Enhancer of zeste homolog 2 modulates oxidative stress‐mediated pyroptosis in vitro and in a mouse kidney ischemia‐reperfusion injury model

Abstract

contributed equally to this work.Abbreviations: AKI, acute kidney injury; ALK5, activin receptor-like kinase; ASC, apoptosis-associated speck-like protein containing a caspase-recruitment domain; BUN, blood urea nitrogen; Cr, creatinine; EZH2, enhancer of zeste homolog 2; FLICA, fluorescent-labeled inhibitors of caspases; H/R, hypoxia/reoxygenation; H3K27me3, histone H3 lysine 27 trimethylation; HK-2, human renal proximal tubular epithelial cell line; I/R, ischemia/reperfusion; IL-1β, interleukin-1beta; LDH, lactate dehydrogenase; MDA, malondialdehyde; NAC, N-acetyl-cysteine; NADPH, nicotinamide adenine dinucleotide phosphate; NLRP3, NOD-like receptor family pyrin domain-containing protein 3; Nox4, NADPH oxidase 4; PRC2, polycomb repressive complex 2; ROS, reactive oxygen species; RT-PCR, real-time polymerase chain reaction; siRNA, small interfering RNA; SOD, superoxide dismutase; TGF-β, transforming growth factor beta; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling. AbstractEnhancer of zeste homolog 2 (EZH2), a well-known methyltransferase, mediates histone H3 lysine 27 trimethylation (H3K27me3) and plays a crucial role in several kidney disease models. However, its role in renal ischemia/reperfusion (I/R) injury still remains unclear. In this study, we found that EZH2 was positively related to renal I/R injury and inhibition of EZH2 with DZNeP alleviated I/R injury and blocked the activation of oxidative stress and pyroptosis in vivo. Similarly, inhibition of EZH2 with either DZNeP or si-RNA also exerted an inhibitory effect on hypoxia/reoxygenation (H/R)-induced oxidative stress and pyroptosis in vitro. Moreover, further study revealed that ablation of reactive oxygen species (ROS) with N-acetyl-cysteine (NAC) suppressed pyroptosis in human renal proximal tubular epithelial cell line cells exposed to H/R stimulation. Furthermore, Nox4, which was positively related to the generation of ROS, was upregulated during H/R process, while it could be reversed by EZH2 inhibition. Consistently, Nox4-mediated ROS generation was attenuated upon inhibition of EZH2 with DZNeP or si-RNA. Additionally, the transcriptional activity of Nox4 was enhanced by the activation of ALK5/Smad2/3 signaling pathway, which was abolished by ALK5 knockdown in vitro. Finally, EZH2 inhibition blocked H/R and I/R-activated ALK5/Smad2/3 pathway and also resulted in an obvious decrease in the transcriptional activity and protein expression levels of Nox4. In conclusion, our results proved that EZH2 inhibition alleviated renal pyroptosis by blocking Nox4-dependent ROS generation through ALK5/Smad2/3 signaling pathway, indicating that EZH2 could be a potential therapeutic target for renal I/R injury. K E Y W O R D Senhancer of zeste homolog 2, ischemia-reperfusion injury, oxidative stress, pyroptosis, Nox4, reactive oxygen species 836 | LIU et aL.

Tisp40 Induces Tubular Epithelial Cell GSDMD-Mediated Pyroptosis in Renal Ischemia-Reperfusion Injury via NF-κB Signaling

“…Pyroptosis, characterized by excessive cell death and inflammation, is a specific cell death program distinct from apoptosis and necrosis (Miao et al, 2010). In this study, we found the injury induced by I/R and OGD/R resulted in excessive TEC death, which always went with pyroptosis in renal IRI (Tajima et al, 2019;Wang et al, 2019;Liu et al, 2020). As the main executor of cell pyroptosis, GSDMD, is widely expressed in different cells and tissues (Shi et al, 2015;Liu et al, 2016).…”

“…It forms pores in the plasma membrane that ultimately cause cell swelling, membrane lysis, and inflammatory cytokines release (Shi et al, 2015). GSDMD-mediated pyroptosis of renal TECs is an essential process in renal ischemia-reperfusion (I/R) injury (Diao et al, 2019;Tajima et al, 2019;Liu et al, 2020). Thus, studies are needed to determine the biological networks involved in GSDMD-mediated pyroptosis in TEC injury induced by renal IR.…”

IP3R1 regulates Ca2+ transport and pyroptosis through the NLRP3/Caspase-1 pathway in myocardial ischemia/reperfusion injury

“…IP3R1 expression is increased in H/R injury of spinal cord white matter 9 . Recent studies have shown that cell pyroptosis is related to I/R injury 22,29 . Pyroptosis is defined as a Caspase-1-dependent mechanism, and NLRP3 is indispensable for the activation of Caspase-1 30,31 .…”

The Suppression of Pin1‐Alleviated Oxidative Stress through the p38 MAPK Pathway in Ischemia‐ and Reperfusion‐Induced Acute Kidney Injury

“…ROS Production Detection. The ROS levels were determined based on our previous study [16] through the commercial kit instruction (Nanjing Jiancheng, China). The samples were incubated with dichloro-dihydro-fluorescein diacetate (20 μM) at room temperature for 30 min, and the result of the ROS level was quantified through flow cytometry.…”