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

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Rhabdomyolysis (Fe)-induced acute renal failure (ARF) causes renal inflammation, and, with repetitive insults, progressive renal failure can result. To gain insights into these phenomena, we assessed the impact of a single episode of glycerol-induced rhabdomyolysis on proinflammatory/profibrotic [TNF-alpha, monocyte chemoattractant protein-1 (MCP-1), and transforming growth factor-beta1 (TGF-beta1)] gene expression and the time course of these changes. CD-1 mice were studied 1-7 days after glycerol injection. Normal mice served as controls. RNA polymerase II (Pol II) binding to the TNF-alpha, MCP-1, and TGF-beta1 genes, "gene-activating" histone modifications [histone 3 lysine 4 (H3K4) trimethylation (H3K4m3) and histone 2 variant H2A.Z], and cognate mRNA levels were assessed. Results were contrasted to changes in anti-inflammatory heme oxygenase-1 (HO-1). Glycerol produced severe ARF (blood urea nitrogen approximately 150-180 mg/dl) followed by marked improvement by day 7 (blood urea nitrogen approximately 40 mg/dl). Early increases in TNF-alpha, MCP-1, and TGF-beta1 mRNAs, Pol II gene binding, and H3K4m3/H2A.Z levels were observed. These progressed with time, despite resolution of azotemia. Comparable early HO-1 changes were observed. However, HO-1 mRNA normalized by day 7, and progressive Pol II binding/histone alterations did not occur. Fe-mediated injury to cultured proximal tubule (HK-2) cells recapitulated these in vivo results. Hence, this in vitro model was used for mechanistic assessments. On the basis of these studies, it was determined that 1) the H3K4m3/H2A.Z increases are early events (i.e., they precede mRNA increases), 2) subsequent mRNA elevations reflect transcription, not mRNA stabilization (actinomycin D assessments), and 3) increased transcription, per se, helps sustain elevated H2A.Z levels. We conclude that 1) Fe/glycerol-induced tubular injury causes sustained proinflammatory gene activation, 2) decreasing HO-1 expression, as reflected by mRNA levels, may facilitate this proinflammatory state, and 3) gene-activating histone modifications are early injury events and progressively increase at selected proinflammatory genes. Thus they may help sustain a proinflammatory state, despite resolving ARF.