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Fluorofenidone Attenuates Inflammation by Inhibiting the NF-κB Pathway
BASIC INVESTIGATION
Fluorofenidone Attenuates Inflammation by Inhibiting the NF-кB Pathway Ling Huang, MD, Fangfang Zhang, MD, Yiting Tang, MD, Jiao Qin, MD, Yu Peng, MD, Lin Wu, MD, Fang Wang, MD, Qiongjing Yuan, MD, Zhangzhe Peng, MD, Jishi Liu, MD, Jie Meng, MD and Lijian Tao, MD
Abstract: Background: Accumulated evidence indicates that inflammation plays a critical role in the progression of many renal diseases. Fluorofenidone (AKF-PD) has been shown to attenuate renal fibrosis in a number of experimental renal fibrosis models. The aim of this study was to assess the anti-inflammatory effect of AKF-PD. Methods: Human proximal tubule (HK-2) cells were stimulated with tumor necrosis factor (TNF)-a in the presence or absence of AKF-PD. Mouse peritoneal macrophages were incubated with necrotic MES-13 cells in the presence or absence of AKF-PD. The production of pro-inflammatory cytokines and chemokines was measured by enzyme-linked immunosorbent assay, and the activation of Nuclear factor kB (NF-kB) pathway was assessed by Western blot analysis. Results: AKF-PD significantly inhibited TNF-a–induced expression of interleukin-6, monocyte chemoattractant protein-1 and interleukin-8 and nuclear translocation of p65 in HK-2 cells. Addition of AKF-PD also significantly suppressed necrotic cell–induced TNF-a expression and p65 nuclear translocation in mouse peritoneal macrophages. Conclusions: These results demonstrated that AKF-PD exerts anti-inflammatory effect, at least in part, through inhibition of the NF-kB pathway. Key Indexing Terms Fluorofenidone; Inflammation; NF-kB pathway. [Am J Med Sci 2014;348(1):75–80.]
enal fibrosis is the end stage of a variety of chronic kidney diseases.1 Chronic nonresolving tubulointerstitial inflammation, caused by persistent infections, autoimmune responses or tissue injuries, is pivotal for the initiation and progression of renal fibrosis.2 Recruitment of immune cells in the renal interstitium is a prominent feature of many renal diseases. Infiltrated immune cells produce a variety of cytokines, chemokines, and reactive oxygen species, resulting in tissue damage and activation of tubular epithelial cells. These tissue resident cells in turn enhance inflammation by the expression of cytokines and chemokines. Growing body of evidence shows that macrophages and proximal tubular epithelial cells are the major source of inflammatory mediators in kidney diseases.3–5 Therefore, therapeutic interventions that attenuate the expression of inflammatory mediators and facilitate inflammation resolution might prevent fibrosis and improve renal function in chronic kidney diseases.
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From the Division of Nephrology (LH, FZ, YT, JQ, YP, LW, FW, QY, ZP, JL, LT), Department of Internal Medicine, Xiangya Hospital, Central South University; and State Key Laboratory of Medical Genetics of China (LT), Central South University, Changsha, Hunan, China. Submitted March 13, 2013; accepted in revised form July 30, 2013. Ling Huang, MD and Fangfang Zhang, MD, contributed equally. Supported by National Natural Science Foundation of China Grant No. 30873110, National Natural Science Foundation of China Grant No. 81273575, National Natural Science Foundation of China Grant No. 81001467, Natural Science Foundation of Hunan Province, China Grant No. 11JJ22051 and The PhD Programs Foundation of Ministry of Education of China from the Ministry of Education Grant No. 20120162130001. The authors have no financial or other conflicts of interest to disclose. Correspondence: Lijian Tao, MD, Division of Nephropathy, Department of Internal Medicine, Xiangya Hospital, Central South University, Changsha, Hunan, China, 410008 (E-mail: [email protected]).
The American Journal of the Medical Sciences
The NF-kB pathway plays a key role in the expression of proinflammatory cytokines and chemokines during inflammation.6 In mammals, the NF-kB family of transcription factor is comprised of 5 subunits: p65 (Rel A), Rel B, c-Rel, p50 and p52. The transcription factors NF-kB are the homodimers or heterodimers of those subunits. In unstimulated cells, NF-kB locates in the cytoplasm because of its binding to the IkB inhibitory complex, which prevents the nuclear translocation of NF-kB. On stimulation by pathogens, molecules released by damaged cells, or proinflammatory cytokines, IkB kinase is activated and phosphorylates its downstream IkBs, which then undergo rapid ubiquitination, culminating in degradation of IkBs by the proteasome, and subsequent liberation of NF-kB. The free NF-kB dimmers then translocate into the nucleus and induce the expression of proinflammatory cytokines and chemokines.7,8 Because of the essential role of NF-kB in inflammation, pharmacological inhibition of the NF-kB pathway might be beneficial for chronic kidney diseases. We previously found that fluorofenidone (AKF-PD), a novel pyridone agent synthesized by our group, attenuates renal fibrosis in a number of experimental renal fibrosis models.9–11 To investigate the mechanisms by which AKF-PD attenuates renal fibrosis, we assessed the effects of AKF-PD on the production of proinflammatory cytokines and the activation of the NF-kB pathway. Indeed, AKF-PD significantly inhibited the expression of proinflammatory cytokines and the nuclear translocation of p65 in both human proximal tubule (HK-2) cells and mouse macrophages. Thus, this study provides an insight into how AKF-PD inhibits renal fibrosis and sterile inflammation.
MATERIALS AND METHODS Macrophage Culture Peritoneal mouse macrophage: male BALB/c mice purchased from Slac Laboratory Animal (Shanghai, China) were used at 6 to 8 weeks of ages (18–22 g). BALB/c mice were injected intraperitoneally with 2 mL of 3% thioglycollate broth 3 days before euthanization. Cells were cultured in RPMI-1640 supplemented with 10% fetal bovine serum, 1% of penicillin– streptomycin in a humidified incubator with 5% CO2 at 37°C. After incubation for 2 to 4 hours, the cells were washed twice with prewarmed sterile D–Hanks buffered saline to remove nonadherent cells. All animal studies were approved by the Institutional Animal Care and Use Committee of Xiangya School of Medicine, Central South University. HK-2 Cell Culture HK-2 cells were purchased from American Type Culture Collection (Rockville, MD), grown in Dulbecco’s modification of eagle’s medium (DMEM)/F12 supplemented with 10% heatinactivated fetal bovine serum (Invitrogen, Carlsbad, CA) and 1% of antibiotics and cultured at 37°C in a humidified 5% CO2
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incubator. HK-2 cells were made quiescent by serum deprivation for 24 hours before treatment. Enzyme-linked Immunosorbent Assay of Tumor Necrosis Factor-a in Macrophages Culture Macrophages were pretreated with 2 mM AKF-PD for 1 hour and then incubated with necrotic MES-13 cell (a murine mesangial cell line) or lipopolysaccharide (Sigma Chem. Co., St. Louis, MO) for 18 hours of stimulation. After treatment, cells supernatants were collected and stored at 280°C until used for tumor necrosis factor (TNF)-a. The levels of TNF-a released from macrophages were determined using a commercially available enzyme-linked immunosorbent assay (ELISA) kit (Invitrogen, Carlsbad, CA). The levels of TNF-a were calculated with reference to standard curves of purified recombinant TNF-a at various dilutions. ELISA of IL-6, IL-8 and Monocyte Chemoattractant Protein-1 in HK-2 Cells Culture HK-2 cells were respectively pretreated with 2 mM AKF-PD or 1 mM dexamethasone (Sigma, St. Louis, WA) for 1 hour and then exposed to 10 ng/mL TNF-a (Biosource, Camarillo, CA) for 24 hours. Cell supernatants were collected and stored at 280°C until used for cytokine determination. The levels of interleukin-6 (IL-6), IL-8 and monocyte chemoattractant protein-1 (MCP-1) of HK-2 cells were determined using a commercially available ELISA kit (R&D Systems, Minneapolis, MN) and were calculated with reference to standard curves of purified recombinant IL-6, IL-8 and MCP-1 at various dilutions. Nuclear Extract Preparation and Western Blotting Macrophages were pretreated with 2 mM AKF-PD for 24 hours and then incubated with 6 3 106 cells/mL necrotic MES-13 cells for 1 hour stimulation. Macrophages were harvested for IкB-a and p65 determination. HK-2 cells were pretreated with 2 mM AKF-PD for 24 hours or 10 mM BAY11-7082 (Calbiochem, San Diego, CA) for 1 hour and then incubated with 10 ng/ mL TNF-a for 1 hour stimulation. HK-2 cells were harvested for p-IкB-a and p65 determination. The nuclear extract was prepared according to the manufacturer’s instruction using NE-PER Nuclear and Cytoplasmic Extracts Reagents kits (Pierce Biotechnology, Rockford, IL). The protein concentration of each sample was measured with micro-BCA protein assay reagent (Pierce, Rockford, IL). Whole cell extracts (20 mg) or nuclear protein was analyzed by a 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis, electro transferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA) and blocked with a solution of trisbuffered-saline (TBS) containing 5% (w/v) bovine serum albumin and 0.1% Tween-20 (TBST) for 1 hour at room temperature. The membranes were washed with 13 TBST 3 times. Blots were probed overnight at 4°C with primary antibodies: p-IкB-a (1:1000; Cell Signaling, Beverly, MA), IкB-a (1:1000; Cell Signaling, Beverly, MA), NF-kB p65 (1:1000; Cell Signaling, Beverly, MA), anti-Sp1 (1:10,000; Millipore, Billerica, MA), laminA/C (1:1000; BD Transduction Laboratories, Santa Cruz, CA), and b-actin (1:5000; Sigma-Aldrich, St. Louis, MO). b-Actin, anti-Sp1 and laminA/C were used as internal controls for equal protein loading. The blots were washed 3 times with TBST. After washing, they were incubated with Horseradish peroxide(HRP)-conjugated secondary antibodies for 1 hour at room temperature and washed 3 additional times with TBST. The membrane was then visualized using an enhanced chemiluminescent technique according to the manufacturer’s instructions.
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Transfection and Luciferase NF-kB Reporter Assay HK-2 cells were transfected as described previously.12 In brief, HK-2 cells were seeded in fresh media to a final cell density of 6 3 105 cells per well in a 24-well plate for transient transfection. Reporter plasmid pNF-kB-Luc (0.25 mg; generously provided by the Department of Pathophysiology, Xiangya School of Medicine, Central South University, Changsha, Hunan, China) and pRL-SV40 (0.25 mg; Promega, Madison, WI) were transfected into HK-2 cells with 1 mL of lipofectamine 2000 (Invitrogen) for 6 hours of incubation and the medium was then replaced with fresh medium supplemented with 10% serum. pRL-SV40 was used as an internal control. After incubation overnight, HK-2 cells were pretreated with 2 mM AKF-PD for 24 hours and then stimulated with 10 ng/mL TNF-a for 1 hour. After treatment, cells were washed in ice-cold phosphate-buffered saline before lysis in 100 mL of cell lysis buffer (Luciferase Assay Kit; Promega). Luciferase activity was assessed using a luciferase assay substrate (Promega) according to the manufacturer’s instructions. All transfection experiments were performed in triplicate for at least 3 individual experiments with similar results. Statistical Analyses All data are presented as mean 6 standard deviation values. Significant differences (P , 0.05) between groups were evaluated using 1-way analysis of variance with SPSS 16.0 software (SPSS Inc, Chicago, IL). Each experiment was repeated at least 3 times with similar results.
RESULTS AKF-PD Inhibits Necrotic Cell–induced TNF-a Production in Macrophages Sterile inflammatory response, triggered by tissue damage, is a key player in renal fibrosis.2–5,13–16 To investigate whether AKF-PD could inhibit sterile inflammatory response, we examined the effect of AKF-PD on necrotic cell–induced TNF-a production in macrophages. Mouse peritoneal macrophages were incubated with necrotic MES-13 cells, generated by freeze/thaw cycles, with cell density ranged from 1.5 3 106 to 12 3 106 cells/mL for 18 hours. As revealed by ELISA, stimulation of macrophages with necrotic MES-13 cells induces robust TNF-a production in a cell density–dependent manner, with the maximum effect when necrotic cell density is at 6 3 106 cells/mL (Figure 1). Notably, addition of AKF-PD significantly inhibited necrotic cell–induced TNF-a production in mouse macrophages (Figure 2). We also noticed that necrotic cells induce TNF-a production in
FIGURE 1. Necrotic cells induce tumor necrosis factor (TNF)-a production in macrophages. Mouse peritoneal macrophages were stimulated with necrotic cells at the indicated cell density (ranged from 1.5 3 106 to 12 3 106 cells/mL). The TNF-a levels in cell culture medium were measured by enzyme-linked immunosorbent assay. *P , 0.05 versus control group. Volume 348, Number 1, July 2014
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FIGURE 2. Fluorofenidone (AKF-PD) treatment inhibits necrotic cell–induced production of tumor necrosis factor (TNF)-a in mouse peritoneal macrophages. Mouse peritoneal macrophages were stimulated with necrotic cells (6 3 106 cells/mL) or lipopolysaccharide (LPS, 500 ng/mL) in the presence or absence of AKF-PD (2 mM). The TNF-a levels in culture medium were measured by enzyme-linked immunosorbent assay. *P , 0.05 versus control group, #P , 0.05 versus necrotic cell–stimulated group.
macrophages in a similar pattern as lipopolysaccharide (Figure 2). Together, these results indicate that AKF-PD inhibits TNF-a production in macrophages response to necrotic cells. AKF-PD Inhibits Necrotic Cell–induced NF-кB Nuclear Translocation in Macrophages In the unstimulated cells, the inhibitory protein IkB-a binds NF-кB and keeps NF-кB in an inactive state in the cytoplasm. On stimulation, IkB-a is phosphorylated and subsequently degraded, which allows the translocation of NF-кB from the cytoplasm to the nucleus.17,18 To study the mechanisms by which AKF-PD inhibits necrotic cell–induced TNF-a production in macrophages, we next determined whether AKF-PD could suppress TNF-a expression through the inhibition of the NF-кB pathway. First, we assessed whether AKF-PD could prevent necrotic cell–induced degradation of IkB-a. As revealed by Western blot analysis, necrotic cell (6 3 106 cells/mL) stimulation induces significant IkB-a degradation within 1 hour. Surprisingly, addition of AKF-PD failed to inhibit
FIGURE 3. Effect of fluorofenidone (AKF-PD) treatment on the necrotic cell–induced IkB-a degradation in macrophages. Mouse peritoneal macrophages were stimulated with necrotic cells (6 3 106 cells/mL) in the presence or absence of AKF-PD (2 mM). IkB-a and b-actin levels were measured by Western blot. Shown in the upper panel (A) are the representative blots. Shown in the lower panel (B) are mean 6 standard deviation values. IkB-a/b-actin ratio was determined by densitometry. *P , 0.05 versus control group. Ó 2014 Lippincott Williams & Wilkins
necrotic cell–induced degradation of IkB-a (Figure 3), indicating that AKF-PD inhibits necrotic cell–induced TNF-a expression in an IkB-a–independent manner. Next, we sought to investigate whether AKF-PD could inhibit the nuclear translocation of the NF-kB. Necrotic cell (6 3 106 cells/mL) stimulation significantly increased the nuclear accumulation of the NF-kB subunit p65 (Figure 4), which is consistent with TNF-a production (Figure 1). Notably, addition of AKF-PD completely blocked the nuclear accumulation of p65 induced by necrotic cells (Figure 4). Collectively, these observations clearly suggest that AKF-PD inhibits TNF-a production, at least in part, through preventing p65 nuclear translocation in macrophages. Effect of AKF-PD on TNF-a–mediated MCP-1, IL-8 and IL-6 Expression in HK-2 Cells Proximal tubular epithelial cells are important source of cytokines and chemokines in kidney diseases.3–5 Next, we determined whether AKF-PD could inhibit the production of cytokines and chemokines in stimulated proximal tubular epithelial cells. HK-2 cells were stimulated with TNF-a (10 ng/mL) for 24 hours in the presence or absence of AKF-PD (2 mM) or dexamethasone (1 mM). We observed that levels of MCP-1, IL-6 and IL-8 in the cell culture medium were significantly increased by TNF-a stimulation. Importantly, addition of AKF-PD or dexamethasone significantly inhibited the production of MCP-1, IL-6 and IL-8 as shown by ELISA (Figure 5). These results demonstrated that AKF-PD inhibits the production of cytokines and chemokines in activated proximal tubular epithelial cells. AKF-PD Inhibits TNF-a–Induced NF-кB Nuclear Translocation in HK-2 Cells To determine whether AKF-PD could inhibit TNF-a– induced activation of the NF-кB pathway, we first examined the effects of AKF-PD on TNF-a–induced phosphorylation of IкB-a, an upstream activating signal for the NF-кB nuclear translocation. TNF-a stimulation significantly increased the phosphorylation of
FIGURE 4. Effect of fluorofenidone (AKF-PD) treatment on necrotic cell–induced nuclear translocation of the subunits of NF-kB (p65) in macrophages. Mouse peritoneal macrophages were stimulated with necrotic cells (6 3 106 cells/mL) in the presence or absence of AKF-PD (2 mM). Levels of p65 and lamin A/C in the nuclear extracts were determined by Western blot. Shown in the upper panel (A) are the representative blots. Shown in the lower panel (B) are mean 6 standard deviation values. NF-kB (p65)/lamin A/C ratio was determined by densitometry. *P , 0.05 versus control group, #P , 0.05 versus necrotic cell–stimulated group.
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FIGURE 5. Effect of fluorofenidone (AKF-PD) treatment on the productions of chemokines and pro-inflammatory cytokines in tumor necrosis factor (TNF)-a–stimulated HK-2 cells. HK-2 cells were stimulated with TNF-a in the presence or absence of AKF-PD (2 mM) or dexamethasone (1 mM). Levels of MCP-1, interleukin (IL)-8 and IL-6 in the cell culture medium were determined by enzyme-linked immunosorbent assay *P , 0.05 versus control group, #P , 0.05 versus TNF-a–stimulated group.
IкB-a, which was abrogated by addition of NF-кB inhibitor BAY 11-7082 (10 mM) (Figure 6). However, addition of AKF-PD failed to affect the phosphorylation of IкB-a induced by TNF-a (Figure 6). Next, we tested whether AKF-PD could inhibit TNFa–induced NF-кB nuclear translocation in HK-2 cells. Notably, AKF-PD treatment completely blocked the accumulation of p65 in the nucleus induced by TNF-a (Figure 7). These results suggested that AKF-PD inhibits TNF-a–induced MCP-1, IL-8 and IL-6 induction in HK-2 cells, at least in part, through blocking NF-кB nuclear translocation. AKF-PD Inhibits TNF-a–induced NF-кB Activation in HK-2 Cells To investigate whether AKF-PD could inhibit enhanced NF-kB transcriptional activity induced by TNF-a, HK-2 cells were transiently transfected with the pNF-кB-Luc plasmids. These genetically modified HK-2 cells were then stimulated with TNF-a in the presence or absence of AKF-PD (2 mM) or dexa-
FIGURE 6. Effect of fluorofenidone (AKF-PD) treatment on tumor necrosis factor (TNF)-a–induced phosphorylation of IкB-a in HK-2 cells. HK-2 cells were stimulated with TNF-a in the presence or absence of BAY 11-7082 (10 mM) or AKF-PD (2 mM). p-IkB and b-actin levels were measured by Western blot. Shown in the upper panel (A) are the representative blots. Shown in the lower panel (B) are mean 6 standard deviation values. p-IkB-a/b-actin ratio was determined by densitometry. *P , 0.05 versus control group, #P , 0.05 versus TNF-a–stimulated group.
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FIGURE 7. Effect of fluorofenidone (AKF-PD) on tumor necrosis factor (TNF)-a–induced nuclear translocation p65 in HK-2 cells. HK-2 cells were stimulated with TNF-a in the presence or absence of AKF-PD (2 mM). Levels of p65 and Sp1 in the nuclear extracts were determined by Western blot. Shown in the upper panel (A) are the representative blots. Shown in the lower panel (B) are mean 6 standard deviation values. NF-kB (p65)/Sp1 ratio was determined by densitometry. *P , 0.05 versus control group, #P , 0.05 versus TNF-a–stimulated group.
methasone (1 mM) for 24 hours. We observed that TNF-a (10 ng/mL) stimulation significantly enhanced the luciferase activity. Moreover, addition of AKF-PD or dexamethasone significantly reduced the luciferase activity in TNF-a–stimulated cells (Figure 8). These observations further confirm the inhibitory role of AKF-PD in the activation of the NF-kB pathway.
DISCUSSION In the present study, our results demonstrated that treatment with AKF-PD markedly reduced necrotic cell–induced production of proinflammatory cytokines and chemokines, at least in part, through blocking NF-кB nuclear translocation in both peritoneal macrophages and proximal tubular epithelial cells. Together with our previous observation that AKF-PD inhibits TNF-a and IL-1b production during lethal endotoxemia,19 here we propose that AKF-PD is an anti-inflammatory agent. Accumulating evidence implicates an essential role of inflammation in various types of progressive renal diseases and
FIGURE 8. Effect of fluorofenidone (AKF-PD) on NF-kB transcriptional activity in HK-2 cells. HK-2 cells were transiently transfected with the pNF-кB-Luc plasmids. These genetically modified HK-2 cells were then stimulated with TNF-a in the presence or absence of AKF-PD (2 mM) or dexamethasone (1 mM) for 24 hours. The luciferase activity in cell lysates was measured. *P , 0.05 versus control group, #P , 0.05 versus TNF-a–stimulated group. Volume 348, Number 1, July 2014
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renal fibrosis.20–24 TNF-a is a multifunctional acute phase protein, which promotes vascular permeability and initiates the production of cytokines and chemokines.20,21 Increased TNF-a levels are associated with macrophage accumulation and subsequent renal fibrosis.22,23 The infiltration of immune cells in kidney is a key feature of the early stage of renal fibrosis. These immune cells migrate to the site of infection or tissue injury in interstitium of kidney and produce a variety of inflammatory mediators, which in turn trigger the activation of tubular cells. The activated tubular cells are the important source of inflammatory cytokines and chemokines and critically contribute to renal inflammation.20–24 Based on these findings, the results in current study suggest that the anti-inflammatory effect of AKFPD might be an important mechanism by which AKF-PD attenuates renal inflammation and fibrosis. It is important to note that that AKF-PD inhibits necrotic cell–induced production of cytokines and chemokines in both macrophages and proximal tubular epithelial cells. Sterile inflammatory response, triggered by necrotic cells, is a key player in renal fibrosis.2–5,13–16,25–28 Necrotic cells lose membrane integrity and subsequently release damage-associated molecular patterns, which are able to recruit neutrophils and induce inflammation.29 Consistent with early observation,27 we found that necrotic cells stimulate macrophages to secrete TNF-a. Furthermore, AKF-PD treatment significantly inhibits necrotic cell–induced TNF-a production and activation of the NF-kB pathway. NF-kB plays a key role in inflammatory responses and contributes to the development of a wide range of human disorders, such as cancers, neurodegenerative diseases, arthritis, asthma, inflammatory bowel diseases and sepsis.30–33 In the present study, we found that AKF-PD effectively inhibits necrotic cells–induced or TNF-a–induced NF-кB (p65) nuclear translocation. Interestingly, AKF-PD has no effect on the phosphorylation of IkBa induced by either TNF-a or necrotic cells, suggesting that AKF-PD inhibits the NF-кB pathway by blocking NF-кB nuclear translocation rather than inhibition of the phosphorylation of IkB-a, which is the upstream activating signal for the NF-кB nuclear translocation. In conclusion, our findings suggest that AKF-PD exerts beneficial effect in chronic renal diseases, at least in part, because of its anti-inflammatory properties. This study also provides novel sights into how AKF-PD inhibits sterile inflammatory responses in both immune and nonimmune cells. REFERENCES 1. Meguid El, Nahas A, Bello AK. Chronic kidney disease: the global challenge. Lancet 2005;365:331–40. 2. Lopez-Novoa JM, Nieto MA. Inflammation and EMT: an alliance towards organ fibrosis and cancer progression. EMBO Mol Med 2009;1:303–14. 3. Deckers JG, Van Der Woude FJ, Van Der Kooij SW, et al. Synergistic effect of IL-1alpha, IFN-gamma, and TNF-alpha on RANTES production by human renal tubular epithelial cells in vitro. J Am Soc Nephrol 1998;9: 194–202. 4. van Kooten C, Gerritsma JS, Paape ME, et al. Possible role for CD40-CD40L in the regulation of interstitial infiltration in the kidney. Kidney Int 1997;51:711–21. 5. Diamond JR. Macrophages and progressive renal disease in experimental hydronephrosis. Am J Kidney Dis 1995;26:133–40. 6. Li Q, Verma IM. NF-kappaB regulation in the immune system. Nat Rev Immunol 2002;2:725–34. 7. Baeuerle PA, Henkel T. Function and activation of NF-kappa B in the immune system. Annu Rev Immunol 1994;12:141–79.
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8. Yamamoto Y, Gaynor RB. IkappaB kinases: key regulators of the NF-kappaB pathway. Trends Biochem Sci 2004;29:72–9. 9. Wang L, Hu GY, Shen H, et al. Fluorofenidone inhibits TGF-beta1 induced CTGF via MAPK pathways in mouse mesangial cells. Pharmazie 2009;64:680–4. 10. Peng ZZ, Hu GY, Shen H, et al. Fluorofenidone attenuates collagen I and transforming growth factor-beta1 expression through a nicotinamide adenine dinucleotide phosphate oxidase-dependent way in NRK-52E cells. Nephrology (Carlton) 2009;14:565–72. 11. Yuan Q, Wang R, Peng Y, et al. Fluorofenidone attenuates tubulointerstitial fibrosis by inhibiting TGF-beta (1) -induced fibroblast activation. Am J Nephrol 2011;34:181–94. 12. Huang L, Tang Y, Qin J, et al. Vasoactive intestinal peptide enhances TNF-alpha-induced IL-6 and IL-8 synthesis in human proximal renal tubular epithelial cells by NF-kappaB-dependent mechanism. Inflammation 2012;35:1154–60. 13. Chen CJ, Kono H, Golenbock D, et al. Identification of a key pathway required for the sterile inflammatory response triggered by dying cells. Nat Med 2007;13:851–6. 14. Majno G, La Gattuta M, Thompson TE. Cellular death and necrosis: chemical, physical and morphologic changes in rat liver. Virchows Arch Pathol Anat Physiol Klin Med 1960;333:421–65. 15. Iyer SS, Pulskens WP, Sadler JJ, et al. Necrotic cells trigger a sterile inflammatory response through the Nlrp3 inflammasome. Proc Natl Acad Sci U S A 2009;106:20388–93. 16. Raucci A, Palumbo R, Bianchi ME. HMGB1: a signal of necrosis. Autoimmunity 2007;40:285–9. 17. Sosne G, Qiu P, Christopherson PL, et al. Thymosin beta 4 suppression of corneal NFkappaB: a potential anti-inflammatory pathway. Exp Eye Res 2007;84:663–9. 18. Li X, Stark GR. NFkappaB-dependent signaling pathways. Exp Hematol 2002;30:285–96. 19. Tang Y, Li B, Wang N, et al. Fluorofenidone protects mice from lethal endotoxemia through the inhibition of TNF-alpha and IL-1beta release. Int Immunopharmacol 2010;10:580–3. 20. Carrero JJ, Park SH, Axelsson J, et al. Cytokines, atherogenesis, and hypercatabolism in chronic kidney disease: a dreadful triad. Semin Dial 2009;22:381–6. 21. Tonnel AB, Gosset P, Molet S, et al. Interactions between endothelial cells and effector cells in allergic inflammation. Ann N Y Acad Sci 1996;796:9–20. 22. Ma FY, Liu J, Kitching AR, et al. Targeting renal macrophage accumulation via c-fms kinase reduces tubular apoptosis but fails to modify progressive fibrosis in the obstructed rat kidney. Am J Physiol Renal Physiol 2009;296:F177–185. 23. Li ZI, Chung AC, Zhou L, et al. C-reactive protein promotes acute renal inflammation and fibrosis in unilateral ureteral obstructive nephropathy in mice. Lab Invest 2011;91:837–51. 24. Nolasco FE, Cameron JS, Hartley B, et al. Intraglomerular T cells and monocytes in nephritis: study with monoclonal antibodies. Kidney Int 1987;31:1160–6. 25. Luo F, Liu X, Li S, et al. Melatonin promoted chemotaxins expression in lung epithelial cell stimulated with TNF-alpha. Respir Res 2004;5:20. 26. Thorburn E, Kolesar L, Brabcova E, et al. CXC and CC chemokines induced in human renal epithelial cells by inflammatory cytokines. APMIS 2009;117:477–87. 27. Scaffidi P, Misteli T, Bianchi ME. Release of chromatin protein HMGB1 by necrotic cells triggers inflammation. Nature 2002;418: 191–5.
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28. Li M, Carpio DF, Zheng Y, et al. An essential role of the NF-kappa B/Toll-like receptor pathway in induction of inflammatory and tissuerepair gene expression by necrotic cells. J Immunol 2001;166:7128–35. 29. Searle J, Kerr JF, Bishop CJ, et al. Necrosis and apoptosis: distinct modes of cell death with fundamentally different significance. Pathol Annu 1982;17 (Pt 2):229–59. 30. Blackwell TS, Blackwell TR, Holden EP, et al. In vivo antioxidant treatment suppresses nuclear factor-kappa B activation and neutrophilic lung inflammation. J Immunol 1996;157:1630–7.
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31. Pierce JW, Schoenleber R, Jesmok G, et al. Novel inhibitors of cytokine-induced IkappaBalpha phosphorylation and endothelial cell adhesion molecule expression show anti-inflammatory effects in vivo. J Biol Chem 1997;272:21096–103. 32. Lewis AJ, Manning AM. New targets for anti-inflammatory drugs. Curr Opin Chem Biol 1999;3:489–94. 33. Segain JP, de la Bletiere DR, Bourreille A, et al. Butyrate inhibits inflammatory responses through NF kappa B inhibition: implications for Crohn’s disease. Gut 2000;47:397–403.
Volume 348, Number 1, July 2014
Fluorofenidone Attenuates Inflammation by Inhibiting the NF-кB Pathway Ling Huang, MD, Fangfang Zhang, MD, Yiting Tang, MD, Jiao Qin, MD, Yu Peng, MD, Lin Wu, MD, Fang Wang, MD, Qiongjing Yuan, MD, Zhangzhe Peng, MD, Jishi Liu, MD, Jie Meng, MD and Lijian Tao, MD
Abstract: Background: Accumulated evidence indicates that inflammation plays a critical role in the progression of many renal diseases. Fluorofenidone (AKF-PD) has been shown to attenuate renal fibrosis in a number of experimental renal fibrosis models. The aim of this study was to assess the anti-inflammatory effect of AKF-PD. Methods: Human proximal tubule (HK-2) cells were stimulated with tumor necrosis factor (TNF)-a in the presence or absence of AKF-PD. Mouse peritoneal macrophages were incubated with necrotic MES-13 cells in the presence or absence of AKF-PD. The production of pro-inflammatory cytokines and chemokines was measured by enzyme-linked immunosorbent assay, and the activation of Nuclear factor kB (NF-kB) pathway was assessed by Western blot analysis. Results: AKF-PD significantly inhibited TNF-a–induced expression of interleukin-6, monocyte chemoattractant protein-1 and interleukin-8 and nuclear translocation of p65 in HK-2 cells. Addition of AKF-PD also significantly suppressed necrotic cell–induced TNF-a expression and p65 nuclear translocation in mouse peritoneal macrophages. Conclusions: These results demonstrated that AKF-PD exerts anti-inflammatory effect, at least in part, through inhibition of the NF-kB pathway. Key Indexing Terms Fluorofenidone; Inflammation; NF-kB pathway. [Am J Med Sci 2014;348(1):75–80.]
enal fibrosis is the end stage of a variety of chronic kidney diseases.1 Chronic nonresolving tubulointerstitial inflammation, caused by persistent infections, autoimmune responses or tissue injuries, is pivotal for the initiation and progression of renal fibrosis.2 Recruitment of immune cells in the renal interstitium is a prominent feature of many renal diseases. Infiltrated immune cells produce a variety of cytokines, chemokines, and reactive oxygen species, resulting in tissue damage and activation of tubular epithelial cells. These tissue resident cells in turn enhance inflammation by the expression of cytokines and chemokines. Growing body of evidence shows that macrophages and proximal tubular epithelial cells are the major source of inflammatory mediators in kidney diseases.3–5 Therefore, therapeutic interventions that attenuate the expression of inflammatory mediators and facilitate inflammation resolution might prevent fibrosis and improve renal function in chronic kidney diseases.
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From the Division of Nephrology (LH, FZ, YT, JQ, YP, LW, FW, QY, ZP, JL, LT), Department of Internal Medicine, Xiangya Hospital, Central South University; and State Key Laboratory of Medical Genetics of China (LT), Central South University, Changsha, Hunan, China. Submitted March 13, 2013; accepted in revised form July 30, 2013. Ling Huang, MD and Fangfang Zhang, MD, contributed equally. Supported by National Natural Science Foundation of China Grant No. 30873110, National Natural Science Foundation of China Grant No. 81273575, National Natural Science Foundation of China Grant No. 81001467, Natural Science Foundation of Hunan Province, China Grant No. 11JJ22051 and The PhD Programs Foundation of Ministry of Education of China from the Ministry of Education Grant No. 20120162130001. The authors have no financial or other conflicts of interest to disclose. Correspondence: Lijian Tao, MD, Division of Nephropathy, Department of Internal Medicine, Xiangya Hospital, Central South University, Changsha, Hunan, China, 410008 (E-mail: [email protected]).
The American Journal of the Medical Sciences
The NF-kB pathway plays a key role in the expression of proinflammatory cytokines and chemokines during inflammation.6 In mammals, the NF-kB family of transcription factor is comprised of 5 subunits: p65 (Rel A), Rel B, c-Rel, p50 and p52. The transcription factors NF-kB are the homodimers or heterodimers of those subunits. In unstimulated cells, NF-kB locates in the cytoplasm because of its binding to the IkB inhibitory complex, which prevents the nuclear translocation of NF-kB. On stimulation by pathogens, molecules released by damaged cells, or proinflammatory cytokines, IkB kinase is activated and phosphorylates its downstream IkBs, which then undergo rapid ubiquitination, culminating in degradation of IkBs by the proteasome, and subsequent liberation of NF-kB. The free NF-kB dimmers then translocate into the nucleus and induce the expression of proinflammatory cytokines and chemokines.7,8 Because of the essential role of NF-kB in inflammation, pharmacological inhibition of the NF-kB pathway might be beneficial for chronic kidney diseases. We previously found that fluorofenidone (AKF-PD), a novel pyridone agent synthesized by our group, attenuates renal fibrosis in a number of experimental renal fibrosis models.9–11 To investigate the mechanisms by which AKF-PD attenuates renal fibrosis, we assessed the effects of AKF-PD on the production of proinflammatory cytokines and the activation of the NF-kB pathway. Indeed, AKF-PD significantly inhibited the expression of proinflammatory cytokines and the nuclear translocation of p65 in both human proximal tubule (HK-2) cells and mouse macrophages. Thus, this study provides an insight into how AKF-PD inhibits renal fibrosis and sterile inflammation.
MATERIALS AND METHODS Macrophage Culture Peritoneal mouse macrophage: male BALB/c mice purchased from Slac Laboratory Animal (Shanghai, China) were used at 6 to 8 weeks of ages (18–22 g). BALB/c mice were injected intraperitoneally with 2 mL of 3% thioglycollate broth 3 days before euthanization. Cells were cultured in RPMI-1640 supplemented with 10% fetal bovine serum, 1% of penicillin– streptomycin in a humidified incubator with 5% CO2 at 37°C. After incubation for 2 to 4 hours, the cells were washed twice with prewarmed sterile D–Hanks buffered saline to remove nonadherent cells. All animal studies were approved by the Institutional Animal Care and Use Committee of Xiangya School of Medicine, Central South University. HK-2 Cell Culture HK-2 cells were purchased from American Type Culture Collection (Rockville, MD), grown in Dulbecco’s modification of eagle’s medium (DMEM)/F12 supplemented with 10% heatinactivated fetal bovine serum (Invitrogen, Carlsbad, CA) and 1% of antibiotics and cultured at 37°C in a humidified 5% CO2
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incubator. HK-2 cells were made quiescent by serum deprivation for 24 hours before treatment. Enzyme-linked Immunosorbent Assay of Tumor Necrosis Factor-a in Macrophages Culture Macrophages were pretreated with 2 mM AKF-PD for 1 hour and then incubated with necrotic MES-13 cell (a murine mesangial cell line) or lipopolysaccharide (Sigma Chem. Co., St. Louis, MO) for 18 hours of stimulation. After treatment, cells supernatants were collected and stored at 280°C until used for tumor necrosis factor (TNF)-a. The levels of TNF-a released from macrophages were determined using a commercially available enzyme-linked immunosorbent assay (ELISA) kit (Invitrogen, Carlsbad, CA). The levels of TNF-a were calculated with reference to standard curves of purified recombinant TNF-a at various dilutions. ELISA of IL-6, IL-8 and Monocyte Chemoattractant Protein-1 in HK-2 Cells Culture HK-2 cells were respectively pretreated with 2 mM AKF-PD or 1 mM dexamethasone (Sigma, St. Louis, WA) for 1 hour and then exposed to 10 ng/mL TNF-a (Biosource, Camarillo, CA) for 24 hours. Cell supernatants were collected and stored at 280°C until used for cytokine determination. The levels of interleukin-6 (IL-6), IL-8 and monocyte chemoattractant protein-1 (MCP-1) of HK-2 cells were determined using a commercially available ELISA kit (R&D Systems, Minneapolis, MN) and were calculated with reference to standard curves of purified recombinant IL-6, IL-8 and MCP-1 at various dilutions. Nuclear Extract Preparation and Western Blotting Macrophages were pretreated with 2 mM AKF-PD for 24 hours and then incubated with 6 3 106 cells/mL necrotic MES-13 cells for 1 hour stimulation. Macrophages were harvested for IкB-a and p65 determination. HK-2 cells were pretreated with 2 mM AKF-PD for 24 hours or 10 mM BAY11-7082 (Calbiochem, San Diego, CA) for 1 hour and then incubated with 10 ng/ mL TNF-a for 1 hour stimulation. HK-2 cells were harvested for p-IкB-a and p65 determination. The nuclear extract was prepared according to the manufacturer’s instruction using NE-PER Nuclear and Cytoplasmic Extracts Reagents kits (Pierce Biotechnology, Rockford, IL). The protein concentration of each sample was measured with micro-BCA protein assay reagent (Pierce, Rockford, IL). Whole cell extracts (20 mg) or nuclear protein was analyzed by a 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis, electro transferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA) and blocked with a solution of trisbuffered-saline (TBS) containing 5% (w/v) bovine serum albumin and 0.1% Tween-20 (TBST) for 1 hour at room temperature. The membranes were washed with 13 TBST 3 times. Blots were probed overnight at 4°C with primary antibodies: p-IкB-a (1:1000; Cell Signaling, Beverly, MA), IкB-a (1:1000; Cell Signaling, Beverly, MA), NF-kB p65 (1:1000; Cell Signaling, Beverly, MA), anti-Sp1 (1:10,000; Millipore, Billerica, MA), laminA/C (1:1000; BD Transduction Laboratories, Santa Cruz, CA), and b-actin (1:5000; Sigma-Aldrich, St. Louis, MO). b-Actin, anti-Sp1 and laminA/C were used as internal controls for equal protein loading. The blots were washed 3 times with TBST. After washing, they were incubated with Horseradish peroxide(HRP)-conjugated secondary antibodies for 1 hour at room temperature and washed 3 additional times with TBST. The membrane was then visualized using an enhanced chemiluminescent technique according to the manufacturer’s instructions.
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Transfection and Luciferase NF-kB Reporter Assay HK-2 cells were transfected as described previously.12 In brief, HK-2 cells were seeded in fresh media to a final cell density of 6 3 105 cells per well in a 24-well plate for transient transfection. Reporter plasmid pNF-kB-Luc (0.25 mg; generously provided by the Department of Pathophysiology, Xiangya School of Medicine, Central South University, Changsha, Hunan, China) and pRL-SV40 (0.25 mg; Promega, Madison, WI) were transfected into HK-2 cells with 1 mL of lipofectamine 2000 (Invitrogen) for 6 hours of incubation and the medium was then replaced with fresh medium supplemented with 10% serum. pRL-SV40 was used as an internal control. After incubation overnight, HK-2 cells were pretreated with 2 mM AKF-PD for 24 hours and then stimulated with 10 ng/mL TNF-a for 1 hour. After treatment, cells were washed in ice-cold phosphate-buffered saline before lysis in 100 mL of cell lysis buffer (Luciferase Assay Kit; Promega). Luciferase activity was assessed using a luciferase assay substrate (Promega) according to the manufacturer’s instructions. All transfection experiments were performed in triplicate for at least 3 individual experiments with similar results. Statistical Analyses All data are presented as mean 6 standard deviation values. Significant differences (P , 0.05) between groups were evaluated using 1-way analysis of variance with SPSS 16.0 software (SPSS Inc, Chicago, IL). Each experiment was repeated at least 3 times with similar results.
RESULTS AKF-PD Inhibits Necrotic Cell–induced TNF-a Production in Macrophages Sterile inflammatory response, triggered by tissue damage, is a key player in renal fibrosis.2–5,13–16 To investigate whether AKF-PD could inhibit sterile inflammatory response, we examined the effect of AKF-PD on necrotic cell–induced TNF-a production in macrophages. Mouse peritoneal macrophages were incubated with necrotic MES-13 cells, generated by freeze/thaw cycles, with cell density ranged from 1.5 3 106 to 12 3 106 cells/mL for 18 hours. As revealed by ELISA, stimulation of macrophages with necrotic MES-13 cells induces robust TNF-a production in a cell density–dependent manner, with the maximum effect when necrotic cell density is at 6 3 106 cells/mL (Figure 1). Notably, addition of AKF-PD significantly inhibited necrotic cell–induced TNF-a production in mouse macrophages (Figure 2). We also noticed that necrotic cells induce TNF-a production in
FIGURE 1. Necrotic cells induce tumor necrosis factor (TNF)-a production in macrophages. Mouse peritoneal macrophages were stimulated with necrotic cells at the indicated cell density (ranged from 1.5 3 106 to 12 3 106 cells/mL). The TNF-a levels in cell culture medium were measured by enzyme-linked immunosorbent assay. *P , 0.05 versus control group. Volume 348, Number 1, July 2014
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FIGURE 2. Fluorofenidone (AKF-PD) treatment inhibits necrotic cell–induced production of tumor necrosis factor (TNF)-a in mouse peritoneal macrophages. Mouse peritoneal macrophages were stimulated with necrotic cells (6 3 106 cells/mL) or lipopolysaccharide (LPS, 500 ng/mL) in the presence or absence of AKF-PD (2 mM). The TNF-a levels in culture medium were measured by enzyme-linked immunosorbent assay. *P , 0.05 versus control group, #P , 0.05 versus necrotic cell–stimulated group.
macrophages in a similar pattern as lipopolysaccharide (Figure 2). Together, these results indicate that AKF-PD inhibits TNF-a production in macrophages response to necrotic cells. AKF-PD Inhibits Necrotic Cell–induced NF-кB Nuclear Translocation in Macrophages In the unstimulated cells, the inhibitory protein IkB-a binds NF-кB and keeps NF-кB in an inactive state in the cytoplasm. On stimulation, IkB-a is phosphorylated and subsequently degraded, which allows the translocation of NF-кB from the cytoplasm to the nucleus.17,18 To study the mechanisms by which AKF-PD inhibits necrotic cell–induced TNF-a production in macrophages, we next determined whether AKF-PD could suppress TNF-a expression through the inhibition of the NF-кB pathway. First, we assessed whether AKF-PD could prevent necrotic cell–induced degradation of IkB-a. As revealed by Western blot analysis, necrotic cell (6 3 106 cells/mL) stimulation induces significant IkB-a degradation within 1 hour. Surprisingly, addition of AKF-PD failed to inhibit
FIGURE 3. Effect of fluorofenidone (AKF-PD) treatment on the necrotic cell–induced IkB-a degradation in macrophages. Mouse peritoneal macrophages were stimulated with necrotic cells (6 3 106 cells/mL) in the presence or absence of AKF-PD (2 mM). IkB-a and b-actin levels were measured by Western blot. Shown in the upper panel (A) are the representative blots. Shown in the lower panel (B) are mean 6 standard deviation values. IkB-a/b-actin ratio was determined by densitometry. *P , 0.05 versus control group. Ó 2014 Lippincott Williams & Wilkins
necrotic cell–induced degradation of IkB-a (Figure 3), indicating that AKF-PD inhibits necrotic cell–induced TNF-a expression in an IkB-a–independent manner. Next, we sought to investigate whether AKF-PD could inhibit the nuclear translocation of the NF-kB. Necrotic cell (6 3 106 cells/mL) stimulation significantly increased the nuclear accumulation of the NF-kB subunit p65 (Figure 4), which is consistent with TNF-a production (Figure 1). Notably, addition of AKF-PD completely blocked the nuclear accumulation of p65 induced by necrotic cells (Figure 4). Collectively, these observations clearly suggest that AKF-PD inhibits TNF-a production, at least in part, through preventing p65 nuclear translocation in macrophages. Effect of AKF-PD on TNF-a–mediated MCP-1, IL-8 and IL-6 Expression in HK-2 Cells Proximal tubular epithelial cells are important source of cytokines and chemokines in kidney diseases.3–5 Next, we determined whether AKF-PD could inhibit the production of cytokines and chemokines in stimulated proximal tubular epithelial cells. HK-2 cells were stimulated with TNF-a (10 ng/mL) for 24 hours in the presence or absence of AKF-PD (2 mM) or dexamethasone (1 mM). We observed that levels of MCP-1, IL-6 and IL-8 in the cell culture medium were significantly increased by TNF-a stimulation. Importantly, addition of AKF-PD or dexamethasone significantly inhibited the production of MCP-1, IL-6 and IL-8 as shown by ELISA (Figure 5). These results demonstrated that AKF-PD inhibits the production of cytokines and chemokines in activated proximal tubular epithelial cells. AKF-PD Inhibits TNF-a–Induced NF-кB Nuclear Translocation in HK-2 Cells To determine whether AKF-PD could inhibit TNF-a– induced activation of the NF-кB pathway, we first examined the effects of AKF-PD on TNF-a–induced phosphorylation of IкB-a, an upstream activating signal for the NF-кB nuclear translocation. TNF-a stimulation significantly increased the phosphorylation of
FIGURE 4. Effect of fluorofenidone (AKF-PD) treatment on necrotic cell–induced nuclear translocation of the subunits of NF-kB (p65) in macrophages. Mouse peritoneal macrophages were stimulated with necrotic cells (6 3 106 cells/mL) in the presence or absence of AKF-PD (2 mM). Levels of p65 and lamin A/C in the nuclear extracts were determined by Western blot. Shown in the upper panel (A) are the representative blots. Shown in the lower panel (B) are mean 6 standard deviation values. NF-kB (p65)/lamin A/C ratio was determined by densitometry. *P , 0.05 versus control group, #P , 0.05 versus necrotic cell–stimulated group.
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FIGURE 5. Effect of fluorofenidone (AKF-PD) treatment on the productions of chemokines and pro-inflammatory cytokines in tumor necrosis factor (TNF)-a–stimulated HK-2 cells. HK-2 cells were stimulated with TNF-a in the presence or absence of AKF-PD (2 mM) or dexamethasone (1 mM). Levels of MCP-1, interleukin (IL)-8 and IL-6 in the cell culture medium were determined by enzyme-linked immunosorbent assay *P , 0.05 versus control group, #P , 0.05 versus TNF-a–stimulated group.
IкB-a, which was abrogated by addition of NF-кB inhibitor BAY 11-7082 (10 mM) (Figure 6). However, addition of AKF-PD failed to affect the phosphorylation of IкB-a induced by TNF-a (Figure 6). Next, we tested whether AKF-PD could inhibit TNFa–induced NF-кB nuclear translocation in HK-2 cells. Notably, AKF-PD treatment completely blocked the accumulation of p65 in the nucleus induced by TNF-a (Figure 7). These results suggested that AKF-PD inhibits TNF-a–induced MCP-1, IL-8 and IL-6 induction in HK-2 cells, at least in part, through blocking NF-кB nuclear translocation. AKF-PD Inhibits TNF-a–induced NF-кB Activation in HK-2 Cells To investigate whether AKF-PD could inhibit enhanced NF-kB transcriptional activity induced by TNF-a, HK-2 cells were transiently transfected with the pNF-кB-Luc plasmids. These genetically modified HK-2 cells were then stimulated with TNF-a in the presence or absence of AKF-PD (2 mM) or dexa-
FIGURE 6. Effect of fluorofenidone (AKF-PD) treatment on tumor necrosis factor (TNF)-a–induced phosphorylation of IкB-a in HK-2 cells. HK-2 cells were stimulated with TNF-a in the presence or absence of BAY 11-7082 (10 mM) or AKF-PD (2 mM). p-IkB and b-actin levels were measured by Western blot. Shown in the upper panel (A) are the representative blots. Shown in the lower panel (B) are mean 6 standard deviation values. p-IkB-a/b-actin ratio was determined by densitometry. *P , 0.05 versus control group, #P , 0.05 versus TNF-a–stimulated group.
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FIGURE 7. Effect of fluorofenidone (AKF-PD) on tumor necrosis factor (TNF)-a–induced nuclear translocation p65 in HK-2 cells. HK-2 cells were stimulated with TNF-a in the presence or absence of AKF-PD (2 mM). Levels of p65 and Sp1 in the nuclear extracts were determined by Western blot. Shown in the upper panel (A) are the representative blots. Shown in the lower panel (B) are mean 6 standard deviation values. NF-kB (p65)/Sp1 ratio was determined by densitometry. *P , 0.05 versus control group, #P , 0.05 versus TNF-a–stimulated group.
methasone (1 mM) for 24 hours. We observed that TNF-a (10 ng/mL) stimulation significantly enhanced the luciferase activity. Moreover, addition of AKF-PD or dexamethasone significantly reduced the luciferase activity in TNF-a–stimulated cells (Figure 8). These observations further confirm the inhibitory role of AKF-PD in the activation of the NF-kB pathway.
DISCUSSION In the present study, our results demonstrated that treatment with AKF-PD markedly reduced necrotic cell–induced production of proinflammatory cytokines and chemokines, at least in part, through blocking NF-кB nuclear translocation in both peritoneal macrophages and proximal tubular epithelial cells. Together with our previous observation that AKF-PD inhibits TNF-a and IL-1b production during lethal endotoxemia,19 here we propose that AKF-PD is an anti-inflammatory agent. Accumulating evidence implicates an essential role of inflammation in various types of progressive renal diseases and
FIGURE 8. Effect of fluorofenidone (AKF-PD) on NF-kB transcriptional activity in HK-2 cells. HK-2 cells were transiently transfected with the pNF-кB-Luc plasmids. These genetically modified HK-2 cells were then stimulated with TNF-a in the presence or absence of AKF-PD (2 mM) or dexamethasone (1 mM) for 24 hours. The luciferase activity in cell lysates was measured. *P , 0.05 versus control group, #P , 0.05 versus TNF-a–stimulated group. Volume 348, Number 1, July 2014
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renal fibrosis.20–24 TNF-a is a multifunctional acute phase protein, which promotes vascular permeability and initiates the production of cytokines and chemokines.20,21 Increased TNF-a levels are associated with macrophage accumulation and subsequent renal fibrosis.22,23 The infiltration of immune cells in kidney is a key feature of the early stage of renal fibrosis. These immune cells migrate to the site of infection or tissue injury in interstitium of kidney and produce a variety of inflammatory mediators, which in turn trigger the activation of tubular cells. The activated tubular cells are the important source of inflammatory cytokines and chemokines and critically contribute to renal inflammation.20–24 Based on these findings, the results in current study suggest that the anti-inflammatory effect of AKFPD might be an important mechanism by which AKF-PD attenuates renal inflammation and fibrosis. It is important to note that that AKF-PD inhibits necrotic cell–induced production of cytokines and chemokines in both macrophages and proximal tubular epithelial cells. Sterile inflammatory response, triggered by necrotic cells, is a key player in renal fibrosis.2–5,13–16,25–28 Necrotic cells lose membrane integrity and subsequently release damage-associated molecular patterns, which are able to recruit neutrophils and induce inflammation.29 Consistent with early observation,27 we found that necrotic cells stimulate macrophages to secrete TNF-a. Furthermore, AKF-PD treatment significantly inhibits necrotic cell–induced TNF-a production and activation of the NF-kB pathway. NF-kB plays a key role in inflammatory responses and contributes to the development of a wide range of human disorders, such as cancers, neurodegenerative diseases, arthritis, asthma, inflammatory bowel diseases and sepsis.30–33 In the present study, we found that AKF-PD effectively inhibits necrotic cells–induced or TNF-a–induced NF-кB (p65) nuclear translocation. Interestingly, AKF-PD has no effect on the phosphorylation of IkBa induced by either TNF-a or necrotic cells, suggesting that AKF-PD inhibits the NF-кB pathway by blocking NF-кB nuclear translocation rather than inhibition of the phosphorylation of IkB-a, which is the upstream activating signal for the NF-кB nuclear translocation. In conclusion, our findings suggest that AKF-PD exerts beneficial effect in chronic renal diseases, at least in part, because of its anti-inflammatory properties. This study also provides novel sights into how AKF-PD inhibits sterile inflammatory responses in both immune and nonimmune cells. REFERENCES 1. Meguid El, Nahas A, Bello AK. Chronic kidney disease: the global challenge. Lancet 2005;365:331–40. 2. Lopez-Novoa JM, Nieto MA. Inflammation and EMT: an alliance towards organ fibrosis and cancer progression. EMBO Mol Med 2009;1:303–14. 3. Deckers JG, Van Der Woude FJ, Van Der Kooij SW, et al. Synergistic effect of IL-1alpha, IFN-gamma, and TNF-alpha on RANTES production by human renal tubular epithelial cells in vitro. J Am Soc Nephrol 1998;9: 194–202. 4. van Kooten C, Gerritsma JS, Paape ME, et al. Possible role for CD40-CD40L in the regulation of interstitial infiltration in the kidney. Kidney Int 1997;51:711–21. 5. Diamond JR. Macrophages and progressive renal disease in experimental hydronephrosis. Am J Kidney Dis 1995;26:133–40. 6. Li Q, Verma IM. NF-kappaB regulation in the immune system. Nat Rev Immunol 2002;2:725–34. 7. Baeuerle PA, Henkel T. Function and activation of NF-kappa B in the immune system. Annu Rev Immunol 1994;12:141–79.
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8. Yamamoto Y, Gaynor RB. IkappaB kinases: key regulators of the NF-kappaB pathway. Trends Biochem Sci 2004;29:72–9. 9. Wang L, Hu GY, Shen H, et al. Fluorofenidone inhibits TGF-beta1 induced CTGF via MAPK pathways in mouse mesangial cells. Pharmazie 2009;64:680–4. 10. Peng ZZ, Hu GY, Shen H, et al. Fluorofenidone attenuates collagen I and transforming growth factor-beta1 expression through a nicotinamide adenine dinucleotide phosphate oxidase-dependent way in NRK-52E cells. Nephrology (Carlton) 2009;14:565–72. 11. Yuan Q, Wang R, Peng Y, et al. Fluorofenidone attenuates tubulointerstitial fibrosis by inhibiting TGF-beta (1) -induced fibroblast activation. Am J Nephrol 2011;34:181–94. 12. Huang L, Tang Y, Qin J, et al. Vasoactive intestinal peptide enhances TNF-alpha-induced IL-6 and IL-8 synthesis in human proximal renal tubular epithelial cells by NF-kappaB-dependent mechanism. Inflammation 2012;35:1154–60. 13. Chen CJ, Kono H, Golenbock D, et al. Identification of a key pathway required for the sterile inflammatory response triggered by dying cells. Nat Med 2007;13:851–6. 14. Majno G, La Gattuta M, Thompson TE. Cellular death and necrosis: chemical, physical and morphologic changes in rat liver. Virchows Arch Pathol Anat Physiol Klin Med 1960;333:421–65. 15. Iyer SS, Pulskens WP, Sadler JJ, et al. Necrotic cells trigger a sterile inflammatory response through the Nlrp3 inflammasome. Proc Natl Acad Sci U S A 2009;106:20388–93. 16. Raucci A, Palumbo R, Bianchi ME. HMGB1: a signal of necrosis. Autoimmunity 2007;40:285–9. 17. Sosne G, Qiu P, Christopherson PL, et al. Thymosin beta 4 suppression of corneal NFkappaB: a potential anti-inflammatory pathway. Exp Eye Res 2007;84:663–9. 18. Li X, Stark GR. NFkappaB-dependent signaling pathways. Exp Hematol 2002;30:285–96. 19. Tang Y, Li B, Wang N, et al. Fluorofenidone protects mice from lethal endotoxemia through the inhibition of TNF-alpha and IL-1beta release. Int Immunopharmacol 2010;10:580–3. 20. Carrero JJ, Park SH, Axelsson J, et al. Cytokines, atherogenesis, and hypercatabolism in chronic kidney disease: a dreadful triad. Semin Dial 2009;22:381–6. 21. Tonnel AB, Gosset P, Molet S, et al. Interactions between endothelial cells and effector cells in allergic inflammation. Ann N Y Acad Sci 1996;796:9–20. 22. Ma FY, Liu J, Kitching AR, et al. Targeting renal macrophage accumulation via c-fms kinase reduces tubular apoptosis but fails to modify progressive fibrosis in the obstructed rat kidney. Am J Physiol Renal Physiol 2009;296:F177–185. 23. Li ZI, Chung AC, Zhou L, et al. C-reactive protein promotes acute renal inflammation and fibrosis in unilateral ureteral obstructive nephropathy in mice. Lab Invest 2011;91:837–51. 24. Nolasco FE, Cameron JS, Hartley B, et al. Intraglomerular T cells and monocytes in nephritis: study with monoclonal antibodies. Kidney Int 1987;31:1160–6. 25. Luo F, Liu X, Li S, et al. Melatonin promoted chemotaxins expression in lung epithelial cell stimulated with TNF-alpha. Respir Res 2004;5:20. 26. Thorburn E, Kolesar L, Brabcova E, et al. CXC and CC chemokines induced in human renal epithelial cells by inflammatory cytokines. APMIS 2009;117:477–87. 27. Scaffidi P, Misteli T, Bianchi ME. Release of chromatin protein HMGB1 by necrotic cells triggers inflammation. Nature 2002;418: 191–5.
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28. Li M, Carpio DF, Zheng Y, et al. An essential role of the NF-kappa B/Toll-like receptor pathway in induction of inflammatory and tissuerepair gene expression by necrotic cells. J Immunol 2001;166:7128–35. 29. Searle J, Kerr JF, Bishop CJ, et al. Necrosis and apoptosis: distinct modes of cell death with fundamentally different significance. Pathol Annu 1982;17 (Pt 2):229–59. 30. Blackwell TS, Blackwell TR, Holden EP, et al. In vivo antioxidant treatment suppresses nuclear factor-kappa B activation and neutrophilic lung inflammation. J Immunol 1996;157:1630–7.
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