P0515BRD4 PROTEIN REGULATES THE RESPONSES TO HYPOXIA TRIGGERED IN THE EXPERIMENAL RENAL DAMAGE
Authors: Diaz-Bulnes Paula , Rodriguez Ramon M , Corte Viviana , Banon Elisenda , Lazo Marta , Ramirez Maria Jose , Lopez-Larrea Carlos , Suarez-Alvarez Beatriz 
Published: June 2020

Abstract

Background and Aims
Renal hypoxia is not only one of the most common causes of acute kidney injury, but also a critical mediator in the transition to chronic kidney disease. When the kidney is exposed to an insufficient supply of oxygen to meet demand, some adaptive mechanisms are triggered by the cells to maintain homeostasis. Induction of HIF-1α transcription factor and activation of unfolded protein response (UPR) pathway, as consequence of ER dysfunction, are both essential to mediate the cell survival. The UPR pathway is regulated by three major protein sensors (IRE1α, PERK and ATF6) which under ER stress initiate the activation of the XBP1, ATF4 and ATF6 transcription factors, respectively. However, inappropriate activation of these mechanisms could lead to the enhanced transcription of genes involved in key processes in renal damage (inflammation, cell death or autophagy). On the other hand, BRD4 is an epigenetic reader that recognizes acetylated lysine residues on histone and other proteins, and mediates the binding of transcription factors to the transcriptional machinery. Our aim was to investigate whether BRD4-mediated epigenetic mechanisms could modulate the response to hypoxia triggered in acute renal damage.

Method
Tubular epithelial cell line, HK2, was cultured with thapsigargin (Tg) or in hypoxia chamber (1% O2, 5% CO2). In addition, these cells were treated with specific BET proteins inhibitors (JQ1, I-BET762) and with small interfering RNAs (siRNA BRD4, p300), or were subjected to knockdown of ATF4 and XBP1 by CRISPR/cas9 technology. Transcriptional changes were analyzed in each condition by RNA-sequencing. The binding of BRD4 to target genes and recruitment of the transcriptional machinery was analyzed by chromatin immunoprecipitation (ChIP) with specific antibodies against BRD4, RNA PolII, AcH3 and AcH4. Effect of JQ1 inhibitor was assayed in an ischemia/reperfusion injury (IRI) model, and analysis of gene expression, inflammatory cell infiltration, and epigenetic remodeling was carried out by quantitative PCR, IHQ and ChIP assay, respectively.

Results
Treatment of HK2 cells with BETs inhibitors, previously cultured with Tg or under hypoxia conditions, inhibits the gene expression of the GPR78 ER chaperon, and the XBP1 and ATF4 transcription factors modulating the downstream signaling pathways. Meanwhile, ATF6 expression remains unchanged. Gene silencing with siRNA and ChIP assays reveal that under activation of the UPR pathway or hypoxia, BRD4 recognizes acetylated histones in the GPR78, ATF4 and XBP1 promoters, recruits the pTEF-b complex and activates RNA-pol II allowing the gene transcription. Additionally, inhibition of BRD4 impairs the HIF-1α stabilization, downregulating the expression of hypoxia-induced genes. Results from whole-genome gene expression assays after stable knockdown of XBP1 and ATF4 reveal that most (86%) of the UPR genes regulated by BET proteins are dependent of XBP1 and only 32% by ATF4. Moreover, almost all genes regulated by ATF4 are also XBP1-dependent. This result may be due to the fact that ATF4 regulates IRE1α expression and thus modulates the XBP1 mRNA splicing. Administration of JQ1 in an IRI model supports that blockage of BRD4 ameliorates the renal damage (reducing BUN and creatinine levels) due to a decreased UPR activation and expression of HIF-1α target genes. As consequence, the expression of inflammatory genes and the inflammatory cell infiltration is diminished.

Conclusion
Our results show that BRD4 protein regulates two key processes, induction of HIF-1α transcription factor and UPR pathway activation triggered by renal hypoxia. Pharmacological inhibition of BET proteins reduces the activation these pathways, ameliorating renal damage and avoiding its progression.