The Epigenetic Regulator BRD4 Is Involved in Cadmium-triggered Inflammatory Response in Rat Kidney

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Zhongguo Gong et al

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ABSTRACT

Cadmium (Cd) has been described as a potential inflammatory inducer while increasing evidence shows that inappropriate inflammation is a contributing factor to kidney injury. Hence, research on Cd-triggered inflammatory response is of great significance for elucidating the mechanism of Cd-induced nephrotoxicity. Bromodomain-containing 4 (BRD4) is an important epigenetic regulator involved in the development of many inflammatory diseases, but its regulatory roles in Cd-triggered inflammatory response remain to be clarified. Here, we found that treatment with Cd in Sprague-Dawley rats (2 mg/kg BW, i.p., 5 consecutive days) and in rat kidney cell line (NRK-52E, 0–10 μM, 12 h) induced the transcription of inflammatory cytokines, which could be reduced by JQ1 (BRD4 inhibitor, 25 mg/kg BW, i.p., 3 consecutive days in vivo; 0.5 μM, 12 h in vitro) or BRD4 small interfering RNA (siRNA, in vitro), suggesting that BRD4 participates in Cd-triggered inflammatory response. Next, our study clarified the roles of BRD4 in Cd-triggered inflammatory response. The inhibition of BRD4 decreased Cd-promoted NF-κB nuclear translocation and activation in vivo and in vitro. Cd increased the acetylation level of RelA K310 and enhanced BRD4 binding to acetylated NF-κB RelA in vivo and in vitro, which were abrogated by inhibiting BRD4. In summary, our study suggests that BRD4 is involved in Cd-triggered transcription of inflammatory cytokines by mediating the activation of NF-κB signaling pathway and increasing itself binding to acetylated NF-κB RelA in rat kidney, therefore, BRD4could be a potential therapeutic target for Cd-induced renal diseases.

Keywords: BRD4 Cadmium Kidney Inflammatory cytokines NF-κB JQ1

1. Introduction

Cadmium (Cd) is a heavy metal pollutant with high toxicity and a long half-life. With the development of industrial production, the increased threat of Cd pollution to environmental security and human health has drawn concern (Wang et al., 2020; Horiguchi and Oguma, 2016). After it is absorbed by the body, Cd induces multi-organ damage and the kidney is the main target organ (Fernando et al., 2020; Zhao et al., 2021). Previously, we systematically verified that autophagy inhibition, oxidative stress, and apoptosis synergistically contribute to Cd-caused nephrotoxicity in rats (Liu et al., 2017; Wang et al., 2017). Inflammation is a physiological response that acts as a defensive reaction against infection or injury, but Cd-induced uncontrolled and inappropriate inflammatory responses can cause harm such as damaging bystander normal tissue and promoting autoimmune diseases (Hossein-Khannazer et al., 2020; Zhang et al., 2020). Cd-triggered inflammatory response exacerbated cardiovascular diseases, and liver damage, suggesting that inflammation may play important roles in Cd-mediated kidney pathological processes (Fagerberg et al., 2017; Almeer et al., 2019).

Epigenetics refers to heritable modifications in gene expression based on non-DNA sequence changes, and the reversibility of these modifications enables the discovery of new therapeutic targets (Vendetti and Rudin, 2013). The bromodomain and extra-terminal domain (BET) family includes bromodomain-containing 2 (BRD2), BRD3, BRD4, and bromodomain testis-specific (BRDT), which characterized by two bromodomains and binding to acetylated lysines of histones and non-histone (Lochrin et al., 2014; Chatterjee and Bohmann, 2018). BET proteins act as the transcriptional co-activator of many genes, thus regulating cell cycle, inflammatory response, oxidative stress, and other physiological processes in various pathological models (Jiao et al., 2020; Wang et al., 2019b). BRD4 is a particularly well-studied member of the BET protein family, and accumulating investigations have reported it as a novel epigenetic target in regulating varied kidney diseases, including chronic nephritis, diabetic nephropathy, and experimental renal damage (Zeng and Zhou, 2002; Morgado-Pascual et al., 2019). BRD4 has been confirmed to participate in the nuclear factor-κB (NF-κB)–dependent transcription process to regulate inflammatory responses in many diseases (Xu and Vakoc, 2014; Suarez-Alvarez et al., 2017). BRD4 can be recruited by acetylated RelA (NF-κB subunit, a protein-coding gene) at lysine 310 (RelA-K310ac) via its bromodomains, thus activating cyclin-dependent kinase 9 (CDK9) and phosphorylated the RNA polymerase II to promote the transcription of NF-κB target genes (Hajmirza et al., 2018). Recently studies suggest that BRD4 inhibitors could be a potential therapeutic option for inflammatory diseases. In rat spinal cord injury models, inhibition of BRD4 attenuated the inflammatory response and promoted the functional recovery of microglia (Dey et al., 2019). Also, inhibition of BRD4 abrogated experimental renal inflammation in murine models of unilateral ureteral obstruction, anti membrane basal GN, and infusion of Angiotensin II (Suarez-Alvarez et al., 2017).

In view of the potential pro-inflammatory effects of Cd and the regulatory effects of BRD4 on inflammation, we hypothesized that BRD4 is involved in Cd-triggered inflammatory response. As expected, BRD4 mediates the transcription process of inflammatory cytokines in Cdexposed rat kidney models. Our study reveals a potential mechanism in the Cd-triggered inflammatory response and provides new insights into the therapy of Cd-induced nephrotoxicity.

2. Materials and methods

2.1. Chemicals and antibodies

Cadmium chloride (CdCl2, anhydrous, 439800) was purchased from Sigma-Aldrich (Carlsbad, CA, USA). (+)-JQ1 (HY-13030) was obtained from MedChemExpress (Monmouth Junction, NJ, USA). A nuclear protein extraction kit was purchased from Beyotime Institute of Biotechnology (Shanghai, China, P0027). An efficient chemiluminescence kit (ECL, 32209) and bicinchoninic acid protein assay reagent (BCA, 23225) were obtained from Thermo Fisher Scientific (Madison, WI, USA). Primary antibodies against the following proteins were used: NF-κB p65 (10745-1- AP), IL-1β (26048-1-AP), TNF-α (17590-1-AP) were purchased from Proteintech Group (Wuhan, China); β-actin (3700 s), Histone H3 (His3, 4499), Sirtuin 1 (Sirt1, 8469), normal rabbit IgG (IgG, 4394) were purchased from Cell Signaling Technology (Danvers, MA, USA); BRD4 (ab128874), K (lysine) acetyltransferase 3 (KAT3B/Ep300, ab14984), NF-κB RelA (acetyl K310) (RelA-K310ac, ab19870), Alexa Fluor® 488–conjugated donkey anti-rabbit (ab150073) were purchased from Abcam (Cambridge, UK). Second antibodies: Goat Anti-Mouse IgG (H+L) (115-035-003) and Goat Anti-Rabbit IgG (H+L) (111-035-003) were purchased from Jackson ImmunoResearch (West Grove, PA, USA).

2.2. Animals and experiments

Twenty-four Sprague-Dawley rats (male, 6 weeks old, 110–120 g) were purchased from Pengyue Experimental Animal Breeding Co., Ltd (Jinan, Shandong, China). The rats were allowed free access to food and water in an environmentally controlled room (24 ± 5 ℃, 12 h light/dark cycle). The experimental procedures were applicable to the European Communities Council Directive 2010/63/EU for animal experiments, and all procedures were approved by the Yangzhou University Animal Care and Use Committee. After one week of acclimatization, the rats were randomly divided into four groups: (1) Control group: injected intraperitoneally with the corresponding solvent (physiological saline or 2-hydroxypropyl-β-cyclodextrin solution). (2) Cd group: CdCl2 (2 mg/kg body weight, dissolved in physiological saline) injected intraperitoneally for 5 consecutive days to establish the Cd intoxication model. (3) Cd+JQ1 group: CdCl2 (2 mg/kg body weight) injected intraperitoneally for 5 consecutive days to establish the Cd intoxication model, and JQ1 (25 mg/kg body weight, dissolved in 2-hydroxypropyl-β-cyclodextrin solution) injected intraperitoneally on day 6–8. (4) JQ1 group: JQ1 (25 mg/kg body weight) injected intraperitoneally on days 6–8. Statistics of Cd content in rat serums and kidney tissues were listed in Table S1, reflecting the successful establishment of Cd-exposed rat models.

Following 8 days of treatment, the rats were killed by cervical dislocation under deep anesthesia after 12 h of fasting. The kidney tissues were dissected and 1 g tissue of each kidney was quickly stored at −80 ◦C for following western blot and quantitative PCR (qPCR) analysis. The remaining amount of tissue was quickly fixed in 4% paraformaldehyde (PFA) for immunohistochemical (IHC) staining.

2.3. Cell culture and treatment

The rat kidney cell line (NRK-52E) was obtained from the Shanghai Cell Bank of China Academy of Sciences. NRK-52E cells were cultured with Dulbecco’s modified Eagle’s medium (DMEM, Gibco, 12800-017) containing 5% fetal bovine serum (FBS, Gibco, 10437-028), penicillin, and streptomycin (100 U/mL) in a humidified condition of 5% CO2 and 95% air at 37 ℃. CdCl2 (ultrapure water–dissolved) and JQ1 (dimethyl sulfoxide-dissolved) were separately stored at 4 ℃ and − 20 ℃, and diluted into working solutions before use. The experimental design was as follows: (1) Cells were treated with 0, 2.5, 5, 10 μM Cd for 12 h, or 5 μM Cd for 0, 6, 12, 24 h to perform the subsequent assays. (2) Cells were treated with 5 μM Cd and/or 0.5 μM JQ1 for 12 h to perform the subsequent assays. (3) Cells were transfected with siBRD4 and/or treated with 5 μM Cd for another 12 h to perform the subsequent assays.

2.4. Small interfering RNA transfection

We transfected 20 nM BRD4 small interfering RNA (siBRD4, Invitrogen, CA, USA) into the NRK-52E cells by Lipofectamine RNAiMAX transfection reagent (Thermo Fisher Scientific, Cat# 13778150) for 24 h according to the product manuals. The following sense siRNAs were used:

siBRD4, with a target sequence of 5′-CCGTCAAGCTGAACCTCCCTGATTA-3′;

siCt (siRNA negative control), with a target sequence of 5′- UUCUCCGAACGUGUCACGUTT-3′.

2.5. Western blotting

Kidney tissue samples and NRK-52E cells were lysed in RIPA buffer containing protease inhibitors to extract total protein. The nuclear protein was prepared from fresh tissues and cells in advance and stored at − 80 ℃. Protein samples (20–30 μg per sample) were separated by SDS-PAGE electrophoresis, then transferred to the polyvinylidene fluoride membranes (Millipore, ISEQ00010). The membranes were blocked with 5% skim milk for 90 min, and incubated 12 h at 4 ℃ with following primary antibodies: β-actin (1:5000), NF-κB p65 (1:1000), IL-1β (1:600), TNF-α (1:600), BRD4 (1:1000), Ep300 (1:1000), Sirt1 (1:1000), His3 (1:1000), RelA-K310ac (1:1000). Then the membranes were washed with TBST and incubated with corresponding secondary antibodies (1:10000) for 90 min at room temperature (RT). The membranes were incubated with electrochemiluminescence (ECL, ThermoFisher Scientific) and determined on the Chemidoc XRS (Bio-Rad, Marnes-LaCoquette, France), and the optical density was analyzed using ImageJ (NIH, Bethesda, MD, USA).

2.6. Immunofluorescence (IF) staining

NRK-52E cells seeded in a 24-wells plate were grown to about 60% confluence. The cells were treated with 5 μM Cd and/or 0.5 μM JQ1 for 12 h, then fixed with PFA (4%, 10 min), permeabilized with Triton X- 100 (0.1%, 15 min), blocked with BSA (2%, 90 min) at RT, and incubated with primary antibody (NF-κB p65, 1:50 dilution) overnight at 4 ℃. After being washed with PBS three times, the cells were incubated with secondary antibody (1:500 dilution) for 90 min, and with DAPI for 5 min at RT, then the cells were observed under a confocal microscope. NF-κB nuclear translocation was analyzed in three independent studies.

2.7. qPCR

The total RNA of kidney tissues and NRK-52E cells were extracted with RNAiso Plus kit (Takara Bio, Shiga, Japan). 1 μg of total RNA was used to synthesize 1st chain cDNA according to the manufacturer’s manual (Roche, Basel, Switzerland). 2 μL complementary DNA (cDNA) per well were taken to detected the relative mRNA levels using a LightCycler® 96 Real-Time PCR System (Roche). Calculate the relative mRNA levels according to equation 2-△△CT. The primers were listed in Table S2. β-actin was a rat reference gene.

2.8. Co-immunoprecipitation (Co-IP)

The kidney tissues and NRK-52E cells were lysed in fresh-prepared cell lysis buffer (20 mM HEPES–KOH pH 7.5, 150 mmol/L NaCl, 2 mmol/L EDTA, 1% Triton X-100) containing PMSF. Protein G beads (100 μL per sample, Bio-Rad, Hercules, CA, USA) were mixed with 2 μg BRD4, RelA-K310ac, or IgG antibody per sample, and rotated for 10 min at RT. Then, the total protein lysates were incubated with beads–antibody complex overnight at 4 ℃. After being washed with cell lysis buffer three times, the immunoprecipitates were resuspended with 1 × SDSPAGE sample buffer to configure the samples. The target proteins were detected using western blotting with anti-BRD4 and anti-RelAK310ac primary antibodies.

2.9. Statistical analysis

All data were obtained from at least three independent experiments and expressed as the mean ± SEM unless mentioned otherwise. Experimental groups were compared by unpaired two-tailed Student’s t-test or one-way analysis of variance (ANOVA) using SPSS 22.0 (SPSS Inc., Chicago, IL, USA). The significance was set at p < 0.05.

3. Results

3.1. Cd induces the expression of inflammatory cytokines in vivo and in vitro

As an environmental pollutant, Cd has been proven to cause kidney damage by regulating a series of biological processes (Wang et al., 2019c). In this study, the changes in the transcription levels of inflammatory cytokines were examined after Cd exposure to investigate whether inflammation is involved in Cd-induced nephrotoxicity. Data showed that Cd exposure significantly increased interleukin (IL)-1β and tumor necrosis factor (TNF)-α protein levels in NRK-52E cells (Fig. 1A-B) and rat kidney tissues (Fig. 1C-D). Meanwhile, as shown in Fig. 1E-F, Cd exposure enhanced the transcriptional levels of inflammatory cytokines (IL-1β, IL-6, TNF-α, and (Monocyte chemoattractant protein-1) MCP-1) in vivo and in vitro. These findings suggest that Cd induces the expression of inflammatory cytokines in rat kidneys.

3.2. BRD4 participates in the Cd-induced transcription process of inflammatory cytokines

BRD4 is a newly described epigenetic regulator that binds to acetylated histones or non-histone to regulate inflammation processes in many diseases. Here, BRD4 inhibitor JQ1 and BRD4-siRNA were used to examine the role of BRD4 on Cd-triggered inflammatory response. Data showed that Cd-elevated IL-1β and TNF-α protein levels were down-regulated by JQ1 treatment in vivo and in vitro (Fig. 2A–D) and by BRD4 knockdown in vitro (Fig. S1A-B). Meanwhile, JQ1 treatment significantly inhibited Cd-enhanced transcription levels of IL-1β, IL-6, TNF-α, and MCP-1 in NRK-52E cells (Fig. 2E) and rat kidney tissues (Fig. 2F). Also, BRD4 knockdown significantly decreased Cd-increased transcription levels of inflammatory cytokines in NRK-52E cells (Fig. S1C). Collectively, these findings suggest that BRD4 is a critical regulator of Cd-triggered inflammatory response in rat kidneys.

3.3. BRD4 expression levels remain unchanged after Cd exposure

Next, changes in the expression levels of BRD4 were detected after Cd exposure. NRK-52E cells were treated with Cd under a concentration gradient, and rats were intraperitoneally injected with Cd for 5 days to elucidate the effect of Cd on BRD4 expression in the kidney in vivo and in vitro. Results showed that the BRD4 protein levels were unaltered in both the Cd-exposed NRK-52E cells (Fig. 3A-B) and rat kidney tissues (Fig. 3C-D). Also, BRD4 mRNA levels were unaltered after Cd exposure (Fig. 3E-F). These results indicate that BRD4 mediates Cd-triggered inflammatory response does not depend on changes in expression level.

3.4. BRD4 regulates Cd-promoted NF-κB nuclear translocation

The roles of BRD4 in Cd-triggered inflammatory response were explored in our study. NF-κB signaling pathway alteration is closely related to the transcription of inflammatory cytokines (Chi et al., 2021). Our study found that BRD4 is involved in Cd-regulated nuclear translocation and activation of NF-κB. First, the subcellular localization of NF-κB was determined by IF staining and IHC staining. Data in Fig. 4A and D showed that JQ1 inhibits Cd-promoted NF-κB nuclear translocation in NRK-52E cells and rat kidney tissues. Meanwhile, western blot results showed that JQ1 significantly decreased Cd-increased nuclear protein levels of NF-κB in vivo (Fig. 4B-C) and in vitro (Fig. 4E-F). Similarly, BRD4 knockdown also significantly inhibited Cd-promoted NF-κB nuclear translocation (Fig. S2A) and decreased Cd-increased nuclear protein levels of NF-κB (Fig. S2B-C) in NRK-52E cells. Taken together, inhibition of BRD4 can reduce Cd-promoted NF-κB nuclear translocation, suggesting that BRD4 mediates the activation of the NF-κB signaling pathway in the kidney after Cd exposure.

3.5. Cd increases RelA K310 acetylation levels

Studies have confirmed that BRD4 binds to acetylated RelA K310 through its bromine domains to regulate NF-κB–dependent transcription (Morgado-Pascual et al., 2019; Zhong et al., 2018). Thus, the acetylation level of RelA K310 and the expression of two enzymes were detected. Data showed that RelA-K310ac and acetylase Ep300 protein levels were decreased continually with the increased concentration of Cd, while deacetylase Sirt1 protein level gradually increased in NRK-52E cells (Fig. 5A-B) and rat kidney tissues (Fig. 5C-D). Also, Ep300 and Sirt1 mRNA levels showed the same trends (Fig. 5E-F). These results suggest that Cd promotes the acetylation level of RelA K310, thus BRD4 may be involved in Cd-triggered inflammatory cytokines transcription in the kidney.

3.6. Cd increases BRD4 binding to RelA-K310ac to enhance its transcriptional function

Binding to acetylation sites is the basis for the transcriptional regulation of BRD4 (Huang et al., 2009). To verify whether Cd regulates BRD4 function by increasing BRD4 binding to RelA-K310ac, the interaction between RelA-K310ac and BRD4 was detected via Co-IP. Data in Fig. 6A showed that Cd significantly enhanced the interaction between RelA-K310ac and BRD4 in NRK-52E cells, which was alleviated by JQ1 treatment or BRD4 knockdown. Also, the same trend was observed in rat kidney tissues (Fig. 6B). These results demonstrate that Cd enhances the binding of BRD4 to RelA-K310ac, which promotes NF-κB–dependent transcription and subsequently contributes to the Cd-triggered inflammatory response.

4. Discussion

Cd has been reported as a potential trigger of inflammation because of its high toxicity, while the exact mechanism is still not fully understood (Arab-Nozari et al., 2020; Ghosh, 2018). Our study confirmed that Cd induces inflammatory cytokines expression in rat renal cells, and found that BRD4, an epigenetic target that has critical functions in a series of cellular processes including inflammation, contributes to Cd-triggered inflammatory response. Further experiments indicated that BRD4 is involved in Cd-promoted NF-κB signaling pathway activation. Also, Cd increased the acetylation level of RelA K310, thereby increasing BRD4 binding to acetylated NF-κB RelA to promote NF-κB–dependent inflammatory cytokines transcription.

Inflammation is one of the natural defense mechanisms against exogenous chemicals, whereas the uncontrolled and inappropriate inflammatory response can damage normal tissues and induce autoimmune diseases (Jian et al., 2018). Cd-increased biological markers of inflammation were often associated with various diseases. Studies have shown that Cd-induced inflammation is involved in atherogenesis (Tinkov et al., 2018). Moreover, Cd promoted the expression of inflammatory cytokines, which contributed to lung and testicular tissue damage and induces hepatotoxicity (Koopsamy Naidoo et al., 2019; Arafa et al., 2014). These harmful effects are consistent with our results, that the inflammatory response is involved in Cd-induced kidney injury. Increasing researches have focused on the occurrence mechanisms of Cd-induced inflammation. As a well-studied BET protein family member, BRD4 plays a critical role in many biological processes including inflammation. Here, JQ1 (an inhibitor of BRD4) and siRNA were used to deregulate the functional activity of BRD4 and found that the inhibition of BRD4 reduced Cd-induced transcription of inflammatory cytokines in rat kidneys, suggesting that BRD4 participates in the Cd-triggered inflammatory response. It is generally believed that the dysregulation of BRD4 expression is a crucial event in the occurrence of inflammatory response. In spinal cord injury, pathological cardiac hypertrophy, and other inflammation-associated diseases, the disordered BRD4 expression contributed to the expression of inflammatory cytokines (Zhu et al., 2020; Marazzi et al., 2018; Ren et al., 2019). However, Cd had no effect on the expression levels of BRD4 under the present experimental conditions, which led us to consider whether BRD4 mediates the Cd-triggered inflammatory response through other pathways.

NF-κB is an important regulatory factor in inflammatory processes and is responsible for controlling the expression of many inflammatory mediators (Lawrence, 2009). Studies have been reported that BRD4 is involved in the regulation of the NF-κB signaling pathway. Huang et al. (2017) suggested that BRD4 regulated IKK-mediated NF-κB activation through the p38 and JNK–MAPK pathways. Wang et al. (2019a) found that BRD4 knockdown or JQ1 treatment blocked lipopolysaccharide (LPS)-activated NF-κB signaling pathway in microglia. Meng et al. (2014) demonstrated that JQ1 treatment blocked the expression of the inflammatory cytokines by inhibiting the activation of NF-κB in LPS-treated RAW 264.7 cells. These reports indicated that BRD4 mediates the activation of NF-κB in many inflammation models while inhibiting BRD4 is an effective therapeutic approach to anti-inflammation. In the current study, the inhibition of BRD4 blocked the nuclear translocation of NF-κB in Cd-exposed rat kidney tissues and NRK-52E cells, suggesting that BRD4 mediates the Cd-activated NF-κB signaling pathway.

Typically, after NF-κB was activated by various stimuli and translocates into the nucleus, BRD4 binds to acetylated NF-κB RelA at the K310 site, which increases its stability and transcriptional activity in the nucleus (Hajmirza et al., 2018). Thus the acetylation level of RelA K310 affects the regulatory function of BRD4 on the inflammatory response. In hippocampal neurons, the deacetylase Sirt1 mediated RelA K310 deacetylation to regulate BACE1 expression, which contributed to the production of neuronal amyloid (Flores-Leon et al., 2019). In glioblastoma cells, photodynamic therapy stress down-regulated the deacetylase Sirt1 and activated the acetylase Ep300, which increased RelA-K310ac levels and led to elevated production of the pro-survival nitric oxide (Fahey et al., 2019). Our study found that Cd promotes the acetylation level of RelA K310 by increasing Ep300 expression and reducing Sirt1 expression, indicating that Cd affects the regulatory function BRD4 by increasing the acetylation levels of RelA K310.

Notably, our results showed that JQ1 treatment was more effective than BRD4 knockdown in alleviating Cd-triggered inflammatory response. JQ1 has a high binding affinity with BRD4 and can almost completely block BRD4 binding to the chromosome, while siBRD4 can only interfere with BRD4 protein expression to reduce BRD4 binding to the target site, suggesting that BRD4 mediates Cd-triggered inflammatory response may depend on the binding with acetylated NF-κB RelA. Recent studies have shown a similar regulatory mechanism: after NF-κB nuclear translocation and activation, BRD4 then interacted with acetylated RelA to coactivate the transcriptional activation of NF-κB (Huang et al., 2009). RelA–BRD4 was recruited to NF-κB target promoters in various inflammatory stimulation conditions, while JQ1 treatment acted by preventing BRD4 binding to acetylated NF-κB RelA, thus reducing the transcriptional activity of NF-κB (Zou et al., 2014). Our results showed that the inhibition of BRD4 blocks Cd-enhanced interaction between BRD4 and RelA-K310ac in rat kidneys, supporting our above results that inhibiting BRD4 suppresses the Cd-induced transcriptional activity of NF-κB, indicating that Cd enhances BRD4 binding to acetylated NF-κB RelA to increase the transcription of inflammatory cytokines.

In summary, our study revealed the regulatory mechanisms of BRD4 in Cd-triggered inflammatory response in rat kidneys: (1) BRD4 mediates Cd-induced NF-κB nuclear translocation and activation. (2) Cd increases RelA K310 acetylation levels and enhances BRD4 binding to acetylated NF-κB RelA, which promotes NF-κB–dependent inflammatory cytokines transcription. In addition, the inhibition of BRD4 significantly alleviated Cd-elevated inflammatory cytokine levels, suggesting that targeted BRD4 has the potential to be developed as an effective means of treating inflammatory diseases including Cd-induced nephritis.

CRediT authorship contribution statement

Zhongguo Gong: Conceptualization, Methodology, Visualization, Formal analysis, Validation, Investigation, Formal analysis, Data curation, Writing – original draft. Gang Liu: Conceptualization, Methodology, Visualization, Data curation, Writing – original draft, Project administration, Funding acquisition. Wenjing Liu: Software, Resources, Methodology, Formal analysis. Hui Zou: Project administration, Supervision. Ruilong Song: Software, Formal analysis, Supervision. Hongyan Zhao: Software, Formal analysis, Supervision. Yan Yuan: Supervision. Jianhong Gu: Supervision. Jianchun Bian: Funding acquisition, Supervision. Jiaqiao Zhu: Writing – review & editing, Supervision. Zongping Liu: Writing – review & editing, Supervision,

Project administration, Funding acquisition, Conceptualization.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgment

This work was supported by the National Natural Science Foundation of China (Nos. 31872533, 31902329, 32072933), the National Key Research and Development Program of China (No. 2016YFD0501208), and the project of the Priority Academic Program Development of Jiangsu Higher Education Institution (PADP). The manuscript was edited for proper English language, grammar, punctuation, spelling, and overall style by one or more of the highly qualified native English-speaking editors at ELIXIGEN.

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