Endotoxin Mediates Recruitment of RNA Polymerase II to Target Genes in Acute Renal Failure

Abstract

Acute renal failure (ARF) sensitizes the kidney to endotoxin (LPS)-driven production of cytokines and chemokines. This study assessed whether this LPS hyperresponsiveness exists at the genomic level. Three heterogeneous mouse models of ARF were studied: Maleate nephrotoxicity, unilateral ureteral obstruction, and LPS preconditioning. In all cases, LPS was injected approximately 18 h after injury was induced, and over the next 0 to 90 min, RNA polymerase II recruitment to the genome at three LPS-responsive genes (TNF-␣, monocyte chemoattractant-1 [MCP-1], and heme oxygenase-1 [HO-1]) was assessed by chromatin immunoprecipitation. LPS hyperresponsiveness was noted in each model, measured by exaggerated increases in TNF-␣ and MCP-1 mRNA (approximately two to 10 times higher than LPS-injected controls). Corresponding increases in the recruitment of RNA polymerase II to the TNF-␣ and MCP-1 genes were observed, and increased trimethylation of histone 3 lysine 4 (H3K4m3) at these sites may have played a role in this recruitment. Conversely, recruitment of RNA polymerase II to the HO-1 gene was suppressed ("tolerance"), and no increase in H3K4m3 was observed at HO-1 exons. The ARF-induced changes in mRNA did not correlate with mRNA stability, suggesting the mechanistic importance of RNA polymerase II-mediated transcriptional events. In conclusion, LPS hyperresponsiveness after ARF is likely mediated at the genomic level, possibly by H3K4m3.

Progressive histone alterations and proinflammatory gene activation: consequences of heme protein/iron-mediated proximal tubule injury

“…Thus it is difficult to ascertain which of these two processes initiated and maintained the in vivo mRNA elevations, as discussed above. Assessments of Pol II binding to target genes can help resolve this issue, given that Pol II is the enzyme that drives transcription and its binding to gene loci serves as a semiquantitative index of transcription rates (16,21,24,26). Given these considerations, we assessed Pol II binding to each of the four target genes.…”

Detrimental Cross-Talk Between Sepsis and Acute Kidney Injury: New Pathogenic Mechanisms, Early Biomarkers and Targeted Therapies

“…These authors demonstrated in vivo increased RNApolymerase II density at TNF-α, monocyte chemotactic protein-1 (MCP-1) and heme oxygenase-1 (HO-1) loci after LPS injection in mice. This effect was related to selective histone methylation and was enhanced by different nephrotoxic stimuli [20]. These AKI-associated epigenetic alterations of tubular cells may be responsible for the above-mentioned increase in circulating inflammatory mediators.…”

Renal cortical hemopexin accumulation in response to acute kidney injury

“…Mice were injected with either LPS or cisplatin, as noted above. Either 24 h (LPS-injected mice; n ϭ 5) or 48 h later (cisplatin-injected mice; n ϭ 4), proximal tubules were isolated as previously described (16). The tubules were then extracted for both RNA and total protein and assayed for both Hpx mRNA and Hpx protein levels, as noted above.…”

“…The above LPS experiment was repeated, but following tubule isolation, the tubules were incubated in the presence or absence of actinomycin D (2 g/ml) to block de novo RNA synthesis, as previously described (16). By so doing, changes in Hpx mRNA levels reflect Hpx degradation rates.…”