Renal ischemia-reperfusion injury upregulates histone-modifying enzyme systems and alters histone expression at proinflammatory/profibrotic genes

Abstract

Ischemic renal injury can produce chronic renal inflammation and fibrosis. This study tested whether ischemia-reperfusion (I/R) activates histone-modifying enzyme systems and alters histone expression at selected proinflammatory/profibrotic genes. CD-1 mice were subjected to 30 min of unilateral I/R. Contralateral kidneys served as controls. At 1, 3, or 7 days of reflow, bilateral nephrectomy was performed. Renal cortices were probed for monocyte chemoattractant protein-1 (MCP-1), transforming growth factor-beta1 (TGF-beta1), and collagen III mRNAs and cytokine levels. RNA polymerase II (Pol II) binding, which initiates transcription, was quantified at exon 1 of the MCP-1, TGF-beta1, collagen III genes (chromatin immunoprecipitation assay). Two representative gene-activating histone modifications [histone 3 lysine 4 (H3K4) trimethylation (m3) (H3K4m3); histone 2 variant H2A.Z] were sought. Degrees of binding of two relevant histone-modifying enzymes (Set1, BRG1) to target genes were assessed. Renal cortical Set1, BRG1, and H2A.Z mRNAs were measured. Finally, the potential utility of urinary mRNA concentrations as noninvasive markers of these in vivo processes was tested. I/R caused progressive increases in Pol II binding to MCP-1, TGF-beta1, and collagen III genes. Parallel increases in cognate mRNAs also were expressed. Progressive increases in renal cortical Set1, BRG1, H2A.Z mRNAs, and increased Set1/BRG1 binding to target genes occurred. These changes corresponded with: 1) progressive elevations of H3K4m3 and H2A.Z at each test gene; 2) increases in renal cortical TGF-beta1/MCP-1 cytokines; and 3) renal collagen deposition (assessed by histomorphology). Postischemic increases in urinary TGF-beta1, MCP-1, Set1, and BRG1 mRNAs were also observed. We conclude that: 1) I/R upregulates histone-modifying enzyme systems, 2) histone modifications at proinflammatory/profibrotic genes can result, and 3) urinary mRNA assessments may have utility for noninvasive monitoring of these in vivo events.

Fig. 1.

Polymerase II (Pol II) binding at the monocyte chemoattractant protein-1 (MCP-1) gene (exon 1), and corresponding MCP-1 mRNA and MCP-1 protein levels, assessed at either 1, 3, or 7 days postischemia. A progressive increase in Pol II binding was observed from 1–7 days (d) postischemia, compared with the values observed in the contralateral, nonischemic (control) kidneys. This corresponded with significant and progressive increases in both MCP-1 mRNA (middle) and MCP-1 protein (right) levels.

Fig. 2.

Assessments of histone 3 lysine 4 (H3K4) trimethylation (m3) and histone variant H2A.Z at exon 1 of the MCP-1 gene. A progressive postischemic increase in H3K4m3 and of H2A.Z levels were observed over the course of the 7-day experiments. These changes became statistically significant at the 3-day (H3K4m3) and 7-day (H2A.Z) time points.

Fig. 3.

Pol II binding at the transforming growth factor-β1 (TGF-β1) gene (exon 1), and corresponding TGF-β1 mRNA and protein levels 1, 3, or 7 days postischemia. Renal ischemia induced a progressive time-dependent increase in Pol II binding at the TGF-β1 gene (exon 1; left) and a corresponding increase in TGF-β1 mRNA (middle). These changes resulted in a progressive increase in TGF-β1 protein levels. Thus, these results mirrored those observed above for MCP-1 (as shown in Fig. 1).

Fig. 4.

Assessments of H3K4m3 and histone variant H2A.Z at exon 1 of the TGF-β1 gene. Progressive increases in both histone marks were apparent over the course of the 7-day experiment.

Fig. 5.

Assessments of collagen expression in the aftermath of ischemic renal injury. A progressive ischemia-induced increase in Pol II binding was observed at exon 1 of the collagen III gene. This was mirrored by increased collagen III mRNA expression. No direct assessment of collagen III protein could be made. As a substitute, collagen deposition was assessed by Masson trichrome staining at 2-wk postischemia (right, bottom) and in control kidney (right, top). Marked (blue) collagen staining was observed in the aftermath of ischemia, confirming that increasing fibrosis corresponded with the preceding collagen III-mRNA and Pol II results.

Fig. 6.

Assessments of H3K4m3 and histone variant H2A.Z at exon 1 of the collagen III gene. The results obtained at collagen III recapitulated those observed at MCP-1 and TGF-β1: a time-dependent progressive increase of both H3K4m3 and H2A.Z levels were observed in postischemic kidneys vs. their contralateral nonischemic controls.

Fig. 7.

Set1 (a histone 3-lysine 4-methylating enzyme), BRG1 (the central catalytic ATPase of the SWI/SNF chromatin remodeling complex), and H2A.Z mRNAs were assessed in postischemic and control kidneys at 1, 3, and 7 days postsurgery. The mRNA for Set1 rose progressively with time in postischemic kidneys. Similarly, the mRNA for BRG1 progressively rose postischemia. H2A.Z mRNA was elevated at each assessed time point, but progressive increases from 3 to 7 days were not observed.

Fig. 8.

Measurements of BRG1 and Set1 levels at exon 1 of the MCP-1, TGF-β1, and collagen III genes in 7-day postischemic and control kidneys. Significant increases in both chromatin remodeling enzymes were observed at each of the 3 assessed genes.

Fig. 9.

TGF-β1 mRNA, MCP-1 mRNA, Set1, and BRG1 mRNAs in urine pellets obtained just prior to and 18 h after the induction of bilateral ischemic renal injury (25 min). TGF-β1, Set1, and BRG1 mRNAs were detectable in baseline urine samples, and each rose dramatically in response to bilateral ischemia. Conversely, MCP-1 mRNA was barely detectable in baseline urine samples but was readily observed postischemia. All values are expressed as a ratio with simultaneously determined urine pellet GAPDH mRNA. To ascertain whether a corresponding increase in the Set1 product H3K4m3 could also be detected, pre- and postischemic urine samples were probed by Western blot analysis. As depicted just above the Set1 mRNA data columns, H3K4m3 was readily detected in postischemic (i) urine, but not in baseline [control (c)] urine samples.

Fig. 10.

MCP-1, TGF-β1, and collagen III mRNA responses to reversible ATP depletion in cultured proximal tubule human kidney (HK-2) cells. Reversible ATP depletion × 6 h + overnight recovery increased both MCP-1 and TGF-β1 mRNAs. Collagen III mRNA also rose in response to reversible ATP depletion, but only after an 18-h challenge, followed by 4 h of recovery.