As a consequence of acute kidney injury (AKI), proximal tubular cells hyperrespond to endotoxin (lipopolysaccharide, LPS) by exaggerated renal Tnf-α Production. This LPS hyperresponsiveness is transcriptionally mediated. The epigenetic pathways that control these responses are unknown.
We applied multiplex chromatin immunoprecipitation platform (Matrix ChIP) to explore epigenetic pathways that underlie endotoxin hyperresponsiveness in the setting of preceding unilateral renal ischemia/reperfusion (I/R) in mouse AKI model. Endotoxin exposure after I/R resulted in enhanced transcription, manifested by hyperresponsive recruitment of RNA polymerase II (Pol II) at the Tnf-α gene. At this locus, LPS but not I/R increased levels of Pol II C-terminal domain (CTD) phosho-serine2 &5 and induced dephosphorylation of the transcription-repressive histone H4 phospho-serine-1. In contrast, I/R but not LPS increased the transcription-permissive histone phosphorylation (H3 phospho-serine-10, H3.3 phospho-serine-31) at the Tnf-α gene. In agreement with these observations, I/R but not LPS increased activity of cognate kinases (Erk1/2, Msk1/2 and Aurora A) at the Tnf-α locus. Cross-talk of histone phosphorylation and acetylation synergize to active gene expression. I/R and LPS increased histone acetylation. (H3K9/14Ac, H4K5/8/12/16Ac, H2KA5Ac, H2BK4/7Ac). Levels of some histone acetyltransferases at this gene (PCAF and MOF) were increased by I/R but not by LPS, while others were induced by either I/R or LPS and exhibited endotoxin hyperresponsive patterns (GCN5, CBP and p300). The adaptor protein 14-3-3 couples histone phosphorylation with acetylation, and tethers chromatin modifiers/transcription elongation factors to target genes. Both I/R and LPS increased levels of 14-3-3 and several chromatin/transcription modifiers (BRD4, BRG1, HP-1γ and IKKα) at the Tnf-α gene, all exhibiting endotoxin hyperresponsive recruitment patterns similar to Pol II.
Our results suggest that I/R and LPS differentially trigger phosphorylation (Pol II and histone) and acetylation (histone) epigenetic pathways that interact at the Tnf-α gene to generate endotoxin hyperresponse in AKI.