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

Abstract

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.

Fig. 1.

Time course of renal cortical TNF-α mRNA levels and RNA polymerase II (Pol II) binding to the TNF-α gene. After glycerol injection, TNF-α mRNA was markedly elevated and continued to increase over the 7 days of observation. A corresponding progressive increase in Pol II binding to exon 1 of the TNF-α gene was also observed. Open bars, controls; solid bars, glycerol-treated mice.

Fig. 2.

Time course of renal cortical monocyte chemoattractant protein-1 (MCP-1) mRNA expression and Pol II binding to the MCP-1 gene. MCP-1 mRNA was markedly increased by 1 day after glycerol injection and continued to increase over the course of the experiments. mRNA increases were paralleled by a progressive rise in Pol II binding to exon 1 of the MCP-1 gene. Open bars, controls; solid bars, glycerol-treated mice.

Fig. 3.

Time course of renal cortical transforming growth factor-β1 (TGF-β1) mRNA levels and Pol II binding to the TGF-β1 gene. Similar to TNF-α and MCP-1, TGF-β1 mRNA levels continued to increase over the 7 days after glycerol injection. Results were paralleled by progressive increases in Pol II binding to exon 1 of the TGF-β1 gene. Open bars, controls; solid bars, glycerol-treated mice.

Fig. 4.

Time course of renal cortical heme oxygenase-1 (HO-1) mRNA expression and Pol II binding to the HO-1 gene. At 1 and 2 days after glycerol injection, HO-1 mRNA was markedly increased compared with control values. By day 7, mRNA levels had returned to control values. Increased Pol II binding was also observed at each time point. Unlike the Pol II binding results for TNF-α, MCP-1, and TGF-β1, no further increase in Pol II binding to the HO-1 gene was observed on day 7. Open bars, controls; solid bars, glycerol-treated mice. NS, not significant.

Fig. 5.

Time course of histone 2 variant H2A.Z and histone 3 lysine 4 (H3K4) methylation at exon 1 of the TNF-α gene. A progressive increase in levels of H2A.Z was observed 1, 2, and 7 days after glycerol injection. H3K4 was probed with an antibody that recognizes mono-, di-, and trimethylation, and a progressive increase in the sum of these 3 moieties was observed over the 7 days of observation. Open bars, controls; solid bars, glycerol-treated mice.

Fig. 8.

Time course of H2A.Z expression and H3K4 methylation at the HO-1 gene. Significant increases in H2A.Z and H3K4m1,2,3 were observed 1 and 7 days (but not 2 days) after glycerol injection. Open bars, controls; solid bars, glycerol-treated mice.

Fig. 6.

Time course of H2A.Z and H3K4 methylation at exon 1 of the MCP-1 gene. Similar to TNF-α, progressive increases in H2A.Z and H3K4 methylation were observed after glycerol treatment at the MCP-1 gene. Open bars, controls; solid bars, glycerol-treated mice.

Fig. 7.

Time course of H2A.Z and H3K4 methylation at exon 1 of the TGF-β1 gene. At each time point, a significant increase in H2A.Z and H3K4 methylation was observed in the glycerol-treated animals at the start exons of the TGF-β1 gene. Unlike TNF-α and MCP-1, increases at TGF-β1 reached maximal or near-maximal levels on day 1 and were not progressive thereafter. Open bars, controls; solid bars, glycerol-treated mice.

Fig. 9.

H3K4m3 7 days after glycerol treatment. To assess whether H3K4m3, in particular, contributed to the overall increase in H3K4 methylation observed with the anti-H3K4m1 + m2 + m3 antibody, samples were probed with an antibody specific for H3K4m3 (a gene-activating histone mark). Assessments were restricted to kidney samples obtained on day 7. H3K4m3 was increased at each of the 4 genes. Open bars, controls; solid bars, glycerol-treated mice.

Fig. 10.

mRNA levels in HK-2 cells incubated for 18 h under control conditions or subjected to 18 h of Fe [Fe gluconate (FeG) or Fe sucrose (FeS)] challenge. Both Fe challenges raised each of the mRNA levels. The increase was greater with FeS than FeG, consistent with the fact that FeS gains greater intracellular HK-2 access than does FeG.

Fig. 11.

Effects of Fe on mRNA stability in HK-2 cells incubated overnight under control conditions or with FeG or FeS. Half of the cells in each group were then treated for 6 h with actinomycin D (AD) to inhibit new mRNA synthesis. Thus, changing mRNA levels in the presence of actinomycin D reflects mRNA degradation rates. Percentage of mRNA remaining in actinomycin D-treated cells was then calculated as follows: (mRNA in actinomycin-treated cells ÷ amount of mRNA in control incubated cells) × 100%. Extent of mRNA reductions in control cells (C) greatly varied (TNF-α manifested the greatest decline, followed in order by HO-1, MCP-1, and TGF-β1 mRNAs). In no instance, did Fe pretreatment affect these results (indicating no Fe-mediated mRNA stabilization).

Fig. 12.

Pol II binding to TNF-α, MCP-1, TGF-β1, and HO-1 genes in HK-2 cells incubated overnight under control conditions or with FeS. In each instance, Fe significantly increased Pol II binding to each test gene.

Fig. 13.

H3K4m3 levels at TNF-α, MCP-1, TGF-β1, and HO-1 genes in HK-2 cells. Cells were treated as described in Fig. 12 legend and then assayed for H3K4m3 levels. Fe significantly increased the H3K4m3 histone mark compared with control incubated cells at each gene.

Fig. 14.

H2A.Z levels at TNF-α, MCP-1, TGF-β1, and HO-1 genes in HK-2 cells. Cells were treated as described in Fig. 12 legend and then assayed for H2A.Z levels at each test gene. Fe significantly increased the H2A.Z histone variant at each gene compared with control incubated cells.

Fig. 15.

Effects of actinomycin D on HK-2 cell H2A.Z expression in the presence and absence of FeS. Under control incubation conditions, actinomycin D reduced H2A.Z expression at TNF, MCP-1, and TGF-β1 genes by approximately one-half. Actinomycin also significantly blunted the extent to which the Fe challenge raised H2A.Z expression at these 3 genes. Conversely, actinomycin failed to suppress control H2A.Z levels at the HO-1 gene, although there was a trend (P < 0.07) toward H2A.Z suppression in the presence of Fe.