BRD4 contributes to high-glucose-induced podocyte injury by modulating Keap1/Nrf2/ARE signaling
Hong Zuo*, Shujin Wang, Jia Feng, Xufeng Liu
Abstract
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 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 39 polymerase chain reaction; CCK-8, Cell Counting Kit-8; OD, optical density; TUNEL, terminal 40
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 oxidoreductase 1 [16]. Kelch-like ECH-associated protein (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
2. Materials and methods
Cell culture and HG exposure
15 min at room temperature and then added to cells at 50 µl/well. After 48 h-transfection, the transfection efficacy was determined by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) or Western blot analysis.
RNA extraction and RT-qPCR analysis
Total RNA was extracted from cells by using TRIzol reagent (Invitrogen) and converted into 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.
Cell viability assay
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 were observed and counted under a fluorescence microscope.
ROS measurement assay
Cells were treated with 10 µM 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 525nm.
3. Results
3.1. BRD4 upregulation in HG-stimulated podocytes 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 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, BRD4-inhibition-mediated protection on HG-induced apoptosis and ROS production was partially 204 reversed by Nrf2 inhibition (Fig. 6C-E). Overall, these results suggest that BRD4 inhibition protects 205 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 (NF)-κB kidney protection. Consistent with these findings, our study reveals that BRD4 inhibition protects podocytes from HG-induced injury. Since HG-induced 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 [28-30]. Hypoxia/reoxygenation-induced apoptosis and ROS production are significantly decreased by BRD4 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 [36-38]. HG induces excessive ROS production and apoptosis, both of which contribute to the development of diabetic nephropathy [39,40]. Notably, Nrf2/ARE signaling activation attenuates HG-induced injury in
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