- Email: [email protected]
ARE signaling
Biochimie 165 (2019) 100e107
Contents lists available at ScienceDirect
Biochimie journal homepage: www.elsevier.com/locate/biochi
Research paper
BRD4 contributes to high-glucose-induced podocyte injury by modulating Keap1/Nrf2/ARE signaling Hong Zuo*, Shujin Wang, Jia Feng, Xufeng Liu Department of Endocrinology, the Ninth Hospital of Xian, Xi'an, 710054, Shaanxi Province, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 18 May 2019 Accepted 12 July 2019 Available online 17 July 2019
High glucose (HG)-induced podocyte injury contributes to the pathogenesis of diabetic nephropathy, a severe complication of diabetes. Bromodomain-containing protein 4 (BRD4) has emerged as a critical regulator for cell injury. However, whether BRD4 participates in HG-induced podocyte injury remains unclear. In this study, we aimed to explore the potential role of BRD4 in regulating HG-induced podocyte injury and its underlying molecular mechanism. HG exposure significantly upregulated BRD4 in podocytes. BRD4 inhibition by small interfering RNA or its chemical inhibitor (JQ1) markedly repressed HGinduced apoptosis and reactive oxygen species (ROS) production. By contrast, BRD4 overexpression exacerbated HG-induced podocyte injury. Moreover, BRD4 inhibition potentiated nuclear factor (erythroid-derived 2)-like 2 (Nrf2) signaling associated with suppression of Kelch-like ECH-associated protein (Keap1). BRD4 inhibition promoted Nrf2 nuclear translocation and upregulated the transcriptional activity of Nrf2/antioxidant response element (ARE). However, Nrf2 silencing partially reversed BRD4-inhibition-mediated protection against HG-induced podocyte injury. Overall, these results suggest that BRD4 inhibition confers cytoprotection against HG injury in podocytes through potentiation of Nrf2/ ARE antioxidant signaling. This finding implicates BRD4/Nrf2/ARE signaling in the pathogenesis of diabetic nephropathy. © 2019 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.
Keywords: BRD4 Diabetic nephropathy High glucose Podocyte Nrf2
1. Introduction Diabetic nephropathy is one of the most prevalent chronic complications to diabetes, and it contributes to the development of end-stage renal disease [1,2]. Hyperglycemia-induced cellular injury and dysfunction is the major cause of diabetic nephropathy [3,4]. Podocytes distributed on the outer surface of the glomerular basement membrane help maintain the integrity of the glomerular filtration barrier [5]. Podocytes are highly differentiated cells in the kidney with limited capability to repair and/or regenerate, and the
Abbreviations: HG, high glucose; NG, normal glucose; BRD4, bromodomaincontaining protein 4; Nrf2, nuclear factor (erythroid-derived 2)-like 2; Keap1, Kelch-like ECH-associated protein; ARE, antioxidant response element; ROS, reactive oxygen species; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; CCK-8, Cell Counting Kit-8; OD, optical density; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling. * Corresponding author. Department of Endocrinology, the Ninth Hospital of Xian, No.151 East Section of South Ring, Beilin District, Xi'an, 710054, Shaanxi Province, China. E-mail address: [email protected] (H. Zuo).
reduction of podocytes from high glucose (HG)-induced apoptosis contributes to the development of diabetic nephropathy [6]. Thus, therapies aimed at preventing HG-induced podocyte injury have potential clinical benefits for diabetic nephropathy. Bromodomain-containing protein 4 (BRD4), a member of the bromodomain and extra-terminal protein family, plays an important role in normal development and disease progression [7,8]. BRD4 is a crucial regulator of transcription, and it regulates the expression of various genes [9,10]. Therefore, BRD4 participates in diverse biological processes, including cell cycle, proliferation, apoptosis, and differentiation [11e13]. Interestingly, accumulating evidence suggests that BRD4 inhibition confers a cytoprotective effect against stress-induced apoptosis and oxidative stress [14,15]. Notably, the underlying molecular mechanism is associated with its regulatory effect on nuclear factor (erythroid-derived 2)-like 2 (Nrf2) antioxidant signaling. Nrf2 functions as a redox-sensitive transcription factor that protects cells from oxidative damage via activating the transcription of cytoprotective genes, such as heme oxygenase and NADPH quinone oxidoreductase 1 [16]. Kelch-like ECH-associated protein
https://doi.org/10.1016/j.biochi.2019.07.012 0300-9084/© 2019 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.
H. Zuo et al. / Biochimie 165 (2019) 100e107
(Keap1) regulates Nrf2 protein stabilization. When bound to Keap1, Nrf2 is polyubiquitinated and degraded through the ubiquitin proteasome pathway [17]. In contrast, Nrf2 dissociated from Keap1 translocates to the nucleus where it binds to the antioxidant response element (ARE) within the promoter region of antioxidant and detoxifying enzymes and initiates gene transcription [17]. Therefore, Nrf2 signaling activation facilitates the elimination of reactive oxygen species (ROS) and confers cytoprotection. A recent study documented that Nrf2 plays an important role in the development of diabetic nephropathy [18] and may serve as a potential and promising therapeutic target for this disease. HG induces oxidative stress and apoptosis in podocytes [19e21]. However, whether BRD4 regulates HG-induced podocyte injury remains unclear. Here, our study aimed to explore the potential role of BRD4 in regulating HG-induced podocyte injury and its underlying molecular mechanism.
101
radioimmunoprecipitation assay (RIPA) buffer that contained protease inhibitor (Beyotime Biotechnology, Shanghai, China). The supernatants were harvested by centrifugation, and protein concentrations were determined with the Enhanced BCA Protein Assay Kit (Beyotime Biotechnology). For protein separation, equal amounts of protein from each sample were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The separated proteins were then transferred onto a polyvinylidene fluoride transfer membrane (Thermo Scientific Pierce), and the membrane was incubated in 5% fat-free milk for 1 h at room temperature. Subsequently, the membrane was immunoblotted with primary antibodies against BRD4, Nrf2, Keap1, b-actin and Lamin B2 (Abcam, Cambridge, UK) at 4 C overnight. The membrane was then probed with horseradish-peroxidase-conjugated secondary antibody (Abcam) for 1 h at room temperature. Finally, ECL Plus Substrate (Thermo Scientific Pierce) was utilized to develop target protein bands.
2. Materials and methods 2.5. Cell viability assay 2.1. Cell culture and HG exposure The mouse podocyte cell line MPC5 was purchased from BeNa Culture Collection (Kunshan City, China) and cultured as per the manufacturer's instructions. In brief, cells were cultured in RPMI Medium 1640 (Gibco; Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco), 100 U/ml penicillin and 100 mg/ml streptomycin. Cells were grown at 37 C in a humidified incubator containing 5% CO2. Podocytes were maintained in medium containing normal glucose concentration (NG, 5 mM) or high glucose concentration (HG, 30 mM) for 24 h to induce HG injury. 2.2. Cell transfection The small interfering RNAs (siRNAs) that targeted BRD4 or Nrf2 were purchased from RiboBio (Guangzhou, China). The BRD4 expression vector was constructed by inserting the BRD4 open reading frame into the pcDNA3.1 vector. Cell transfection of siRNAs or vectors was carried out using Lipofectamine 3000 Reagent (Invitrogen; Thermo Fisher Scientific) following the manufacturer's manuals. Briefly, cells were seeded into cultured until cells reached 70e90% confluent at the time of transfection. The siRNA- or plasmid DNA-lipid complexes were prepared by adding siRNAs or plasmids and Lipofectamine 3000 Reagent to the Opti-MEM medium. The mixture was incubated for 15 min at room temperature and then added to cells at 50 ml/well. After 48 h-transfection, the transfection efficacy was determined by reverse transcriptionquantitative polymerase chain reaction (RT-qPCR) or Western blot analysis.
Cell viability was determined with the Enhanced Cell Counting Kit-8 (CCK-8) following the manufacturer's protocols. Briefly, cells were seeded into a 96-well plate and transfected with BRD4 siRNA or expression vector for 24 h followed by HG exposure for 24 h. Thereafter, 10 ml CCK-8 solution was added to each well and incubated for 1 h at 37 C. The optical density (OD) value of the colorimetric solution at 450 nm was measured with a microplate reader (Bio-Rad, Hercules, CA, USA). 2.6. Caspase-3 activity assay Caspase-3 activity was measured with a Colorimetric Caspase-3 Assay Kit (Abcam) as per the standard procedure. After the indicated treatment, cells were collected and resuspended into chilled cell lysis buffer and incubated for 10 min on ice. The supernatants were harvested by centrifugation and transferred to a new 96-well plate at 50 ml/well. Then, 50 ml reaction buffer and 5 ml DEVD-p-NA substrate (4 mM) were added to each well followed by incubation for 2 h at 37 C. The OD value of the colorimetric solution at 405 nm was determined by a microplate reader (Bio-Rad). 2.7. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay Cells on coverslips were fixed with 4% paraformaldehyde for 30 min at room temperature. Next, cells were permeabilized with 0.3% Triton X-100 for 5 min at room temperature. Subsequently, cells were incubated with TUNEL detection solution for 1 h at 37 C in the dark. The TUNEL-positive cells were observed and counted under a fluorescence microscope.
2.3. RNA extraction and RT-qPCR analysis 2.8. ROS measurement assay Total RNA was extracted from cells by using TRIzol reagent (Invitrogen) and converted into complementary DNA (cDNA) using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems; Thermo Fisher Scientific). RT-qPCR was performed using the PowerUp SYBR Green PCR Master Mix (Applied Biosystems) on an Applied Biosystems 7500 Real-Time PCR System. The housekeeping gene b-actin was used for standardizing target gene expression. The data for target gene expression was calculated using the 2DDCt method.
Cells were treated with 10 mM DCFH-DA (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany) in serum-free medium and incubated at 37 C for 30 min. After washing with serum-free medium, intracellular ROS levels were detected by measuring fluorescence intensity. The fluorescence intensity was detected by flow cytometry with excitation wavelength of 488 nm and emission wavelength of 525 nm. 2.9. Luciferase reporter assay
2.4. Western blot For total protein extraction, cells were lysed in ice-cold
Cells were co-transfected with the pARE-luc reporter vector (Beyotime Biotechnology), Renilla luciferase reporter vector pRL-
102
H. Zuo et al. / Biochimie 165 (2019) 100e107
TK, and BRD4 siRNA or expression vector for 24 h, followed by HG exposure for 24 h. The cells were then lysed, and luciferase activity was determined with the Dual-GLO Luciferase Assay System (Promega, Madison, WI, USA), as per the manufacturer's instructions.
t-test or one-way analysis of variance with the Bonferroni post-hoc test (where appropriate). Statistical analyses were performed with SPSS Statistics Version 19.0 (SPSS Inc., Chicago, IL, USA) or GraphPad Prism 6 (GraphPad Software, San Diego, CA, USA). p < 0.05 was considered significant.
2.10. Statistical analysis
3. Results
Results are expressed as the mean ± standard deviation (SD). Statistical significance of differences was determined by Student's
3.1. BRD4 upregulation in HG-stimulated podocytes To investigate the potential relevance of BRD4 in HG-induced podocyte injury, we detected the effect of HG exposure on BRD4 expression. RT-qPCR revealed that HG exposure significantly elevated podocyte BRD4 mRNA expression (Fig. 1A). Consistently, western blot analysis demonstrated that HG also markedly upregulated BRD4 protein expression (Fig. 1B). These results suggest that BRD4 may participate in regulating HG-induced podocyte injury. 3.2. BRD4 inhibition alleviated HG injury in podocytes in vitro
Fig. 1. Effect of HG exposure on BRD4 expression in podocytes. Podocytes were treated with either normal glucose (NG, 5 mM) or high glucose (HG, 30 mM) for 24 h and then harvested for BRD4 detection. (A) Relative BRD4 mRNA expression in HG-exposed podocytes was determined by RT-qPCR and normalized against b-actin. (B) BRD4 protein expression in HG-exposed podocytes was examined by western blot. N ¼ 3, *p < 0.05.
To investigate the function of BRD4 in HG-induced podocyte injury, we performed BRD4 loss-of-function experiments. BRD4 siRNA was utilized to deplete BRD4 expression in podocytes; its downregulation was confirmed by RT-qPCR and western blot analyses (Fig. 2A and B). BRD4 knockdown significantly rescued cell viability, which was inhibited by HG exposure (Fig. 2C). Moreover, BRD4 silencing significantly reduced HG-induced apoptosis (Fig. 2D and E). BRD4 silencing also markedly reduced HG-induced excessive ROS production (Fig. 2F). Consistently, treatment with the
Fig. 2. BRD4 silencing downregulates HG-induced apoptosis and oxidative stress in podocytes. Podocytes were transfected with either BRD4 or NC siRNA for 48 h followed by HG exposure for 24 h. (A) Relative BRD4 mRNA expression was determined by RT-qPCR and normalized against b-actin. (B) BRD4 protein expression was examined by western blot. (C) Effect of BRD4 knockdown on HG-impaired cell viability was assessed by the CCK-8 assay. (D) Effect of BRD4 knockdown on HG-induced caspase-3 activity was evaluated by a colorimetric caspase-3 assay. (E) Effect of BRD4 knockdown on HG-induced cell apoptosis was measured with the TUNEL assay. (F) Effect of BRD4 knockdown on HG-induced ROS production was detected by DCFH-DA staining. N ¼ 3, *p < 0.05.
H. Zuo et al. / Biochimie 165 (2019) 100e107
BRD4 chemical inhibitor JQ1 significantly attenuated HG-induced apoptosis and ROS production (Fig. 3AeD). Downregulation of nephrin and or podocin is an indicator of podocyte injury [22]. Notably, BRD4 inhibition significantly increased the expression of nephrin and or podocin in HG-exposed podocytes (Supplementary Fig. 1). Collectively, these results suggest that BRD4 inhibition protects podocytes from HG injury. 3.3. BRD4 overexpression exacerbated HG injury in podocytes To confirm that BRD4 contributes to HG injury in podocytes, we performed BRD4 gain-of-function experiments by using a BRD4 expression vector. BRD4 overexpression in vector-transfected cells was confirmed by western blot (Fig. 4A). This overexpression significantly decreased cell viability in HG-exposed cells (Fig. 4B). Moreover, BRD4 overexpression exacerbated HG-induced cell apoptosis and ROS production in podocytes (Fig. 4C and D). These results suggest that BRD4 overexpression promotes HG injury in podocytes. 3.4. BRD4 inhibition potentiated Nrf2/ARE signaling in HG-exposed podocytes BRD4 inhibition confers cytoprotection by enhancing Nrf2/ARE signaling. Thus, we next investigated whether BRD4 inhibition contributes to Nrf2/ARE signaling regulation in HG-exposed podocytes. BRD4 knockdown significantly downregulated Keap1 expression and upregulated Nrf2 nuclear translocation (Fig. 5A and B). Moreover, BRD4 knockdown enhanced Nrf2/ARE-dependent transcriptional activity (Fig. 5C). Consistently, BRD4 inhibition by its chemical inhibitor JQ1 also enhanced the activation of Nrf2/ARE
103
signaling (Fig. 5EeF). Overall, our results suggest that BRD4 inhibition potentiates Nrf2/ARE signaling in HG-exposed podocytes. 3.5. BRD4-inhibition-mediated protection was reversed by Nrf2 inhibition To confirm whether Nrf2/ARE signaling contributes to BRD4inhibition-mediated protection, we detected the consequence of Nrf2 inhibition on this effect in podocytes. Transfection of Nrf2 siRNA significantly downregulated Nrf2 expression (Fig. 6A). Nrf2 inhibition significantly abrogated the promoting effect of BRD4 inhibition on Nrf2/ARE signaling (Fig. 6B). Moreover, BRD4inhibition-mediated protection on HG-induced apoptosis and ROS production was partially reversed by Nrf2 inhibition (Fig. 6CeE). Overall, these results suggest that BRD4 inhibition protects podocytes from HG injury by potentiating Nrf2/ARE signaling. 4. Discussion In the present study, we provided convincing evidence that BRD4 contributes to regulation of HG-induced podocyte injury. Our findings revealed that BRD4 inhibition by siRNA or its chemical inhibitor significantly repressed HG-induced apoptosis and ROS production in podocytes. The underlying molecular mechanism was associated with its promoting effect on Nrf2/ARE antioxidant signaling via downregulation of Keap1 (Fig. 6F). Therefore, our findings highlight the importance of BRD4/Nrf2/ARE signaling in regulating podocyte injury. BRD4 dysregulation is implicated in various maladies, particularly in kidney disease [23e25]. It is reported that BRD4 inhibition attenuates experimental renal damage with inactivation of nuclear
Fig. 3. Treatment with the BRD4 inhibitor JQ1 decreased HG injury in podocytes. Podocytes were treated with 5 mM JQ1 for 48 h and then stimulated with HG for 24 h. (A) Cell viability was detected with the CCK-8 assay. (B) Caspase-3 activity was measured by a colorimetric caspase-3 assay. (C) Cell apoptosis was detected with the TUNEL assay. (D) ROS production was determined by DCFH-DA staining. N ¼ 3, *p < 0.05.
104
H. Zuo et al. / Biochimie 165 (2019) 100e107
Fig. 4. BRD4 overexpression exacerbated HG-induced apoptosis and ROS production in podocytes. Podocytes were transfected with BRD4 expression vector (BRD4) or empty vector (EV) for 48 h, stimulated with HG for 24 h, and then (A) BRD4 protein expression was detected by western blot. (B) Effect of BRD4 overexpression on HG-impaired cell viability was detected by the CCK-8 assay. (C) Effect of BRD4 overexpression on HG-induced caspase-3 activity was measured by a colorimetric caspase-3 assay. (D) Effect of BRD4 overexpression on HG-induced ROS production was assessed by DCFH-DA staining. N ¼ 3, *p < 0.05.
Fig. 5. BRD4 inhibition potentiated Nrf2/ARE signaling. Podocytes were transfected with BRD4 siRNA for 48 h followed by HG exposure for 24 h. Effect of BRD4 silencing on (A) Keap1 and (B) nuclear Nrf2 (nu-Nrf2) protein expression was detected by western blot. (C) Effect of BRD4 silencing on Nrf2/ARE-dependent transcriptional activity was determined by an ARE reporter. Podocytes were pretreated with 5 mM JQ1 for 48 h and then stimulated with HG for 24 h. Effect of JQ1 treatment on (D) Keap1 and (E) nuclear Nrf2 (nu-Nrf2) protein expression was detected by western blot. (F) Effect of JQ1 treatment on Nrf2/ARE-dependent transcriptional activity was determined by an ARE reporter. N ¼ 3, *p < 0.05.
H. Zuo et al. / Biochimie 165 (2019) 100e107
105
Fig. 6. BRD4 inhibition protected podocytes from HG injury via Nrf2/ARE signaling. Podocytes were transfected with Nrf2 siRNA in the presence of JQ1 for 48 h, followed by HG exposure for 24 h. (A) Nrf2 protein expression was determined by western blot. (B) Nrf2/ARE-dependent transcriptional activity was determined by an ARE reporter. (C) Cell viability was detected by the CCK-8 assay. (D) Caspase-3 activity was measured by a colorimetric caspase-3 assay. (E) ROS production was determined by DCFH-DA staining. N ¼ 3, *p < 0.05. (F) A graphic model of BRD4/Keap1/Nrf2/ARE signaling in regulating HG-induced injury in podocytes. HG treatment upregulates BRD4 expression which then promotes Keap1 expression, leading to enhanced Nrf2 degradation. Therefore, BRD4 inhibition decreases Keap1 expression, which enhanced Nrf2 accumulation and nuclear translocation, leading to transcription of Nrf2/ARE target genes that protect podocytes from HG-induced injury.
factor (NF)-kB signaling [26]. Moreover, BRD4 inhibition ameliorates human-immunodeficiency-virus-induced inflammation and kidney injury in mouse models via inactivation of NF-kB signaling [27]. Zhou et al. reported that BRD4 inhibition suppresses cystic epithelial cell proliferation, delays kidney enlargement, and preserves renal function in autosomal dominant polycystic kidney disease [28]. BRD4 is highly expressed in fibrotic kidneys, and BRD4 inhibition prevents the activation of renal fibroblasts [29]. Notably, BRD4 inhibition represses ROS production in the kidney induced by unilateral ureteral obstruction and delays the progression of renal fibrosis [29]. Moreover, BRD4 inhibition also alleviates ischemia/ reperfusion injury-induced apoptosis and ROS production in the kidney [30]. Collectively, these findings suggest that BRD4 inhibition protects the kidney under pathological stimuli and BRD4 serves as a potential therapeutic target for kidney protection. Consistent with these findings, our study reveals that BRD4 inhibition protects podocytes from HG-induced injury. Since HGinduced podocyte injury is involved in the pathogenesis of diabetic nephropathy, our study suggests that BRD4 may play an important role in diabetic nephropathy and serve as a potential therapeutic target. The function of BRD4 in the kidney is mainly focused on the kidney epithelial cells [28e30]. Hypoxia/reoxygenation-induced apoptosis and ROS production are significantly decreased by BRD4 inhibition [30]. However, little is known about the role of BRD4 in the podocytes of the kidney. Herein, our results demonstrated that BRD4 inhibition by siRNA or its chemical inhibitor markedly improved cell viability and reduced apoptosis and ROS
production in HG-exposed podocytes. Therefore, our study suggests that BRD4 inhibition also exerts its cytoprotective function in the podocytes. BRD4 has emerged as a critical regulator for oxidative stress [31,32]. Interestingly, the antioxidant effect of BRD4 inhibition is associated with potentiation of Nrf2 antioxidant signaling. BRD4 inhibition promotes the recruitment of Nrf2 to ARE binding sites on antioxidant gene promoters [14]. Keap1 is a transcriptional target of BRD4, and BRD4 inhibition represses Keap1 expression and thereby leads to nuclear Nrf2 translocation and accumulation [15]. Thus, BRD4 inhibition upregulates antioxidant gene expression by increasing Nrf2/ARE signaling via decreasing Keap1 [15,33]. BRD4 inhibition reduces ROS production induced by transforming growth factor-b in corneal myofibroblasts by enhancing Nrf2-dependant antioxidant signaling, and it reverses mechanical injury-induced corneal scarring [34]. In rat chondrocytes, BRD4 inhibition downregulates hydrogen-peroxide-induced apoptosis and ROS production and delays chondrocyte degeneration [35]. Consistent with these findings, our results demonstrated that BRD4 inhibition downregulated Keap1 expression and upregulated Nrf2/ARE antioxidant signaling, changes that abrogated HG-induced podocyte injury. Therefore, the BRD4/Nrf2/ARE signaling axis may play an important role in regulating podocyte injury in diabetic nephropathy. Accumulating evidence suggests an important role for Nrf2 in diabetic nephropathy [18]. Enhancing the Nrf2 antioxidant pathway alleviates the development of diabetic nephropathy [36e38]. HG induces excessive ROS production and apoptosis, both
106
H. Zuo et al. / Biochimie 165 (2019) 100e107
of which contribute to the development of diabetic nephropathy [39,40]. Notably, Nrf2/ARE signaling activation attenuates HGinduced injury in podocytes [41,42]. Here, BRD4 regulated Nrf2/ ARE signaling in podocytes, a finding that reveals a novel function for BRD4/Nrf2/ARE signaling in modulating HG-induced podocyte injury. Considering that HG-induced podocyte injury is thought to be an important early event in diabetic nephropathy [43,44], targeting BRD4 to potentiate Nrf2-mediated antioxidant signaling may have potential applications for treating diabetic nephropathy. Taken together, our study demonstrated that BRD4 inhibition protects podocytes from HG-induced apoptosis and oxidative damage by potentiating Nrf2/ARE antioxidant signaling via downregulating Keap1. These findings highlight an important role for the BRD4/Keap1/Nrf2/ARE signaling axis in regulating podocyte injury in diabetic nephropathy and provide novel insights into the molecular pathogenesis of diabetic nephropathy. Therefore, BRD4 may serve as a promising therapeutic target for preventing diabetic nephropathy. However, the precise role of BRD4 in regulating podocyte injury in diabetic nephropathy needs further in vivo investigation using animal models. Conflict of interest The authors declare no conflict of interest. Contributor Hong Zuo: conception of the work. Hong Zuo: collection of data. Shujin Wang, Jia Feng and Xufeng Liu: analysis of data. Hong Zuo: writing of manuscript. Acknowledgments This study was supported by The Fund of Xi'an City (J201902047). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.biochi.2019.07.012. References [1] K. Reidy, H.M. Kang, T. Hostetter, K. Susztak, Molecular mechanisms of diabetic kidney disease, J. Clin. Investig. 124 (2014) 2333e2340. [2] S.S. Badal, F.R. Danesh, New insights into molecular mechanisms of diabetic kidney disease, Am. J. Kidney Dis. 63 (2014) S63eS83. [3] G. Soldatos, M.E. Cooper, Diabetic nephropathy: important pathophysiologic mechanisms, Diabetes Res. Clin. Pract. 13 (2008) 042. [4] G. Remuzzi, A. Schieppati, P. Ruggenenti, Clinical practice. Nephropathy in patients with type 2 diabetes, N. Engl. J. Med. 346 (2002) 1145e1151. [5] P.W. Mathieson, The podocyte as a target for therapies–new and old, Nat. Rev. Nephrol. 8 (2011) 52e56. [6] P. Anil Kumar, G.I. Welsh, M.A. Saleem, R.K. Menon, Molecular and cellular events mediating glomerular podocyte dysfunction and depletion in diabetes mellitus, Front. Endocrinol. 5 (2014) 151. [7] S.Y. Wu, C.M. Chiang, The double bromodomain-containing chromatin adaptor Brd4 and transcriptional regulation, J. Biol. Chem. 282 (2007) 13141e13145. [8] B.N. Devaiah, D.S. Singer, Two faces of brd4: mitotic bookmark and transcriptional lynchpin, Transcription 4 (2013) 13e17. [9] B. Donati, E. Lorenzini, A. Ciarrocchi, BRD4 and cancer: going beyond transcriptional regulation, Mol. Cancer 17 (2018) 018e0915. [10] M.K. Jang, K. Mochizuki, M. Zhou, H.S. Jeong, J.N. Brady, K. Ozato, The bromodomain protein Brd4 is a positive regulatory component of P-TEFb and stimulates RNA polymerase II-dependent transcription, Mol. Cell 19 (2005) 523e534. [11] T. Xiang, J.Y. Bai, C. She, D.J. Yu, X.Z. Zhou, T.L. Zhao, Bromodomain protein BRD4 promotes cell proliferation in skin squamous cell carcinoma, Cell. Signal. 42 (2018) 106e113.
[12] K.L. Cheung, F. Zhang, A. Jaganathan, R. Sharma, Q. Zhang, T. Konuma, T. Shen, J.Y. Lee, C. Ren, C.H. Chen, G. Lu, M.R. Olson, W. Zhang, M.H. Kaplan, D.R. Littman, M.J. Walsh, H. Xiong, L. Zeng, M.M. Zhou, Distinct roles of Brd2 and Brd4 in potentiating the transcriptional program for Th17 cell differentiation, Mol. Cell 65 (2017) 1068e1080. [13] Y. Tan, L. Wang, Y. Du, X. Liu, Z. Chen, X. Weng, J. Guo, H. Chen, M. Wang, X. Wang, Inhibition of BRD4 suppresses tumor growth in prostate cancer via the enhancement of FOXO1 expression, Int. J. Oncol. 53 (2018) 2503e2517. [14] C. Michaeloudes, N. Mercado, C. Clarke, P.K. Bhavsar, I.M. Adcock, P.J. Barnes, K.F. Chung, Bromodomain and extraterminal proteins suppress NF-E2-related factor 2-mediated antioxidant gene expression, J. Immunol. 192 (2014) 4913e4920. [15] M. Hussong, S.T. Borno, M. Kerick, A. Wunderlich, A. Franz, H. Sultmann, B. Timmermann, H. Lehrach, M. Hirsch-Kauffmann, M.R. Schweiger, The bromodomain protein BRD4 regulates the KEAP1/NRF2-dependent oxidative stress response, Cell Death Dis. 24 (2014) 157. [16] A. Kobayashi, T. Ohta, M. Yamamoto, Unique function of the Nrf2-Keap1 pathway in the inducible expression of antioxidant and detoxifying enzymes, Methods Enzymol. 378 (2004) 273e286. [17] M. McMahon, K. Itoh, M. Yamamoto, J.D. Hayes, Keap1-dependent proteasomal degradation of transcription factor Nrf2 contributes to the negative regulation of antioxidant response element-driven gene expression, J. Biol. Chem. 278 (2003) 21592e21600. [18] W. Cui, X. Min, X. Xu, B. Du, P. Luo, Role of nuclear factor erythroid 2-related factor 2 in diabetic nephropathy, J. Diabetes Res. 3797802 (2017) 23. [19] J.X. Shi, Q.J. Wang, H. Li, Q. Huang, Silencing of USP22 suppresses high glucose-induced apoptosis, ROS production and inflammation in podocytes, Mol. Biosyst. 12 (2016) 1445e1456. [20] F. Thilo, M. Lee, S. Xia, A. Zakrzewicz, M. Tepel, High glucose modifies transient receptor potential canonical type 6 channels via increased oxidative stress and syndecan-4 in human podocytes, Biochem. Biophys. Res. Commun. 450 (2014) 312e317. [21] B.C. Liu, X. Song, X.Y. Lu, D.T. Li, D.C. Eaton, B.Z. Shen, X.Q. Li, H.P. Ma, High glucose induces podocyte apoptosis by stimulating TRPC6 via elevation of reactive oxygen species, Biochim. Biophys. Acta 6 (2013) 13. [22] V. Soetikno, W. Arozal, M. Louisa, R. Setiabudy, New insight into the molecular drug target of diabetic nephropathy, Internet J. Endocrinol. 2014 (2014) 968681. [23] X. Wu, D. Liu, X. Gao, F. Xie, D. Tao, X. Xiao, L. Wang, G. Jiang, F. Zeng, Inhibition of BRD4 suppresses cell proliferation and induces apoptosis in renal cell carcinoma, Cell. Physiol. Biochem. 41 (2017) 1947e1956. [24] Z.Y. Xing, Y. Wang, L. Cheng, J. Chen, X.Z. He, W. Xing, Bromodomain-containing protein 4 (BRD4) inhibition sensitizes palomid 529-induced anti-renal cell carcinoma cell activity in vitro and in vivo, Cell. Physiol. Biochem. 50 (2018) 640e653. [25] H. Zhu, J.H. Mao, Y. Wang, D.H. Gu, X.D. Pan, Y. Shan, B. Zheng, Dual inhibition of BRD4 and PI3K-AKT by SF2523 suppresses human renal cell carcinoma cell growth, Oncotarget 8 (2017) 98471e98481. [26] B. Suarez-Alvarez, J.L. Morgado-Pascual, S. Rayego-Mateos, R.M. Rodriguez, R. Rodrigues-Diez, P. Cannata-Ortiz, A.B. Sanz, J. Egido, P.L. Tharaux, A. Ortiz, C. Lopez-Larrea, M. Ruiz-Ortega, Inhibition of bromodomain and extraterminal domain family proteins ameliorates experimental renal damage, J. Am. Soc. Nephrol. 28 (2017) 504e519. [27] G. Zhang, R. Liu, Y. Zhong, A.N. Plotnikov, W. Zhang, L. Zeng, E. Rusinova, G. Gerona-Nevarro, N. Moshkina, J. Joshua, P.Y. Chuang, M. Ohlmeyer, J.C. He, M.M. Zhou, Down-regulation of NF-kappaB transcriptional activity in HIVassociated kidney disease by BRD4 inhibition, J. Biol. Chem. 287 (2012) 28840e28851. [28] X. Zhou, L.X. Fan, D.J. Peters, M. Trudel, J.E. Bradner, X. Li, Therapeutic targeting of BET bromodomain protein, Brd4, delays cyst growth in ADPKD, Hum. Mol. Genet. 24 (2015) 3982e3993. [29] C. Xiong, M.V. Masucci, X. Zhou, N. Liu, X. Zang, E. Tolbert, T.C. Zhao, S. Zhuang, Pharmacological targeting of BET proteins inhibits renal fibroblast activation and alleviates renal fibrosis, Oncotarget 7 (2016) 69291e69308. [30] H. Liu, L. Wang, X. Weng, H. Chen, Y. Du, C. Diao, Z. Chen, X. Liu, Inhibition of Brd4 alleviates renal ischemia/reperfusion injury-induced apoptosis and endoplasmic reticulum stress by blocking FoxO4-mediated oxidative stress, Redox Biol. 24 (2019) 11. [31] T. Ijaz, M. Jamaluddin, Y. Zhao, Y. Zhang, J. Jay, C.C. Finnerty, D.N. Herndon, R.G. Tilton, A.R. Brasier, Coordinate activities of BRD4 and CDK9 in the transcriptional elongation complex are required for TGFbeta-induced Nox4 expression and myofibroblast transdifferentiation, Cell Death Dis. 8 (2017) 434. [32] Y.M. Yang, R.H. Shi, C.X. Xu, L. Li, BRD4 expression in patients with essential hypertension and its effect on blood pressure in spontaneously hypertensive rats, J. Am. Soc. Hypertens. 12 (2018) e107ee117. [33] M. Huang, L. Zhu, J.S. Garcia, M.X. Li, A.J. Gentles, B.S. Mitchell, Brd4 regulates the expression of essential autophagy genes and Keap1 in AML cells, Oncotarget 9 (2018) 11665e11676. [34] M. Qu, X. Zhang, X. Hu, M. Dong, X. Pan, J. Bian, Q. Zhou, BRD4 inhibitor JQ1 inhibits and reverses mechanical injury-induced corneal scarring, Cell Death Dis. 4 (2018) 5. [35] Q.D. An, Y.Y. Li, H.X. Zhang, J. Lu, X.D. Yu, Q. Lin, Y.G. Zhang, Inhibition of bromodomain-containing protein 4 ameliorates oxidative stress-mediated apoptosis and cartilage matrix degeneration through activation of NF-E2-
H. Zuo et al. / Biochimie 165 (2019) 100e107
[36]
[37]
[38]
[39]
related factor 2-heme oxygenase-1 signaling in rat chondrocytes, J. Cell. Biochem. 119 (2018) 7719e7728. B. Zhang, X. Zhang, C. Zhang, Q. Shen, G. Sun, X. Sun, Notoginsenoside R1 protects db/db mice against diabetic nephropathy via upregulation of Nrf2mediated HO-1 expression, Molecules 24 (2019) E247. Z. Xie, L. Zhong, Y. Wu, X. Wan, H. Yang, X. Xu, P. Li, Carnosic acid improves diabetic nephropathy by activating Nrf2/ARE and inhibition of NF-kappaB pathway, Phytomedicine 47 (2018) 161e173. A. Mathur, V.K. Pandey, P. Kakkar, Activation of GSK3beta/beta-TrCP axis via PHLPP1 exacerbates Nrf2 degradation leading to impairment in cell survival pathway during diabetic nephropathy, Free Radic. Biol. Med. 120 (2018) 414e424. L.N. Sun, X.C. Liu, X.J. Chen, G.J. Guan, G. Liu, Curcumin attenuates high glucose-induced podocyte apoptosis by regulating functional connections between caveolin-1 phosphorylation and ROS, Acta Pharmacol. Sin. 37 (2016) 645e655.
107
[40] C. Dong, H. Zheng, S. Huang, N. You, J. Xu, X. Ye, Q. Zhu, Y. Feng, Q. You, H. Miao, D. Ding, Y. Lu, Heme oxygenase-1 enhances autophagy in podocytes as a protective mechanism against high glucose-induced apoptosis, Exp. Cell Res. 337 (2015) 146e159. [41] C. Wang, C. Li, H. Peng, Z. Ye, J. Zhang, X. Liu, T. Lou, Activation of the Nrf2-ARE pathway attenuates hyperglycemia-mediated injuries in mouse podocytes, Cell. Physiol. Biochem. 34 (2014) 891e902. [42] Q. Zhang, Q. Deng, J. Zhang, J. Ke, Y. Zhu, R.W. Wen, Z. Ye, H. Peng, Z.Z. Su, C. Wang, T. Lou, Activation of the Nrf2-ARE pathway ameliorates hyperglycemia-mediated mitochondrial dysfunction in podocytes partly through Sirt1, Cell. Physiol. Biochem. 48 (2018) 1e15. [43] F.C. Brosius, R.J. Coward, Podocytes, signaling pathways, and vascular factors in diabetic kidney disease, Adv. Chron. Kidney Dis. 21 (2014) 304e310. [44] I.H. Ali, D.P. Brazil, Under the right conditions: protecting podocytes from diabetes-induced damage, Stem Cell Res. Ther. 4 (2013) 119.
Contents lists available at ScienceDirect
Biochimie journal homepage: www.elsevier.com/locate/biochi
Research paper
BRD4 contributes to high-glucose-induced podocyte injury by modulating Keap1/Nrf2/ARE signaling Hong Zuo*, Shujin Wang, Jia Feng, Xufeng Liu Department of Endocrinology, the Ninth Hospital of Xian, Xi'an, 710054, Shaanxi Province, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 18 May 2019 Accepted 12 July 2019 Available online 17 July 2019
High glucose (HG)-induced podocyte injury contributes to the pathogenesis of diabetic nephropathy, a severe complication of diabetes. Bromodomain-containing protein 4 (BRD4) has emerged as a critical regulator for cell injury. However, whether BRD4 participates in HG-induced podocyte injury remains unclear. In this study, we aimed to explore the potential role of BRD4 in regulating HG-induced podocyte injury and its underlying molecular mechanism. HG exposure significantly upregulated BRD4 in podocytes. BRD4 inhibition by small interfering RNA or its chemical inhibitor (JQ1) markedly repressed HGinduced apoptosis and reactive oxygen species (ROS) production. By contrast, BRD4 overexpression exacerbated HG-induced podocyte injury. Moreover, BRD4 inhibition potentiated nuclear factor (erythroid-derived 2)-like 2 (Nrf2) signaling associated with suppression of Kelch-like ECH-associated protein (Keap1). BRD4 inhibition promoted Nrf2 nuclear translocation and upregulated the transcriptional activity of Nrf2/antioxidant response element (ARE). However, Nrf2 silencing partially reversed BRD4-inhibition-mediated protection against HG-induced podocyte injury. Overall, these results suggest that BRD4 inhibition confers cytoprotection against HG injury in podocytes through potentiation of Nrf2/ ARE antioxidant signaling. This finding implicates BRD4/Nrf2/ARE signaling in the pathogenesis of diabetic nephropathy. © 2019 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.
Keywords: BRD4 Diabetic nephropathy High glucose Podocyte Nrf2
1. Introduction Diabetic nephropathy is one of the most prevalent chronic complications to diabetes, and it contributes to the development of end-stage renal disease [1,2]. Hyperglycemia-induced cellular injury and dysfunction is the major cause of diabetic nephropathy [3,4]. Podocytes distributed on the outer surface of the glomerular basement membrane help maintain the integrity of the glomerular filtration barrier [5]. Podocytes are highly differentiated cells in the kidney with limited capability to repair and/or regenerate, and the
Abbreviations: HG, high glucose; NG, normal glucose; BRD4, bromodomaincontaining protein 4; Nrf2, nuclear factor (erythroid-derived 2)-like 2; Keap1, Kelch-like ECH-associated protein; ARE, antioxidant response element; ROS, reactive oxygen species; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; CCK-8, Cell Counting Kit-8; OD, optical density; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling. * Corresponding author. Department of Endocrinology, the Ninth Hospital of Xian, No.151 East Section of South Ring, Beilin District, Xi'an, 710054, Shaanxi Province, China. E-mail address: [email protected] (H. Zuo).
reduction of podocytes from high glucose (HG)-induced apoptosis contributes to the development of diabetic nephropathy [6]. Thus, therapies aimed at preventing HG-induced podocyte injury have potential clinical benefits for diabetic nephropathy. Bromodomain-containing protein 4 (BRD4), a member of the bromodomain and extra-terminal protein family, plays an important role in normal development and disease progression [7,8]. BRD4 is a crucial regulator of transcription, and it regulates the expression of various genes [9,10]. Therefore, BRD4 participates in diverse biological processes, including cell cycle, proliferation, apoptosis, and differentiation [11e13]. Interestingly, accumulating evidence suggests that BRD4 inhibition confers a cytoprotective effect against stress-induced apoptosis and oxidative stress [14,15]. Notably, the underlying molecular mechanism is associated with its regulatory effect on nuclear factor (erythroid-derived 2)-like 2 (Nrf2) antioxidant signaling. Nrf2 functions as a redox-sensitive transcription factor that protects cells from oxidative damage via activating the transcription of cytoprotective genes, such as heme oxygenase and NADPH quinone oxidoreductase 1 [16]. Kelch-like ECH-associated protein
https://doi.org/10.1016/j.biochi.2019.07.012 0300-9084/© 2019 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.
H. Zuo et al. / Biochimie 165 (2019) 100e107
(Keap1) regulates Nrf2 protein stabilization. When bound to Keap1, Nrf2 is polyubiquitinated and degraded through the ubiquitin proteasome pathway [17]. In contrast, Nrf2 dissociated from Keap1 translocates to the nucleus where it binds to the antioxidant response element (ARE) within the promoter region of antioxidant and detoxifying enzymes and initiates gene transcription [17]. Therefore, Nrf2 signaling activation facilitates the elimination of reactive oxygen species (ROS) and confers cytoprotection. A recent study documented that Nrf2 plays an important role in the development of diabetic nephropathy [18] and may serve as a potential and promising therapeutic target for this disease. HG induces oxidative stress and apoptosis in podocytes [19e21]. However, whether BRD4 regulates HG-induced podocyte injury remains unclear. Here, our study aimed to explore the potential role of BRD4 in regulating HG-induced podocyte injury and its underlying molecular mechanism.
101
radioimmunoprecipitation assay (RIPA) buffer that contained protease inhibitor (Beyotime Biotechnology, Shanghai, China). The supernatants were harvested by centrifugation, and protein concentrations were determined with the Enhanced BCA Protein Assay Kit (Beyotime Biotechnology). For protein separation, equal amounts of protein from each sample were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The separated proteins were then transferred onto a polyvinylidene fluoride transfer membrane (Thermo Scientific Pierce), and the membrane was incubated in 5% fat-free milk for 1 h at room temperature. Subsequently, the membrane was immunoblotted with primary antibodies against BRD4, Nrf2, Keap1, b-actin and Lamin B2 (Abcam, Cambridge, UK) at 4 C overnight. The membrane was then probed with horseradish-peroxidase-conjugated secondary antibody (Abcam) for 1 h at room temperature. Finally, ECL Plus Substrate (Thermo Scientific Pierce) was utilized to develop target protein bands.
2. Materials and methods 2.5. Cell viability assay 2.1. Cell culture and HG exposure The mouse podocyte cell line MPC5 was purchased from BeNa Culture Collection (Kunshan City, China) and cultured as per the manufacturer's instructions. In brief, cells were cultured in RPMI Medium 1640 (Gibco; Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco), 100 U/ml penicillin and 100 mg/ml streptomycin. Cells were grown at 37 C in a humidified incubator containing 5% CO2. Podocytes were maintained in medium containing normal glucose concentration (NG, 5 mM) or high glucose concentration (HG, 30 mM) for 24 h to induce HG injury. 2.2. Cell transfection The small interfering RNAs (siRNAs) that targeted BRD4 or Nrf2 were purchased from RiboBio (Guangzhou, China). The BRD4 expression vector was constructed by inserting the BRD4 open reading frame into the pcDNA3.1 vector. Cell transfection of siRNAs or vectors was carried out using Lipofectamine 3000 Reagent (Invitrogen; Thermo Fisher Scientific) following the manufacturer's manuals. Briefly, cells were seeded into cultured until cells reached 70e90% confluent at the time of transfection. The siRNA- or plasmid DNA-lipid complexes were prepared by adding siRNAs or plasmids and Lipofectamine 3000 Reagent to the Opti-MEM medium. The mixture was incubated for 15 min at room temperature and then added to cells at 50 ml/well. After 48 h-transfection, the transfection efficacy was determined by reverse transcriptionquantitative polymerase chain reaction (RT-qPCR) or Western blot analysis.
Cell viability was determined with the Enhanced Cell Counting Kit-8 (CCK-8) following the manufacturer's protocols. Briefly, cells were seeded into a 96-well plate and transfected with BRD4 siRNA or expression vector for 24 h followed by HG exposure for 24 h. Thereafter, 10 ml CCK-8 solution was added to each well and incubated for 1 h at 37 C. The optical density (OD) value of the colorimetric solution at 450 nm was measured with a microplate reader (Bio-Rad, Hercules, CA, USA). 2.6. Caspase-3 activity assay Caspase-3 activity was measured with a Colorimetric Caspase-3 Assay Kit (Abcam) as per the standard procedure. After the indicated treatment, cells were collected and resuspended into chilled cell lysis buffer and incubated for 10 min on ice. The supernatants were harvested by centrifugation and transferred to a new 96-well plate at 50 ml/well. Then, 50 ml reaction buffer and 5 ml DEVD-p-NA substrate (4 mM) were added to each well followed by incubation for 2 h at 37 C. The OD value of the colorimetric solution at 405 nm was determined by a microplate reader (Bio-Rad). 2.7. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay Cells on coverslips were fixed with 4% paraformaldehyde for 30 min at room temperature. Next, cells were permeabilized with 0.3% Triton X-100 for 5 min at room temperature. Subsequently, cells were incubated with TUNEL detection solution for 1 h at 37 C in the dark. The TUNEL-positive cells were observed and counted under a fluorescence microscope.
2.3. RNA extraction and RT-qPCR analysis 2.8. ROS measurement assay Total RNA was extracted from cells by using TRIzol reagent (Invitrogen) and converted into complementary DNA (cDNA) using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems; Thermo Fisher Scientific). RT-qPCR was performed using the PowerUp SYBR Green PCR Master Mix (Applied Biosystems) on an Applied Biosystems 7500 Real-Time PCR System. The housekeeping gene b-actin was used for standardizing target gene expression. The data for target gene expression was calculated using the 2DDCt method.
Cells were treated with 10 mM DCFH-DA (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany) in serum-free medium and incubated at 37 C for 30 min. After washing with serum-free medium, intracellular ROS levels were detected by measuring fluorescence intensity. The fluorescence intensity was detected by flow cytometry with excitation wavelength of 488 nm and emission wavelength of 525 nm. 2.9. Luciferase reporter assay
2.4. Western blot For total protein extraction, cells were lysed in ice-cold
Cells were co-transfected with the pARE-luc reporter vector (Beyotime Biotechnology), Renilla luciferase reporter vector pRL-
102
H. Zuo et al. / Biochimie 165 (2019) 100e107
TK, and BRD4 siRNA or expression vector for 24 h, followed by HG exposure for 24 h. The cells were then lysed, and luciferase activity was determined with the Dual-GLO Luciferase Assay System (Promega, Madison, WI, USA), as per the manufacturer's instructions.
t-test or one-way analysis of variance with the Bonferroni post-hoc test (where appropriate). Statistical analyses were performed with SPSS Statistics Version 19.0 (SPSS Inc., Chicago, IL, USA) or GraphPad Prism 6 (GraphPad Software, San Diego, CA, USA). p < 0.05 was considered significant.
2.10. Statistical analysis
3. Results
Results are expressed as the mean ± standard deviation (SD). Statistical significance of differences was determined by Student's
3.1. BRD4 upregulation in HG-stimulated podocytes To investigate the potential relevance of BRD4 in HG-induced podocyte injury, we detected the effect of HG exposure on BRD4 expression. RT-qPCR revealed that HG exposure significantly elevated podocyte BRD4 mRNA expression (Fig. 1A). Consistently, western blot analysis demonstrated that HG also markedly upregulated BRD4 protein expression (Fig. 1B). These results suggest that BRD4 may participate in regulating HG-induced podocyte injury. 3.2. BRD4 inhibition alleviated HG injury in podocytes in vitro
Fig. 1. Effect of HG exposure on BRD4 expression in podocytes. Podocytes were treated with either normal glucose (NG, 5 mM) or high glucose (HG, 30 mM) for 24 h and then harvested for BRD4 detection. (A) Relative BRD4 mRNA expression in HG-exposed podocytes was determined by RT-qPCR and normalized against b-actin. (B) BRD4 protein expression in HG-exposed podocytes was examined by western blot. N ¼ 3, *p < 0.05.
To investigate the function of BRD4 in HG-induced podocyte injury, we performed BRD4 loss-of-function experiments. BRD4 siRNA was utilized to deplete BRD4 expression in podocytes; its downregulation was confirmed by RT-qPCR and western blot analyses (Fig. 2A and B). BRD4 knockdown significantly rescued cell viability, which was inhibited by HG exposure (Fig. 2C). Moreover, BRD4 silencing significantly reduced HG-induced apoptosis (Fig. 2D and E). BRD4 silencing also markedly reduced HG-induced excessive ROS production (Fig. 2F). Consistently, treatment with the
Fig. 2. BRD4 silencing downregulates HG-induced apoptosis and oxidative stress in podocytes. Podocytes were transfected with either BRD4 or NC siRNA for 48 h followed by HG exposure for 24 h. (A) Relative BRD4 mRNA expression was determined by RT-qPCR and normalized against b-actin. (B) BRD4 protein expression was examined by western blot. (C) Effect of BRD4 knockdown on HG-impaired cell viability was assessed by the CCK-8 assay. (D) Effect of BRD4 knockdown on HG-induced caspase-3 activity was evaluated by a colorimetric caspase-3 assay. (E) Effect of BRD4 knockdown on HG-induced cell apoptosis was measured with the TUNEL assay. (F) Effect of BRD4 knockdown on HG-induced ROS production was detected by DCFH-DA staining. N ¼ 3, *p < 0.05.
H. Zuo et al. / Biochimie 165 (2019) 100e107
BRD4 chemical inhibitor JQ1 significantly attenuated HG-induced apoptosis and ROS production (Fig. 3AeD). Downregulation of nephrin and or podocin is an indicator of podocyte injury [22]. Notably, BRD4 inhibition significantly increased the expression of nephrin and or podocin in HG-exposed podocytes (Supplementary Fig. 1). Collectively, these results suggest that BRD4 inhibition protects podocytes from HG injury. 3.3. BRD4 overexpression exacerbated HG injury in podocytes To confirm that BRD4 contributes to HG injury in podocytes, we performed BRD4 gain-of-function experiments by using a BRD4 expression vector. BRD4 overexpression in vector-transfected cells was confirmed by western blot (Fig. 4A). This overexpression significantly decreased cell viability in HG-exposed cells (Fig. 4B). Moreover, BRD4 overexpression exacerbated HG-induced cell apoptosis and ROS production in podocytes (Fig. 4C and D). These results suggest that BRD4 overexpression promotes HG injury in podocytes. 3.4. BRD4 inhibition potentiated Nrf2/ARE signaling in HG-exposed podocytes BRD4 inhibition confers cytoprotection by enhancing Nrf2/ARE signaling. Thus, we next investigated whether BRD4 inhibition contributes to Nrf2/ARE signaling regulation in HG-exposed podocytes. BRD4 knockdown significantly downregulated Keap1 expression and upregulated Nrf2 nuclear translocation (Fig. 5A and B). Moreover, BRD4 knockdown enhanced Nrf2/ARE-dependent transcriptional activity (Fig. 5C). Consistently, BRD4 inhibition by its chemical inhibitor JQ1 also enhanced the activation of Nrf2/ARE
103
signaling (Fig. 5EeF). Overall, our results suggest that BRD4 inhibition potentiates Nrf2/ARE signaling in HG-exposed podocytes. 3.5. BRD4-inhibition-mediated protection was reversed by Nrf2 inhibition To confirm whether Nrf2/ARE signaling contributes to BRD4inhibition-mediated protection, we detected the consequence of Nrf2 inhibition on this effect in podocytes. Transfection of Nrf2 siRNA significantly downregulated Nrf2 expression (Fig. 6A). Nrf2 inhibition significantly abrogated the promoting effect of BRD4 inhibition on Nrf2/ARE signaling (Fig. 6B). Moreover, BRD4inhibition-mediated protection on HG-induced apoptosis and ROS production was partially reversed by Nrf2 inhibition (Fig. 6CeE). Overall, these results suggest that BRD4 inhibition protects podocytes from HG injury by potentiating Nrf2/ARE signaling. 4. Discussion In the present study, we provided convincing evidence that BRD4 contributes to regulation of HG-induced podocyte injury. Our findings revealed that BRD4 inhibition by siRNA or its chemical inhibitor significantly repressed HG-induced apoptosis and ROS production in podocytes. The underlying molecular mechanism was associated with its promoting effect on Nrf2/ARE antioxidant signaling via downregulation of Keap1 (Fig. 6F). Therefore, our findings highlight the importance of BRD4/Nrf2/ARE signaling in regulating podocyte injury. BRD4 dysregulation is implicated in various maladies, particularly in kidney disease [23e25]. It is reported that BRD4 inhibition attenuates experimental renal damage with inactivation of nuclear
Fig. 3. Treatment with the BRD4 inhibitor JQ1 decreased HG injury in podocytes. Podocytes were treated with 5 mM JQ1 for 48 h and then stimulated with HG for 24 h. (A) Cell viability was detected with the CCK-8 assay. (B) Caspase-3 activity was measured by a colorimetric caspase-3 assay. (C) Cell apoptosis was detected with the TUNEL assay. (D) ROS production was determined by DCFH-DA staining. N ¼ 3, *p < 0.05.
104
H. Zuo et al. / Biochimie 165 (2019) 100e107
Fig. 4. BRD4 overexpression exacerbated HG-induced apoptosis and ROS production in podocytes. Podocytes were transfected with BRD4 expression vector (BRD4) or empty vector (EV) for 48 h, stimulated with HG for 24 h, and then (A) BRD4 protein expression was detected by western blot. (B) Effect of BRD4 overexpression on HG-impaired cell viability was detected by the CCK-8 assay. (C) Effect of BRD4 overexpression on HG-induced caspase-3 activity was measured by a colorimetric caspase-3 assay. (D) Effect of BRD4 overexpression on HG-induced ROS production was assessed by DCFH-DA staining. N ¼ 3, *p < 0.05.
Fig. 5. BRD4 inhibition potentiated Nrf2/ARE signaling. Podocytes were transfected with BRD4 siRNA for 48 h followed by HG exposure for 24 h. Effect of BRD4 silencing on (A) Keap1 and (B) nuclear Nrf2 (nu-Nrf2) protein expression was detected by western blot. (C) Effect of BRD4 silencing on Nrf2/ARE-dependent transcriptional activity was determined by an ARE reporter. Podocytes were pretreated with 5 mM JQ1 for 48 h and then stimulated with HG for 24 h. Effect of JQ1 treatment on (D) Keap1 and (E) nuclear Nrf2 (nu-Nrf2) protein expression was detected by western blot. (F) Effect of JQ1 treatment on Nrf2/ARE-dependent transcriptional activity was determined by an ARE reporter. N ¼ 3, *p < 0.05.
H. Zuo et al. / Biochimie 165 (2019) 100e107
105
Fig. 6. BRD4 inhibition protected podocytes from HG injury via Nrf2/ARE signaling. Podocytes were transfected with Nrf2 siRNA in the presence of JQ1 for 48 h, followed by HG exposure for 24 h. (A) Nrf2 protein expression was determined by western blot. (B) Nrf2/ARE-dependent transcriptional activity was determined by an ARE reporter. (C) Cell viability was detected by the CCK-8 assay. (D) Caspase-3 activity was measured by a colorimetric caspase-3 assay. (E) ROS production was determined by DCFH-DA staining. N ¼ 3, *p < 0.05. (F) A graphic model of BRD4/Keap1/Nrf2/ARE signaling in regulating HG-induced injury in podocytes. HG treatment upregulates BRD4 expression which then promotes Keap1 expression, leading to enhanced Nrf2 degradation. Therefore, BRD4 inhibition decreases Keap1 expression, which enhanced Nrf2 accumulation and nuclear translocation, leading to transcription of Nrf2/ARE target genes that protect podocytes from HG-induced injury.
factor (NF)-kB signaling [26]. Moreover, BRD4 inhibition ameliorates human-immunodeficiency-virus-induced inflammation and kidney injury in mouse models via inactivation of NF-kB signaling [27]. Zhou et al. reported that BRD4 inhibition suppresses cystic epithelial cell proliferation, delays kidney enlargement, and preserves renal function in autosomal dominant polycystic kidney disease [28]. BRD4 is highly expressed in fibrotic kidneys, and BRD4 inhibition prevents the activation of renal fibroblasts [29]. Notably, BRD4 inhibition represses ROS production in the kidney induced by unilateral ureteral obstruction and delays the progression of renal fibrosis [29]. Moreover, BRD4 inhibition also alleviates ischemia/ reperfusion injury-induced apoptosis and ROS production in the kidney [30]. Collectively, these findings suggest that BRD4 inhibition protects the kidney under pathological stimuli and BRD4 serves as a potential therapeutic target for kidney protection. Consistent with these findings, our study reveals that BRD4 inhibition protects podocytes from HG-induced injury. Since HGinduced podocyte injury is involved in the pathogenesis of diabetic nephropathy, our study suggests that BRD4 may play an important role in diabetic nephropathy and serve as a potential therapeutic target. The function of BRD4 in the kidney is mainly focused on the kidney epithelial cells [28e30]. Hypoxia/reoxygenation-induced apoptosis and ROS production are significantly decreased by BRD4 inhibition [30]. However, little is known about the role of BRD4 in the podocytes of the kidney. Herein, our results demonstrated that BRD4 inhibition by siRNA or its chemical inhibitor markedly improved cell viability and reduced apoptosis and ROS
production in HG-exposed podocytes. Therefore, our study suggests that BRD4 inhibition also exerts its cytoprotective function in the podocytes. BRD4 has emerged as a critical regulator for oxidative stress [31,32]. Interestingly, the antioxidant effect of BRD4 inhibition is associated with potentiation of Nrf2 antioxidant signaling. BRD4 inhibition promotes the recruitment of Nrf2 to ARE binding sites on antioxidant gene promoters [14]. Keap1 is a transcriptional target of BRD4, and BRD4 inhibition represses Keap1 expression and thereby leads to nuclear Nrf2 translocation and accumulation [15]. Thus, BRD4 inhibition upregulates antioxidant gene expression by increasing Nrf2/ARE signaling via decreasing Keap1 [15,33]. BRD4 inhibition reduces ROS production induced by transforming growth factor-b in corneal myofibroblasts by enhancing Nrf2-dependant antioxidant signaling, and it reverses mechanical injury-induced corneal scarring [34]. In rat chondrocytes, BRD4 inhibition downregulates hydrogen-peroxide-induced apoptosis and ROS production and delays chondrocyte degeneration [35]. Consistent with these findings, our results demonstrated that BRD4 inhibition downregulated Keap1 expression and upregulated Nrf2/ARE antioxidant signaling, changes that abrogated HG-induced podocyte injury. Therefore, the BRD4/Nrf2/ARE signaling axis may play an important role in regulating podocyte injury in diabetic nephropathy. Accumulating evidence suggests an important role for Nrf2 in diabetic nephropathy [18]. Enhancing the Nrf2 antioxidant pathway alleviates the development of diabetic nephropathy [36e38]. HG induces excessive ROS production and apoptosis, both
106
H. Zuo et al. / Biochimie 165 (2019) 100e107
of which contribute to the development of diabetic nephropathy [39,40]. Notably, Nrf2/ARE signaling activation attenuates HGinduced injury in podocytes [41,42]. Here, BRD4 regulated Nrf2/ ARE signaling in podocytes, a finding that reveals a novel function for BRD4/Nrf2/ARE signaling in modulating HG-induced podocyte injury. Considering that HG-induced podocyte injury is thought to be an important early event in diabetic nephropathy [43,44], targeting BRD4 to potentiate Nrf2-mediated antioxidant signaling may have potential applications for treating diabetic nephropathy. Taken together, our study demonstrated that BRD4 inhibition protects podocytes from HG-induced apoptosis and oxidative damage by potentiating Nrf2/ARE antioxidant signaling via downregulating Keap1. These findings highlight an important role for the BRD4/Keap1/Nrf2/ARE signaling axis in regulating podocyte injury in diabetic nephropathy and provide novel insights into the molecular pathogenesis of diabetic nephropathy. Therefore, BRD4 may serve as a promising therapeutic target for preventing diabetic nephropathy. However, the precise role of BRD4 in regulating podocyte injury in diabetic nephropathy needs further in vivo investigation using animal models. Conflict of interest The authors declare no conflict of interest. Contributor Hong Zuo: conception of the work. Hong Zuo: collection of data. Shujin Wang, Jia Feng and Xufeng Liu: analysis of data. Hong Zuo: writing of manuscript. Acknowledgments This study was supported by The Fund of Xi'an City (J201902047). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.biochi.2019.07.012. References [1] K. Reidy, H.M. Kang, T. Hostetter, K. Susztak, Molecular mechanisms of diabetic kidney disease, J. Clin. Investig. 124 (2014) 2333e2340. [2] S.S. Badal, F.R. Danesh, New insights into molecular mechanisms of diabetic kidney disease, Am. J. Kidney Dis. 63 (2014) S63eS83. [3] G. Soldatos, M.E. Cooper, Diabetic nephropathy: important pathophysiologic mechanisms, Diabetes Res. Clin. Pract. 13 (2008) 042. [4] G. Remuzzi, A. Schieppati, P. Ruggenenti, Clinical practice. Nephropathy in patients with type 2 diabetes, N. Engl. J. Med. 346 (2002) 1145e1151. [5] P.W. Mathieson, The podocyte as a target for therapies–new and old, Nat. Rev. Nephrol. 8 (2011) 52e56. [6] P. Anil Kumar, G.I. Welsh, M.A. Saleem, R.K. Menon, Molecular and cellular events mediating glomerular podocyte dysfunction and depletion in diabetes mellitus, Front. Endocrinol. 5 (2014) 151. [7] S.Y. Wu, C.M. Chiang, The double bromodomain-containing chromatin adaptor Brd4 and transcriptional regulation, J. Biol. Chem. 282 (2007) 13141e13145. [8] B.N. Devaiah, D.S. Singer, Two faces of brd4: mitotic bookmark and transcriptional lynchpin, Transcription 4 (2013) 13e17. [9] B. Donati, E. Lorenzini, A. Ciarrocchi, BRD4 and cancer: going beyond transcriptional regulation, Mol. Cancer 17 (2018) 018e0915. [10] M.K. Jang, K. Mochizuki, M. Zhou, H.S. Jeong, J.N. Brady, K. Ozato, The bromodomain protein Brd4 is a positive regulatory component of P-TEFb and stimulates RNA polymerase II-dependent transcription, Mol. Cell 19 (2005) 523e534. [11] T. Xiang, J.Y. Bai, C. She, D.J. Yu, X.Z. Zhou, T.L. Zhao, Bromodomain protein BRD4 promotes cell proliferation in skin squamous cell carcinoma, Cell. Signal. 42 (2018) 106e113.
[12] K.L. Cheung, F. Zhang, A. Jaganathan, R. Sharma, Q. Zhang, T. Konuma, T. Shen, J.Y. Lee, C. Ren, C.H. Chen, G. Lu, M.R. Olson, W. Zhang, M.H. Kaplan, D.R. Littman, M.J. Walsh, H. Xiong, L. Zeng, M.M. Zhou, Distinct roles of Brd2 and Brd4 in potentiating the transcriptional program for Th17 cell differentiation, Mol. Cell 65 (2017) 1068e1080. [13] Y. Tan, L. Wang, Y. Du, X. Liu, Z. Chen, X. Weng, J. Guo, H. Chen, M. Wang, X. Wang, Inhibition of BRD4 suppresses tumor growth in prostate cancer via the enhancement of FOXO1 expression, Int. J. Oncol. 53 (2018) 2503e2517. [14] C. Michaeloudes, N. Mercado, C. Clarke, P.K. Bhavsar, I.M. Adcock, P.J. Barnes, K.F. Chung, Bromodomain and extraterminal proteins suppress NF-E2-related factor 2-mediated antioxidant gene expression, J. Immunol. 192 (2014) 4913e4920. [15] M. Hussong, S.T. Borno, M. Kerick, A. Wunderlich, A. Franz, H. Sultmann, B. Timmermann, H. Lehrach, M. Hirsch-Kauffmann, M.R. Schweiger, The bromodomain protein BRD4 regulates the KEAP1/NRF2-dependent oxidative stress response, Cell Death Dis. 24 (2014) 157. [16] A. Kobayashi, T. Ohta, M. Yamamoto, Unique function of the Nrf2-Keap1 pathway in the inducible expression of antioxidant and detoxifying enzymes, Methods Enzymol. 378 (2004) 273e286. [17] M. McMahon, K. Itoh, M. Yamamoto, J.D. Hayes, Keap1-dependent proteasomal degradation of transcription factor Nrf2 contributes to the negative regulation of antioxidant response element-driven gene expression, J. Biol. Chem. 278 (2003) 21592e21600. [18] W. Cui, X. Min, X. Xu, B. Du, P. Luo, Role of nuclear factor erythroid 2-related factor 2 in diabetic nephropathy, J. Diabetes Res. 3797802 (2017) 23. [19] J.X. Shi, Q.J. Wang, H. Li, Q. Huang, Silencing of USP22 suppresses high glucose-induced apoptosis, ROS production and inflammation in podocytes, Mol. Biosyst. 12 (2016) 1445e1456. [20] F. Thilo, M. Lee, S. Xia, A. Zakrzewicz, M. Tepel, High glucose modifies transient receptor potential canonical type 6 channels via increased oxidative stress and syndecan-4 in human podocytes, Biochem. Biophys. Res. Commun. 450 (2014) 312e317. [21] B.C. Liu, X. Song, X.Y. Lu, D.T. Li, D.C. Eaton, B.Z. Shen, X.Q. Li, H.P. Ma, High glucose induces podocyte apoptosis by stimulating TRPC6 via elevation of reactive oxygen species, Biochim. Biophys. Acta 6 (2013) 13. [22] V. Soetikno, W. Arozal, M. Louisa, R. Setiabudy, New insight into the molecular drug target of diabetic nephropathy, Internet J. Endocrinol. 2014 (2014) 968681. [23] X. Wu, D. Liu, X. Gao, F. Xie, D. Tao, X. Xiao, L. Wang, G. Jiang, F. Zeng, Inhibition of BRD4 suppresses cell proliferation and induces apoptosis in renal cell carcinoma, Cell. Physiol. Biochem. 41 (2017) 1947e1956. [24] Z.Y. Xing, Y. Wang, L. Cheng, J. Chen, X.Z. He, W. Xing, Bromodomain-containing protein 4 (BRD4) inhibition sensitizes palomid 529-induced anti-renal cell carcinoma cell activity in vitro and in vivo, Cell. Physiol. Biochem. 50 (2018) 640e653. [25] H. Zhu, J.H. Mao, Y. Wang, D.H. Gu, X.D. Pan, Y. Shan, B. Zheng, Dual inhibition of BRD4 and PI3K-AKT by SF2523 suppresses human renal cell carcinoma cell growth, Oncotarget 8 (2017) 98471e98481. [26] B. Suarez-Alvarez, J.L. Morgado-Pascual, S. Rayego-Mateos, R.M. Rodriguez, R. Rodrigues-Diez, P. Cannata-Ortiz, A.B. Sanz, J. Egido, P.L. Tharaux, A. Ortiz, C. Lopez-Larrea, M. Ruiz-Ortega, Inhibition of bromodomain and extraterminal domain family proteins ameliorates experimental renal damage, J. Am. Soc. Nephrol. 28 (2017) 504e519. [27] G. Zhang, R. Liu, Y. Zhong, A.N. Plotnikov, W. Zhang, L. Zeng, E. Rusinova, G. Gerona-Nevarro, N. Moshkina, J. Joshua, P.Y. Chuang, M. Ohlmeyer, J.C. He, M.M. Zhou, Down-regulation of NF-kappaB transcriptional activity in HIVassociated kidney disease by BRD4 inhibition, J. Biol. Chem. 287 (2012) 28840e28851. [28] X. Zhou, L.X. Fan, D.J. Peters, M. Trudel, J.E. Bradner, X. Li, Therapeutic targeting of BET bromodomain protein, Brd4, delays cyst growth in ADPKD, Hum. Mol. Genet. 24 (2015) 3982e3993. [29] C. Xiong, M.V. Masucci, X. Zhou, N. Liu, X. Zang, E. Tolbert, T.C. Zhao, S. Zhuang, Pharmacological targeting of BET proteins inhibits renal fibroblast activation and alleviates renal fibrosis, Oncotarget 7 (2016) 69291e69308. [30] H. Liu, L. Wang, X. Weng, H. Chen, Y. Du, C. Diao, Z. Chen, X. Liu, Inhibition of Brd4 alleviates renal ischemia/reperfusion injury-induced apoptosis and endoplasmic reticulum stress by blocking FoxO4-mediated oxidative stress, Redox Biol. 24 (2019) 11. [31] T. Ijaz, M. Jamaluddin, Y. Zhao, Y. Zhang, J. Jay, C.C. Finnerty, D.N. Herndon, R.G. Tilton, A.R. Brasier, Coordinate activities of BRD4 and CDK9 in the transcriptional elongation complex are required for TGFbeta-induced Nox4 expression and myofibroblast transdifferentiation, Cell Death Dis. 8 (2017) 434. [32] Y.M. Yang, R.H. Shi, C.X. Xu, L. Li, BRD4 expression in patients with essential hypertension and its effect on blood pressure in spontaneously hypertensive rats, J. Am. Soc. Hypertens. 12 (2018) e107ee117. [33] M. Huang, L. Zhu, J.S. Garcia, M.X. Li, A.J. Gentles, B.S. Mitchell, Brd4 regulates the expression of essential autophagy genes and Keap1 in AML cells, Oncotarget 9 (2018) 11665e11676. [34] M. Qu, X. Zhang, X. Hu, M. Dong, X. Pan, J. Bian, Q. Zhou, BRD4 inhibitor JQ1 inhibits and reverses mechanical injury-induced corneal scarring, Cell Death Dis. 4 (2018) 5. [35] Q.D. An, Y.Y. Li, H.X. Zhang, J. Lu, X.D. Yu, Q. Lin, Y.G. Zhang, Inhibition of bromodomain-containing protein 4 ameliorates oxidative stress-mediated apoptosis and cartilage matrix degeneration through activation of NF-E2-
H. Zuo et al. / Biochimie 165 (2019) 100e107
[36]
[37]
[38]
[39]
related factor 2-heme oxygenase-1 signaling in rat chondrocytes, J. Cell. Biochem. 119 (2018) 7719e7728. B. Zhang, X. Zhang, C. Zhang, Q. Shen, G. Sun, X. Sun, Notoginsenoside R1 protects db/db mice against diabetic nephropathy via upregulation of Nrf2mediated HO-1 expression, Molecules 24 (2019) E247. Z. Xie, L. Zhong, Y. Wu, X. Wan, H. Yang, X. Xu, P. Li, Carnosic acid improves diabetic nephropathy by activating Nrf2/ARE and inhibition of NF-kappaB pathway, Phytomedicine 47 (2018) 161e173. A. Mathur, V.K. Pandey, P. Kakkar, Activation of GSK3beta/beta-TrCP axis via PHLPP1 exacerbates Nrf2 degradation leading to impairment in cell survival pathway during diabetic nephropathy, Free Radic. Biol. Med. 120 (2018) 414e424. L.N. Sun, X.C. Liu, X.J. Chen, G.J. Guan, G. Liu, Curcumin attenuates high glucose-induced podocyte apoptosis by regulating functional connections between caveolin-1 phosphorylation and ROS, Acta Pharmacol. Sin. 37 (2016) 645e655.
107
[40] C. Dong, H. Zheng, S. Huang, N. You, J. Xu, X. Ye, Q. Zhu, Y. Feng, Q. You, H. Miao, D. Ding, Y. Lu, Heme oxygenase-1 enhances autophagy in podocytes as a protective mechanism against high glucose-induced apoptosis, Exp. Cell Res. 337 (2015) 146e159. [41] C. Wang, C. Li, H. Peng, Z. Ye, J. Zhang, X. Liu, T. Lou, Activation of the Nrf2-ARE pathway attenuates hyperglycemia-mediated injuries in mouse podocytes, Cell. Physiol. Biochem. 34 (2014) 891e902. [42] Q. Zhang, Q. Deng, J. Zhang, J. Ke, Y. Zhu, R.W. Wen, Z. Ye, H. Peng, Z.Z. Su, C. Wang, T. Lou, Activation of the Nrf2-ARE pathway ameliorates hyperglycemia-mediated mitochondrial dysfunction in podocytes partly through Sirt1, Cell. Physiol. Biochem. 48 (2018) 1e15. [43] F.C. Brosius, R.J. Coward, Podocytes, signaling pathways, and vascular factors in diabetic kidney disease, Adv. Chron. Kidney Dis. 21 (2014) 304e310. [44] I.H. Ali, D.P. Brazil, Under the right conditions: protecting podocytes from diabetes-induced damage, Stem Cell Res. Ther. 4 (2013) 119.