BRD4 plays an antiaging role in the senescence of renal tubular epithelial cells

Abstract

Background: Age-related kidney failure is often induced by a decrease in the bioavailability of tubular epithelial cells in elderly chronic kidney disease (CKD) patients. BRD4, an epigenetic regulator and a member of the bromodomain and extraterminal (BET) protein family, acts as a super-enhancer (SE) organizing and regulating genes expression during embryogenesis and cancer development. But the physiological function of BRD4 in normal cells has been less studied. This study aimed to research certain biological roles of BRD4 in the process of normal cell aging and discuss the potential mechanisms.

Methods: In this study, we investigated the biological functions of BRD4 proteins in the aging of renal tubular cells. At first, we used a D-galactose (D-gal) and BRD4 inhibitor (Abbv-075) to replicate kidney senescence in vivo. D-gal and Abbv-075 were then used to measure the aging-related changes, such as changes in cell cycle, β-galactosidase activity, cell migration, and p16 protein expression in vitro. At last, we knocked down and over-expressed BRD4 to investigate the aging-related physiological phenomena in renal tubular cells.

Results: In vitro, D-gal treatment induced noticeable aging-related changes such as inducing cell apoptosis and cell cycle arrest, increasing β-galactosidase activity as well as up-regulating p16 protein expression in primary human tubular epithelial cells. In the aging mice model, D-gal significantly induced renal function impairment and attenuated BRD4 protein expression. At the same time, the BRD4 inhibitor (Abbv-075) was able to mimic D-gal-induced cell senescence. In vivo, Abbv-075 also decreased kidney function and up-regulated p21 protein expression. When we knocked down the expression of BRD4, the senescence-associated β-galactosidase (SA-β-gal) activity increased dramatically, cell migration was inhibited, and the proportion of cells in the G0/G1 phase increased. Additionally, the knockdown also promoted the expression of the senescence-related proteins p16. When the renal tubular cells were overexpressed with BRD4, cell aging-related indicators were reversed in the D-gal-induced cell aging model.

Conclusions: BRD4 appears to have an active role in the aging of renal tubular cells in vivo and in vitro. The findings also suggest that BRD4 inhibitors have potential nephrotoxic effects for oncology treatment. BRD4 may be a potential therapeutic biomarker and drug target for aging-related kidney diseases, which warrants additional studies.

Keywords: BRD4; renal tubular epithelial cells; senescence.

Figure 1

D-gal induces HRPTEpiC senescence and accelerates kidney function injury. (A) D-gal inhibits the proliferation of primary human tubular epithelial cells; (B) D-gal increases SA-β-gal staining in vitro (200×); (C) D-gal induces G0/G1 cell cycle arrest in vitro; (D) D-gal upregulates the relative expression levels of senescence-related proteins in vitro; (E) D-gal induces renal injury in vivo. The values are expressed as the means ± SD; n=3. *, P<0.05, control vs. D-gal-treated. D-gal, D-galactose; PI, propidium iodide; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Con, control; Cr, creatinine; Cys C, cystatin C; HRPTEpiC, human proximal tubular epithelial cells; SD, standard deviation.

Figure 2

A BRD4 inhibitor induces HRPTEpiC senescence and kidney function injury. (A) D-gal decreases BRD4 protein expression in vivo; (B) D-gal decreases BRD4 protein expression in vivo (IHC) (100×); (C) a BRD4 inhibitor (Abbv-075) decreases BRD4 protein expression in HRPTEpiCs by WB; (D) a BRD4 inhibitor increases SA-β-gal staining in vitro (100×); (E) a BRD4 inhibitor induces renal injury in vivo; (F) a BRD4 inhibitor upregulates the expression levels of senescence-related proteins in vivo (IHC) (100×). The values are expressed as the means ± SD; n=3. *, P<0.05, control vs. treated with a BRD4 inhibitor. BRD, bromodomain; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Con, control; D-gal, D-galactose; HRPTEpiC, human proximal tubular epithelial cells; WB, western blotting; IHC, immunohistochemistry; SD standard deviation.

Figure 3

Knockdown of BRD4 induces HRPTEpiC senescence. (A) Knockdown of BRD4 intensified SA-β-gal staining in vitro (100×); (B) knockdown of BRD4 decreased cell migration in vitro by crystal violet staining (100×); (C) knockdown of BRD4 induced G0/G1 cell cycle arrest in vitro; (D) knockdown of BRD4 regulated the expression levels of senescence-related proteins in vivo (WB). The values are expressed as the means ± SD; n=3. *, P<0.05, control vs. shRNA-BRD4 treated. SH1, BRD4-Sh1; SH2, RD4-Sh2; SH3, RD4-Sh3. Con, control; PI, propidium iodide; BRD, bromodomain; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HRPTEpiC, human proximal tubular epithelial cells; WB, western blotting; SD, standard deviation; shRNA, short hairpin RNA.

Figure 4

Overexpression of BRD4 rescues D-gal-induced HRPTEpiC senescence by de-activating the p16 function. (A) Overexpression of BRD4 increases cell migration in vitro by crystal violet staining; (B) overexpression of BRD4 alleviates D-gal-induced cell apoptosis in vitro; (C) overexpression of BRD4 alleviates D-gal-induced cell cycle arrest in vitro; (D) overexpression of BRD4 induces p16 protein expression in vitro. The values are expressed as the means ± SD; n=3. *, P<0.05, control vs. control + D-gal; ^#, P<0.05, control + D-gal vs. D-gal + BRD4 overexpression. Con, control; PI, propidium iodide; D-gal, D-galactose; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; BRD, bromodomain; HRPTEpiC, human proximal tubular epithelial cells; SD, standard deviation.