell viability of LLC-PK1, HK-2, Caki-1, and Caki-2 cells by 15%, 25%, 8%, and 10%, respectively, compared with cells not treated with CORM-3 (Fig 2B). Inhibition of Elevated TNF-a Messenger RNA Levels by CORM-3 Following IRI or Cisplatin Treatment
We assessed the induction of the inflammation marker TNF-a, which is important in IRI-induced or cisplatinexposed normal renal cells and RCC cells, and used TNFa messenger RNA (mRNA) levels to investigate the effects of CORM-3 on cell inflammation. As shown in Fig 3, IRIinduced or cisplatin-exposed cells exhibited significantly upregulated TNF-a mRNA levels in the different cell lines, whereas CORM-3-pretreated cells showed reduced TNF-a mRNA levels compared with cells not pretreated with CORM-3. Expression of Oxidative Stress-Related Proteins and Cleaved-Caspase 3
The expression of proteins involved in the oxidative stress pathway was evaluated following IRI or cisplatin treatment. Western blot results showed that IRI induced significant upregulation of oxidative stress-related proteins (p-JNK, p-P38) and downregulation of p-Erk1/2 protein expression in renal cells (LLC-PK1, HK-2). These effects were significantly decreased when the cells were pretreated with
CORM-3 (Fig 4). In contrast to the effects in normal kidney cell lines, pretreatment with CORM-3 did not affect oxidative stress markers in the RCC cell lines (Caki-1, Caki-2). However, all cell lines showed decreased cleaved-caspase 3 after CORM-3 treatment. In the cisplatin-induced toxicity experiments, all cell lines showed little difference in expression patterns between groups treated with CORM-3 or iCORM-3 or not treated with CORM-3 (Fig 5). LLCPK1 cells showed slightly decreased p-JNK and p-P38 expression when pretreated with CORM-3.
DISCUSSION
The aim of this study was to evaluate the effects of CORM-3 in renal cells in the settings of IRI and cisplatin-induced cytotoxicity. CORM-3 showed protective effects in not only normal kidney epithelial cells, but also in RCC cell lines in IRI. These findings are consistent with the results of previous studies, which demonstrated that CORM-3 has renoprotective effects in IRI. Sener et al demonstrated that CORM-3 could prevent transplantation-related apoptosis through the upregulation of mitochondrial Bcl-2 survival factors [7]. However, in their in vitro experiments, the authors used human umbilical vein endothelial cells to assess the effects of CORM-3 in transplant-relevant conditions of cold storage [7]. To our knowledge, the current study is the first to use kidney cell lines to assess the effects of CORM-3 in IRI.
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Fig 5. Cisplatin (CP) stimulation of oxidative stress signaling and apoptosis, as well as carbon monoxideereleasing molecule 3 (CORM-3; abbreviated “C” in the figure) inhibition of CP-induced oxidative stress markers and cleaved caspase-3 protein expression, in normal renal cells and renal cell carcinoma (RCC) cells. Normal renal cells and RCC cells were pretreated with CORM-3 (200 mM) for 3 hours, then LLC-PK1 and HK-2 cells were exposed to 50 mM CP and Caki-1 and Caki-2 cells were exposed to 200 mM CP. Inactive iCORM-3 (iC) was used as a negative control (Con). After 24-hour incubation, cell lysates were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Immunoblot analysis was performed using antibodies against Erk1/2, p-Erk1/2, P38, p-P38, JNK, p-JNK, cleavedcaspase 3 (C-caspase 3), and b-actin.
TNF-a is a proinflammatory cytokine that is an important mediator of renal cell injury in IRI and an inducer of apoptosis [14,15]. Normal renal cells and renal cancer cells pretreated with CORM-3 showed lower levels of TNF-a than untreated cells, demonstrating the effects of CORM-3 in protecting against inflammation and subsequent cell death. Our results also indicate that these protective effects could be acting through a reduction of oxidative stress, suggesting that the MAPKs-p53-caspase-3 signaling pathway is involved in the protective effects of CORM-3 [16]. Activation of ERK1/2 improves cell survival through inhibition of apoptosis, and activation of JNK and P38 is linked to cell death [17]. In our results, ERK1/2 was increased and JNK and P38 were decreased, suggesting that CORM-3 acted by preventing cell death. We also performed experiments with cisplatin to evaluate the effects of CORM-3 during cytotoxic injury. In general, the effects of CORM-3 in cisplatin-induced cytotoxicity were less protective than in IRI. Tayem et al demonstrated that CORM-3 could be used as a potent therapeutic adjuvant in the treatment of cisplatin-induced nephrotoxicity [4]. These authors used LLC-PK1, which is renal tubule epithelial cell, and noted that treatment with CORM-3 (1e50 mM) resulted in a remarkable and concentrationdependent decrease in cisplatin-induced caspase-3 activity and cell detachment [4]. In the current study, we demonstrated similar results regarding cell viability and the inflammatory pathway marker TNF-a. We also evaluated the effects of CORM-3 in RCC cells because these cells are
resistant to chemotherapeutic agents such as cisplatin [18]. Although slightly improved cell viability and reduction of TNF-a levels were seen in RCC cells pretreated with CORM-3, CORM-3 did not produce discernible effects during Western blot analysis, including cleaved-caspase 3 levels. Perhaps our model of cisplatin-induced cytotoxicity was insufficient to show the effects of CORM-3; alternatively, CORM-3 may indeed have no effect on RCCs exposed to this type of injury. Additional detailed experiments are required to determine the interaction between RCC cells and CORM-3. We conclude that CORM-3 has protective effects in IRI and cisplatin-induced cytotoxicity models in vitro. Our results suggest that CORM-3 attenuates IRI and cisplatininduced cytotoxicity by amelioration of inflammatory and oxidative stress pathways. These effects were observed in not only normal kidney cells but also in RCC cells.
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