http://informahealthcare.com/rnf ISSN: 0886-022X (print), 1525-6049 (electronic) Ren Fail, 2014; 36(9): 1449–1454 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/0886022X.2014.950934
LABORATORY STUDY
The protective effect of ozone oxidative preconditioning against hypoxia/reoxygenation injury in rat kidney cells Lei Wang, Hui Chen, Xiu-Heng Liu, Zhi-Yuan Chen, Xiao-Dong Weng, Tao Qiu, and Lin Liu
Ren Fail Downloaded from informahealthcare.com by University of Newcastle on 09/26/14 For personal use only.
Department of Urology, Renmin Hospital of Wuhan University, Wuhan, Hubei Province, P.R. China
Abstract
Keywords
Ozone (O3) has been viewed as a novel treatment for different diseases in these years and oxidative stress and apoptosis play a key role in the pathogenesis of kidney diseases including renal ischemia and reperfusion (I/R). In the present study, we investigated the role of ozone oxidative preconditioning (OzoneOP) in attenuating oxidative stress and apoptosis in a hypoxia/reoxygenation (H/R) injury model using rat kidney cells. We induced H/R injury in kidney cells treated with or without OzoneOP. Oxidative stress parameters such as superoxide dismutase (SOD), malondialdehyde (MDA) and lactate dehydrogenase (LDH) were determined, as well as some apoptotic proteins. We observed that oxidative stress and apoptosis were increased in H/R group compared to OzoneOP group; however, these changes were significantly decreased by the treatment with OzoneOP. We concluded that OzoneOP can protect the kidney cells against H/R injury and its mechanism may be through the reduction of oxidative stress and apoptosis.
Apoptosis, hypoxia and reoxygenation, oxidative stress, ozone oxidative preconditioning, rat
Introduction In recent years, ischemia and reperfusion (I/R) injury has elicited an increasing interest of scholars in its impact on such organs as liver, heart and kidney. Renal ischemia is one of the main causes of acute kidney injury (AKI) and often occurs in such surgeries as kidney transplantation, partial nephrectomy, renal artery angioplasty, accidental or iatrogenic trauma, hydronephrosis and elective urological operations.1,2 Oxidative stress and apoptosis play a key role in the pathogenesis of many diseases and the dysregulation of them can lead to a variety of kidney diseases including AKI. As the role of oxidative stress and apoptosis in kidney diseases becoming increasingly clear, therefore, it is essential to develop a therapeutic treatment to block or prevent inappropriate apoptosis and oxidative stress in kidney diseases. Ozone (O3), a reactive gas, has been viewed as a therapeutical material for the treatment of many diseases.3 In recent studies, it has been demonstrated that ozone has the protective effects of anti-apoptosis on renal I/R injury of animal models4, through rectal insufflations. It provides a novel treatment to protect organ from I/R injury and is relatively simple and harmless. It has also been demonstrated that ozone improves the ability of organs subjected to I/R and adapts the organs to slight and transient oxidative stresses,
History Received 14 April 2014 Revised 3 July 2014 Accepted 30 July 2014 Published online 23 September 2014
resulting in the improvement of the anti-oxidant endogenous systems.5,6 It is called OzoneOP, which is brought about by stimulating the endogenous anti-oxidant mechanisms to modulate the cells’ redox state. The majority of the studies of OzoneOP on renal I/R injury are based on animal models, including rats.4 On account of the limitation of many factors, it is difficult to investigate direct effects of renal OzoneOP with these models. In order to eliminate these confounding variables, we seek to develop an in vitro Hypoxia/Reoxygenation (H/R) model of simulating OzoneOP using normal rat kidney epithelial (NRK-52E) cells based on the former study,7,8 and these cells had already been used to investigate H/R injury in previous study.9 Indeed, in vitro methodologies outweigh in vivo experimentation in several aspects. Its main advantage is that the complexity of an in vivo model can be eliminated. It can test the direct effects of the specific stimuli which can only be isolated and controlled in vitro.10 And these advantages can further motivate new in vivo researches in turn and provide deeper insight into the biological phenomena of OzoneOP. This is the unique innovation in our study that implements an in vitro OzoneOP model with NRK-52E cells subjected to H/R injury, which has not been previously described.
Materials and methods Cell culture Address correspondence to Professor Xiu-Heng Liu, Department of Urology, Renmin Hospital of Wuhan University, No. 238 Jiefang Road, Wuhan 430060, Hubei Province, P.R. China. E-mail: [email protected]
The renal tubular epithelial cell line, NRK-52E cells, was obtained from the Cell Bank of Chinese Academy of Sciences (Shanghai, China). The cells were cultured in DMEM
1450
L. Wang et al.
containing 10% FBS and cultured in a chamber at 37 C with 95% air/5% CO2. H/R model
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The H/R injury protocol was performed as previously described.11 Briefly, prior to the experiments, all cells in every group were cultured in 2-mL serum-free medium for 24 h and then seeded on six-well plates for the subsequent treatments. Then, cells were exposed to a bicarbonate-MES buffered Ringer (1 mL per well) at pH 6.6 without glucose in hypoxic (O2 fraction below 1%) condition for 3 h and then cultivated under normal condition (95% air/5% CO2) with fresh oxygenated culture medium (2 mL per well) for 24 h. A hermetic chamber, filled with gas consisting of 95% N2 and 5% CO2, was used to preserve the cells subjected to hypoxic condition. OzoneOP protocols and experimental groups The oxygen–ozone was delivered to biological samples using the method as previously described.17 Ozone generator (YKS1000-type, Zhuhai Yike Medical Co., Ltd, Zhuhai, China) was used to generate oxygen–ozone with pure gaseous oxygen and ultraviolet spectrophotometry was performed to measure its concentration precisely. Then, oxygen–ozone gas mixture of various concentrations (20, 40, 60 mg/mL of gas per ml of complete medium (CM)) with a predetermined volume was collected with a polypropylene, silicone-coated, disposable syringe and then, via a ‘‘Y’’ connector, immediately introduced into a second one. These syringes contained CM with the same volume. The final gas pressure remained at normal atmospheric pressure. As ozone is a very reactive gas, it is extremely important that the handling must be rapid and precise in order to obtain reproducible results. Samples were mixed with the gas gently and continuously for 20 min. Control group was also added CM untreated. All the cells were divided to six groups (n ¼ 7). They were first cultured in 2-mL serum-free medium for 24 h in order to be synchronized and after receiving the following treatment, all the groups were added with 2-mL CM for 24 h in normal condition: Control group (Group C) was only washed twice by D-Hank’s fluid. H/R group (Group H/R) was washed twice and then subjected to the H/R process described above. CM group was first washed by D-Hank’s and then added with 1-mL CM without intervention for 2 h. After being washed again, it was subjected to the H/R process. Ozone 20 preconditioning group (Group O-20), ozone 40 preconditioning group (Group O-40) and ozone 60 preconditioning group (Group O-60) were also washed and then added with 1-mL CM after action of ozone with various concentrations of 20, 40, 60 mg/mL, respectively for 2 h. Then these three groups were subjected to the H/R process. Apoptosis assay A total of 5 105 NRK-52E cells were cultured on a six-well plate and processed in accordance with the above grouping. After 24 h reoxygenation, the cells in all groups were collected and stained using FITC-conjugated annexin V and propidium iodide (KeyGEN, NanJing, China) according to its
Ren Fail, 2014; 36(9): 1449–1454
instructions, and the testing completed within 1 h after being dyed. Then, we performed flow cytometry (FCM) for apoptosis analysis. Hoechst 33258 staining In order to distinguish apoptotic cells from necrotic cells, the cells were stained with Hoechst 33258. After 24 h re-oxygenation, all cells in every group were washed twice by PBS, and kept in 0.5 mL of 4% paraformaldehyde for 10 min or longer. After being washed twice with PBS again, the cells were stained with Hoechst 33258 for 5 min at room temperature. Then, the cells were washed and examined by fluorescence microscopy (Olympus BX-53F, Tokyo, Japan). Measurement of SOD, MDA and LDH leaking ratio in the cells Superoxide dismutase (SOD) activity was measured by the xanthinoxidase method. Malondialdehyde (MDA) (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) concentration was measured by the thiobarbituric acid (TBA) method. Lactate dehydrogenase (LDH) leakage ratio was measured by dinitro-phenyl-hydrazine (DPNH) method, %. All procedures above were performed according to directions of the commercial kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Immunocytochemistry staining NRK-52E cells were grown on poly-L-lysine-coated slides in the six-well plates. After the above treatments, the slides were washed twice with PBS and fixed in 4% paraformaldehyde for 30 min at the room temperature. Immunocytochemistry was carried out using Envision methods. The working concentration of cleaved caspase-3 was 1:1000. And the cells were incubated with these antibodies at 4 C overnight. After washing three times with PBS, the cells were incubated with a secondary antibody for 50 min at room temperature, followed by color reagent DAB. Assays were conducted according to the manufacturers’ instructions. Western blot analysis Besides control group, the cells in Group H/R and Group O-40, were cultivated in reoxygenation condition for 3, 6, 12 and 24 h, respectively. Total protein of NRK-52E cells were extracted, and quantified using Bicinchoninic acid method. Then, equivalent weights of protein (40 mg/Lane) was separated on 10% SDS-PAGE gels and then transferred to nitrocellulose membrane. The membranes were blocked with 5% non-fat milk in TBST buffer (10 mmol/L Tris-HCl, 0.15 mol/L NaCl, and 0.05% Tween 20, pH 7.2) and then incubated with the following rabbit polyclonal primary antibodies: poly ADP ribose polymerase (PARP)-1 (1:1000 dilution; SantaCruz Biotechnology, Santa Cruz, CA), Bax (1:1000 dilution; Cell Signaling Technology, Boston, MA), Bcl-2(1:1000 dilution; Cell Signaling Technology, Boston, MA). Subsequently, after being washed twice with PBS, the membranes were incubated with secondary antibody conjugated with horseradish peroxidase at 1:2000 dilution. Specific bands were visualized by using an enhanced chemiluminescence detection kit.
DOI: 10.3109/0886022X.2014.950934
Statistical analysis Data were presented as mean ± SEM. The means of the different groups were compared using one-way ANOVA 3 Student–Newman–Keuls test. Differences were considered statistically significant when p50.05.
Results Analysis of NRK-52E apoptosis
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As shown in Figure 1, the apoptosis rate in Group H/R was the highest. Compared with Group O-40, the apoptosis rate in Group O-20 and Group O-60 was significantly higher and there was no obvious difference between Group C and Group CM. As the apoptosis rate of Group O-40 was obviously lower than other ozone preconditioning groups, so in the following experiments, we treated Group O-40 as the only preconditioning group.
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shrinkage chromatin condensation is visualized. However, the bright blue emission in Group O-40 was obviously attenuated. SOD, MDA and LDH analysis As shown in Figure 3, MDA content and LDH leaking ratio in Group H/R was significantly higher, whereas this increase induced by H/R injury was significantly attenuated by treatment with OzoneOP in Group O-40. SOD decreased obviously in Group H/R compared with that in Group C, but OzoneOP inhibited the decrease of SOD induced by H/R injury. Immunocytochemistry staining As shown in Figure 2(D–F), the process of H/R injury upregulated expression of cleaved caspase-3. But there was a significant decrease in the number of cleaved caspase3-positive in Group O-40.
Hoechst 33258 staining As shown in Figure 2(A–C), in Group C, the cell nuclei fluoresced faint blue which was homogenous. But the Group H/R had the obvious apoptotic features, as the nuclear
Western blot As shown in Figure 4, PARP-1 and Bax were upregulated in Group H/R and Group O-40 when compared with Group C.
Figure 1. Effect of ozone OP on apoptosis of NRK-52E cells subjected to H/R injury. The apoptosis was examined using annexin V-FITC/PI staining and flow cytometry analysis. The experiment was repeated three times and representative data are shown.
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Figure 2. Staining with Hoechst 33258 and immunocytochemistry. (A and D) cells from control group, (B and E) cells from H/R group, (C and F) cells from ozone 40 preconditioning group. (A–C) Hoechst 33258 staining. (D–F) cleaved caspase-3 immunohistochemical staining. All Hoechst 33258 and immunohistochemical staining, original magnification 200.
Figure 3. (A) Assay of SOD activity in NRK-52E cells of different groups. (B) Assay of MDA content in NRK-52E cells of different groups. (C) Assay of LDH leaking ratio in NRK-52E cells of different groups. Bars represent means ± SEM (n ¼ 4); *p50.05 versus Group C, #p50.05 versus Group H/R.
But treatment with ozone could attenuate these expression induced by H/R. With the preconditioning time extended, the expression of PARP-1 and Bax obviously increased in Group H/R and decreased in Group O-40. Compared with Group C, Bcl-2 was down regulated in cells subjected to H/R, but in Group O-40, the expression was obviously more than that observed in Group H/R.
Discussion The highlights of the study are that we implemented an in vitro OzoneOP model with NRK-52E cells subjected to H/R injury. We detected different concentrations of ozone on NRK-52E cells subjected to H/R injury and found that OzoneOP could protect kidney cells through reduction of oxidative stress and apoptosis. During the last decade, there are some researchers who have been engaged in investigating the possible mechanisms
of action triggered by ozone, which is briefly mixed with human serum.12 Just like oxygen, ozone dissolves in the human serum and reacts rapidly with many substrates,13 generating with an array of products such as H2O2, malondialdehyde and so on. They act as cellular signals and trigger a variety of biological effects. With different ozone concentrations, it must be admitted that these compounds seem to behave as a double-edged sword. Because of it, we have been enthusiastic in exploring the biological active dose in terms of ozone concentration, which ranges between 20 and 60 mg/mL gas per ml of CM. As shown in Figure 1, the result indicated that OzoneOP can reduce apoptosis significantly in comparison with Group H/R and the apoptosis rate of Group O-40 was obviously lower than Group O-20 and Group O-60. So in the following experiments, we treated Group O-40 as the only preconditioning group. Oxidative stress is characterized by increased production of reactive oxygen species (ROS) and plays a key role in
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DOI: 10.3109/0886022X.2014.950934
Ozone protects kidney cells against H/R injury
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Figure 4. Representative Western blots showed the effects of ozone on PARP-1, Bcl-2 and Bax expression in NRK-52E cells of different groups. (A) Representative Western blots showing the effects of ozone preconditioning on PARP-1, Bcl-2 and Bax expression. (B) Relative band densities of PARP1 to the mean value of the control. (C) Relative band densities of Bcl-2 to the mean value of the control. (D) Relative band densities of Bax to the mean value of the control (means ± SEM; *p50.05 vs. Group C; #p50.05 vs. Group H/R 3 h; p50.05 vs. Group H/R 6 h; gp50.05 vs. Group H/R 12 h; ?p50.05 vs. Group H/R 24 h).
mediating pathologic responses in many renal diseases. Normally, the generated ROS from metabolic processes can be scavenged by endogenous anti-oxidant enzymes such as SOD,14 catalyzing the dismutation of the superoxide radical to hydrogen peroxide. However, in pathological conditions just like I/R injury, the overproduction of ROS can cause oxidation of lipids, proteins, and nucleic acids, resulting in cellular dysfunction.15 In addition, the content of MDA reflects the extent of cell injury by oxidative stress and LDH leaking ratio is the reflection of cell membrane permeability. In animal models, it had been already demonstrated that ozone therapy could reduce oxidative stress in I/R injury of different organs, such as heart, liver and kidney.16–18 And our in vitro results were in accordance with those of in vivo. We found that kidney cells subjected to H/R caused an elevation of oxidative stress accompanied by increased MDA production and LDH leaking ratio, and reduction in SOD activities. However, these changes could be inhibited by the treatment with ozone. These findings suggest that overproduction of ROS plays a critical role in the kidney cell injury induced by H/R and ozone might offer renal protection through antioxidant properties. Cellular apoptosis is an important outcome of I/R.19 In order to investigate the possible mechanism by which ozone protects NRK-52E cells against apoptosis, we used ozonized CM to mimic OzoneOP. The proteins of Bcl-2 family, which
are crucial regulatory factors, could either promote cell survival such as Bcl-2, or cell death such as Bax by apoptosis. The ratio of Bcl-2/Bax is a pivotal factor that determines whether or not apoptosis can happen to cells exposed to many injuries.20 Studies have shown that the caspase family could promote and implement cell apoptosis in mammalian cells and caspase-3 is the most crucial downstream apoptosis protease in the caspase cascade ‘‘waterfall’’.21 In response to oxidative load in the mitochondria, the outer membrane of mitochondria becomes permeabilized4, resulting in the decreased ratio of Bcl-2/Bax, which promoted the activation of caspase-3. Also, the activated caspase-3 can hydrolyze PARP, which is a kind of protease with catalytic activity. Our in vitro results correlated with the results of an in vivo study. It certified that OzoneOP has a protective effect on the NRK52E cells subjected to H/R injury. Western blot and immunocytochemistry staining indicated that the expressions of Bax, PARP-1 and cleaved caspase-3 were upregulated in Group H/R when compared with Group C, but ozone treatment could attenuate these expression induced by H/R. These findings suggest that ozone could protect kidney cells against apoptosis induced by H/R. In conclusion, the present study provides definite evidence for the first time that ozone oxidative preconditioning exerts a significant renal protective effect against H/R injury by attenuating oxidant stress and apoptosis in kidney cells.
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Declaration of interest This study was supported by the National Natural Science Foundation of China (Grant No. 30901494) and the Province Natural Science Foundation of Hubei (Grant No. 2013CFB226).
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References 1. Yun Y, Duan WG, Chen P, et al. Ischemic postconditioning modified renal oxidative stress and lipid peroxidation caused by ischemic reperfusion injury in rats. Transplant Proc. 2009;41: 3597–3602. 2. Barri YM, Sanchez EQ, Jennings LW, et al. Acute kidney injury following liver transplantation: Definition and outcome. Liver Transpl. 2009;15:475–483. 3. Martinez-Sanchez G, Al-Dalain SM, Menendez S, et al. Therapeutic efficacy of ozone in patients with diabetic foot. Eur J Pharmacol. 2005;523:151–161. 4. Chen H, Xing B, Liu X, et al. Ozone oxidative preconditioning inhibits inflammation and apoptosis in a rat model of renal ischemia/reperfusion injury. Eur J Pharmacol. 2008;581:306–314. 5. Leon OS, Menendez S, Merino N, et al. Ozone oxidative preconditioning: A protection against cellular damage by free radicals. Mediators Inflamm. 1998;7:289–294. 6. Peralta C, Xaus C, Bartrons R, et al. Effect of ozone treatment on reactive oxygen species and adenosine production during hepatic ischemia-reperfusion. Free Radic Res. 2000;33:595–605. 7. Valacchi G, Bocci V. Studies on the biological effects of ozone: 11. Release of factors from human endothelial cells. Mediators Inflamm. 2000;9:271–276. 8. Zhou NB, Fu ZJ, Sun T. Effects of different concentrations of oxygen-ozone on rats’ astrocytes in vitro. Neurosci Lett. 2008;441: 178–182. 9. Zang X, Zheng F, Hong HJ, et al. Neutrophil gelatinase-associated lipocalin protects renal tubular epithelial cells in hypoxia-reperfusion by reducing apoptosis. Int Urol Nephrol. 2014;46:1673–1679. 10. Russ AL, Haberstroh KM, Rundell AE. Experimental strategies to improve in vitro models of renal ischemia. Exp Mol Pathol. 2007; 83:143–159.
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11. Sauvant C, Schneider R, Holzinger H, et al. Implementation of an in vitro model system for investigation of reperfusion damage after renal ischemia. Cell Physiol Biochem. 2009;24: 567–576. 12. Bocci V. Biological and clinical effects of ozone. Has ozone therapy a future in medicine? Br J Biomed Sci. 1999;56: 270–279. 13. Pryor WA. Mechanisms of radical formation from reactions of ozone with target molecules in the lung. Free Radic Biol Med. 1994;17:451–465. 14. Meng FJ, Hou ZW, Li Y, et al. The protective effect of picroside II against hypoxia/reoxygenation injury in neonatal rat cardiomyocytes. Pharm Biol. 2012;50:1226–1232. 15. Ferrari R, Ceconi C, Curello S, et al. The occurrence of oxidative stress during reperfusion in experimental animals and men. Cardiovasc Drugs Ther. 1991;5(suppl 2):277–287. 16. Peralta C, Leon OS, Xaus C, et al. Protective effect of ozone treatment on the injury associated with hepatic ischemia-reperfusion: Antioxidant–pro-oxidant balance. Free Radic Res. 1999;31: 191–196. 17. Ahmed LA, Salem HA, Mawsouf MN, et al. Cardioprotective effects of ozone oxidative preconditioning in an in vivo model of ischemia/reperfusion injury in rats. Scand J Clin Lab Invest. 2012; 72:345–354. 18. Oztosun M, Akgul EO, Cakir E, et al. The effects of medical ozone therapy on renal ischemia/reperfusion injury. Ren Fail. 2012;34: 921–925. 19. Zhang ZX, Shek K, Wang S, et al. Osteopontin expressed in tubular epithelial cells regulates NK cell-mediated kidney ischemia reperfusion injury. J Immunol. 2010;185:967–973. 20. Liang H, Yu F, Tong Z, et al. Effect of ischemia post-conditioning on skeletal muscle oxidative injury, mTOR, Bax, Bcl-2 proteins expression, and HIF-1alpha/beta-actin mRNA, IL-6/beta-actin mRNA and caveolin-3/beta-actin mRNA expression in ischemiareperfusion rabbits. Mol Biol Rep. 2013;40:507–514. 21. Prabhakar G, Vona-Davis L, Murray D, et al. Phosphocreatine restores high-energy phosphates in ischemic myocardium: Implication for off-pump cardiac revascularization. J Am Coll Surg. 2003;197:786–791.
LABORATORY STUDY
The protective effect of ozone oxidative preconditioning against hypoxia/reoxygenation injury in rat kidney cells Lei Wang, Hui Chen, Xiu-Heng Liu, Zhi-Yuan Chen, Xiao-Dong Weng, Tao Qiu, and Lin Liu
Ren Fail Downloaded from informahealthcare.com by University of Newcastle on 09/26/14 For personal use only.
Department of Urology, Renmin Hospital of Wuhan University, Wuhan, Hubei Province, P.R. China
Abstract
Keywords
Ozone (O3) has been viewed as a novel treatment for different diseases in these years and oxidative stress and apoptosis play a key role in the pathogenesis of kidney diseases including renal ischemia and reperfusion (I/R). In the present study, we investigated the role of ozone oxidative preconditioning (OzoneOP) in attenuating oxidative stress and apoptosis in a hypoxia/reoxygenation (H/R) injury model using rat kidney cells. We induced H/R injury in kidney cells treated with or without OzoneOP. Oxidative stress parameters such as superoxide dismutase (SOD), malondialdehyde (MDA) and lactate dehydrogenase (LDH) were determined, as well as some apoptotic proteins. We observed that oxidative stress and apoptosis were increased in H/R group compared to OzoneOP group; however, these changes were significantly decreased by the treatment with OzoneOP. We concluded that OzoneOP can protect the kidney cells against H/R injury and its mechanism may be through the reduction of oxidative stress and apoptosis.
Apoptosis, hypoxia and reoxygenation, oxidative stress, ozone oxidative preconditioning, rat
Introduction In recent years, ischemia and reperfusion (I/R) injury has elicited an increasing interest of scholars in its impact on such organs as liver, heart and kidney. Renal ischemia is one of the main causes of acute kidney injury (AKI) and often occurs in such surgeries as kidney transplantation, partial nephrectomy, renal artery angioplasty, accidental or iatrogenic trauma, hydronephrosis and elective urological operations.1,2 Oxidative stress and apoptosis play a key role in the pathogenesis of many diseases and the dysregulation of them can lead to a variety of kidney diseases including AKI. As the role of oxidative stress and apoptosis in kidney diseases becoming increasingly clear, therefore, it is essential to develop a therapeutic treatment to block or prevent inappropriate apoptosis and oxidative stress in kidney diseases. Ozone (O3), a reactive gas, has been viewed as a therapeutical material for the treatment of many diseases.3 In recent studies, it has been demonstrated that ozone has the protective effects of anti-apoptosis on renal I/R injury of animal models4, through rectal insufflations. It provides a novel treatment to protect organ from I/R injury and is relatively simple and harmless. It has also been demonstrated that ozone improves the ability of organs subjected to I/R and adapts the organs to slight and transient oxidative stresses,
History Received 14 April 2014 Revised 3 July 2014 Accepted 30 July 2014 Published online 23 September 2014
resulting in the improvement of the anti-oxidant endogenous systems.5,6 It is called OzoneOP, which is brought about by stimulating the endogenous anti-oxidant mechanisms to modulate the cells’ redox state. The majority of the studies of OzoneOP on renal I/R injury are based on animal models, including rats.4 On account of the limitation of many factors, it is difficult to investigate direct effects of renal OzoneOP with these models. In order to eliminate these confounding variables, we seek to develop an in vitro Hypoxia/Reoxygenation (H/R) model of simulating OzoneOP using normal rat kidney epithelial (NRK-52E) cells based on the former study,7,8 and these cells had already been used to investigate H/R injury in previous study.9 Indeed, in vitro methodologies outweigh in vivo experimentation in several aspects. Its main advantage is that the complexity of an in vivo model can be eliminated. It can test the direct effects of the specific stimuli which can only be isolated and controlled in vitro.10 And these advantages can further motivate new in vivo researches in turn and provide deeper insight into the biological phenomena of OzoneOP. This is the unique innovation in our study that implements an in vitro OzoneOP model with NRK-52E cells subjected to H/R injury, which has not been previously described.
Materials and methods Cell culture Address correspondence to Professor Xiu-Heng Liu, Department of Urology, Renmin Hospital of Wuhan University, No. 238 Jiefang Road, Wuhan 430060, Hubei Province, P.R. China. E-mail: [email protected]
The renal tubular epithelial cell line, NRK-52E cells, was obtained from the Cell Bank of Chinese Academy of Sciences (Shanghai, China). The cells were cultured in DMEM
1450
L. Wang et al.
containing 10% FBS and cultured in a chamber at 37 C with 95% air/5% CO2. H/R model
Ren Fail Downloaded from informahealthcare.com by University of Newcastle on 09/26/14 For personal use only.
The H/R injury protocol was performed as previously described.11 Briefly, prior to the experiments, all cells in every group were cultured in 2-mL serum-free medium for 24 h and then seeded on six-well plates for the subsequent treatments. Then, cells were exposed to a bicarbonate-MES buffered Ringer (1 mL per well) at pH 6.6 without glucose in hypoxic (O2 fraction below 1%) condition for 3 h and then cultivated under normal condition (95% air/5% CO2) with fresh oxygenated culture medium (2 mL per well) for 24 h. A hermetic chamber, filled with gas consisting of 95% N2 and 5% CO2, was used to preserve the cells subjected to hypoxic condition. OzoneOP protocols and experimental groups The oxygen–ozone was delivered to biological samples using the method as previously described.17 Ozone generator (YKS1000-type, Zhuhai Yike Medical Co., Ltd, Zhuhai, China) was used to generate oxygen–ozone with pure gaseous oxygen and ultraviolet spectrophotometry was performed to measure its concentration precisely. Then, oxygen–ozone gas mixture of various concentrations (20, 40, 60 mg/mL of gas per ml of complete medium (CM)) with a predetermined volume was collected with a polypropylene, silicone-coated, disposable syringe and then, via a ‘‘Y’’ connector, immediately introduced into a second one. These syringes contained CM with the same volume. The final gas pressure remained at normal atmospheric pressure. As ozone is a very reactive gas, it is extremely important that the handling must be rapid and precise in order to obtain reproducible results. Samples were mixed with the gas gently and continuously for 20 min. Control group was also added CM untreated. All the cells were divided to six groups (n ¼ 7). They were first cultured in 2-mL serum-free medium for 24 h in order to be synchronized and after receiving the following treatment, all the groups were added with 2-mL CM for 24 h in normal condition: Control group (Group C) was only washed twice by D-Hank’s fluid. H/R group (Group H/R) was washed twice and then subjected to the H/R process described above. CM group was first washed by D-Hank’s and then added with 1-mL CM without intervention for 2 h. After being washed again, it was subjected to the H/R process. Ozone 20 preconditioning group (Group O-20), ozone 40 preconditioning group (Group O-40) and ozone 60 preconditioning group (Group O-60) were also washed and then added with 1-mL CM after action of ozone with various concentrations of 20, 40, 60 mg/mL, respectively for 2 h. Then these three groups were subjected to the H/R process. Apoptosis assay A total of 5 105 NRK-52E cells were cultured on a six-well plate and processed in accordance with the above grouping. After 24 h reoxygenation, the cells in all groups were collected and stained using FITC-conjugated annexin V and propidium iodide (KeyGEN, NanJing, China) according to its
Ren Fail, 2014; 36(9): 1449–1454
instructions, and the testing completed within 1 h after being dyed. Then, we performed flow cytometry (FCM) for apoptosis analysis. Hoechst 33258 staining In order to distinguish apoptotic cells from necrotic cells, the cells were stained with Hoechst 33258. After 24 h re-oxygenation, all cells in every group were washed twice by PBS, and kept in 0.5 mL of 4% paraformaldehyde for 10 min or longer. After being washed twice with PBS again, the cells were stained with Hoechst 33258 for 5 min at room temperature. Then, the cells were washed and examined by fluorescence microscopy (Olympus BX-53F, Tokyo, Japan). Measurement of SOD, MDA and LDH leaking ratio in the cells Superoxide dismutase (SOD) activity was measured by the xanthinoxidase method. Malondialdehyde (MDA) (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) concentration was measured by the thiobarbituric acid (TBA) method. Lactate dehydrogenase (LDH) leakage ratio was measured by dinitro-phenyl-hydrazine (DPNH) method, %. All procedures above were performed according to directions of the commercial kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Immunocytochemistry staining NRK-52E cells were grown on poly-L-lysine-coated slides in the six-well plates. After the above treatments, the slides were washed twice with PBS and fixed in 4% paraformaldehyde for 30 min at the room temperature. Immunocytochemistry was carried out using Envision methods. The working concentration of cleaved caspase-3 was 1:1000. And the cells were incubated with these antibodies at 4 C overnight. After washing three times with PBS, the cells were incubated with a secondary antibody for 50 min at room temperature, followed by color reagent DAB. Assays were conducted according to the manufacturers’ instructions. Western blot analysis Besides control group, the cells in Group H/R and Group O-40, were cultivated in reoxygenation condition for 3, 6, 12 and 24 h, respectively. Total protein of NRK-52E cells were extracted, and quantified using Bicinchoninic acid method. Then, equivalent weights of protein (40 mg/Lane) was separated on 10% SDS-PAGE gels and then transferred to nitrocellulose membrane. The membranes were blocked with 5% non-fat milk in TBST buffer (10 mmol/L Tris-HCl, 0.15 mol/L NaCl, and 0.05% Tween 20, pH 7.2) and then incubated with the following rabbit polyclonal primary antibodies: poly ADP ribose polymerase (PARP)-1 (1:1000 dilution; SantaCruz Biotechnology, Santa Cruz, CA), Bax (1:1000 dilution; Cell Signaling Technology, Boston, MA), Bcl-2(1:1000 dilution; Cell Signaling Technology, Boston, MA). Subsequently, after being washed twice with PBS, the membranes were incubated with secondary antibody conjugated with horseradish peroxidase at 1:2000 dilution. Specific bands were visualized by using an enhanced chemiluminescence detection kit.
DOI: 10.3109/0886022X.2014.950934
Statistical analysis Data were presented as mean ± SEM. The means of the different groups were compared using one-way ANOVA 3 Student–Newman–Keuls test. Differences were considered statistically significant when p50.05.
Results Analysis of NRK-52E apoptosis
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As shown in Figure 1, the apoptosis rate in Group H/R was the highest. Compared with Group O-40, the apoptosis rate in Group O-20 and Group O-60 was significantly higher and there was no obvious difference between Group C and Group CM. As the apoptosis rate of Group O-40 was obviously lower than other ozone preconditioning groups, so in the following experiments, we treated Group O-40 as the only preconditioning group.
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shrinkage chromatin condensation is visualized. However, the bright blue emission in Group O-40 was obviously attenuated. SOD, MDA and LDH analysis As shown in Figure 3, MDA content and LDH leaking ratio in Group H/R was significantly higher, whereas this increase induced by H/R injury was significantly attenuated by treatment with OzoneOP in Group O-40. SOD decreased obviously in Group H/R compared with that in Group C, but OzoneOP inhibited the decrease of SOD induced by H/R injury. Immunocytochemistry staining As shown in Figure 2(D–F), the process of H/R injury upregulated expression of cleaved caspase-3. But there was a significant decrease in the number of cleaved caspase3-positive in Group O-40.
Hoechst 33258 staining As shown in Figure 2(A–C), in Group C, the cell nuclei fluoresced faint blue which was homogenous. But the Group H/R had the obvious apoptotic features, as the nuclear
Western blot As shown in Figure 4, PARP-1 and Bax were upregulated in Group H/R and Group O-40 when compared with Group C.
Figure 1. Effect of ozone OP on apoptosis of NRK-52E cells subjected to H/R injury. The apoptosis was examined using annexin V-FITC/PI staining and flow cytometry analysis. The experiment was repeated three times and representative data are shown.
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Figure 2. Staining with Hoechst 33258 and immunocytochemistry. (A and D) cells from control group, (B and E) cells from H/R group, (C and F) cells from ozone 40 preconditioning group. (A–C) Hoechst 33258 staining. (D–F) cleaved caspase-3 immunohistochemical staining. All Hoechst 33258 and immunohistochemical staining, original magnification 200.
Figure 3. (A) Assay of SOD activity in NRK-52E cells of different groups. (B) Assay of MDA content in NRK-52E cells of different groups. (C) Assay of LDH leaking ratio in NRK-52E cells of different groups. Bars represent means ± SEM (n ¼ 4); *p50.05 versus Group C, #p50.05 versus Group H/R.
But treatment with ozone could attenuate these expression induced by H/R. With the preconditioning time extended, the expression of PARP-1 and Bax obviously increased in Group H/R and decreased in Group O-40. Compared with Group C, Bcl-2 was down regulated in cells subjected to H/R, but in Group O-40, the expression was obviously more than that observed in Group H/R.
Discussion The highlights of the study are that we implemented an in vitro OzoneOP model with NRK-52E cells subjected to H/R injury. We detected different concentrations of ozone on NRK-52E cells subjected to H/R injury and found that OzoneOP could protect kidney cells through reduction of oxidative stress and apoptosis. During the last decade, there are some researchers who have been engaged in investigating the possible mechanisms
of action triggered by ozone, which is briefly mixed with human serum.12 Just like oxygen, ozone dissolves in the human serum and reacts rapidly with many substrates,13 generating with an array of products such as H2O2, malondialdehyde and so on. They act as cellular signals and trigger a variety of biological effects. With different ozone concentrations, it must be admitted that these compounds seem to behave as a double-edged sword. Because of it, we have been enthusiastic in exploring the biological active dose in terms of ozone concentration, which ranges between 20 and 60 mg/mL gas per ml of CM. As shown in Figure 1, the result indicated that OzoneOP can reduce apoptosis significantly in comparison with Group H/R and the apoptosis rate of Group O-40 was obviously lower than Group O-20 and Group O-60. So in the following experiments, we treated Group O-40 as the only preconditioning group. Oxidative stress is characterized by increased production of reactive oxygen species (ROS) and plays a key role in
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DOI: 10.3109/0886022X.2014.950934
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Figure 4. Representative Western blots showed the effects of ozone on PARP-1, Bcl-2 and Bax expression in NRK-52E cells of different groups. (A) Representative Western blots showing the effects of ozone preconditioning on PARP-1, Bcl-2 and Bax expression. (B) Relative band densities of PARP1 to the mean value of the control. (C) Relative band densities of Bcl-2 to the mean value of the control. (D) Relative band densities of Bax to the mean value of the control (means ± SEM; *p50.05 vs. Group C; #p50.05 vs. Group H/R 3 h; p50.05 vs. Group H/R 6 h; gp50.05 vs. Group H/R 12 h; ?p50.05 vs. Group H/R 24 h).
mediating pathologic responses in many renal diseases. Normally, the generated ROS from metabolic processes can be scavenged by endogenous anti-oxidant enzymes such as SOD,14 catalyzing the dismutation of the superoxide radical to hydrogen peroxide. However, in pathological conditions just like I/R injury, the overproduction of ROS can cause oxidation of lipids, proteins, and nucleic acids, resulting in cellular dysfunction.15 In addition, the content of MDA reflects the extent of cell injury by oxidative stress and LDH leaking ratio is the reflection of cell membrane permeability. In animal models, it had been already demonstrated that ozone therapy could reduce oxidative stress in I/R injury of different organs, such as heart, liver and kidney.16–18 And our in vitro results were in accordance with those of in vivo. We found that kidney cells subjected to H/R caused an elevation of oxidative stress accompanied by increased MDA production and LDH leaking ratio, and reduction in SOD activities. However, these changes could be inhibited by the treatment with ozone. These findings suggest that overproduction of ROS plays a critical role in the kidney cell injury induced by H/R and ozone might offer renal protection through antioxidant properties. Cellular apoptosis is an important outcome of I/R.19 In order to investigate the possible mechanism by which ozone protects NRK-52E cells against apoptosis, we used ozonized CM to mimic OzoneOP. The proteins of Bcl-2 family, which
are crucial regulatory factors, could either promote cell survival such as Bcl-2, or cell death such as Bax by apoptosis. The ratio of Bcl-2/Bax is a pivotal factor that determines whether or not apoptosis can happen to cells exposed to many injuries.20 Studies have shown that the caspase family could promote and implement cell apoptosis in mammalian cells and caspase-3 is the most crucial downstream apoptosis protease in the caspase cascade ‘‘waterfall’’.21 In response to oxidative load in the mitochondria, the outer membrane of mitochondria becomes permeabilized4, resulting in the decreased ratio of Bcl-2/Bax, which promoted the activation of caspase-3. Also, the activated caspase-3 can hydrolyze PARP, which is a kind of protease with catalytic activity. Our in vitro results correlated with the results of an in vivo study. It certified that OzoneOP has a protective effect on the NRK52E cells subjected to H/R injury. Western blot and immunocytochemistry staining indicated that the expressions of Bax, PARP-1 and cleaved caspase-3 were upregulated in Group H/R when compared with Group C, but ozone treatment could attenuate these expression induced by H/R. These findings suggest that ozone could protect kidney cells against apoptosis induced by H/R. In conclusion, the present study provides definite evidence for the first time that ozone oxidative preconditioning exerts a significant renal protective effect against H/R injury by attenuating oxidant stress and apoptosis in kidney cells.
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Declaration of interest This study was supported by the National Natural Science Foundation of China (Grant No. 30901494) and the Province Natural Science Foundation of Hubei (Grant No. 2013CFB226).
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