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. 2009 Jan;174(1):54-62.
doi: 10.2353/ajpath.2009.080602. Epub 2008 Dec 18.

Renal ischemia-induced cholesterol loading: transcription factor recruitment and chromatin remodeling along the HMG CoA reductase gene

Masayo Naito  1 , Karol Bomsztyk, Richard A Zager
Affiliations

Renal ischemia-induced cholesterol loading: transcription factor recruitment and chromatin remodeling along the HMG CoA reductase gene

Masayo Naito et al. Am J Pathol. 2009 Jan.

Abstract

Acute kidney injury evokes renal tubular cholesterol synthesis. However, the factors during acute kidney injury that regulate HMG CoA reductase (HMGCR) activity, the rate-limiting step in cholesterol synthesis, have not been defined. To investigate these factors, mice were subjected to 30 minutes of either unilateral renal ischemia or sham surgery. After 3 days, bilateral nephrectomy was performed and cortical tissue extracts were prepared. The recruitment of RNA polymerase II (Pol II), transcription factors (SREBP-1, SREBP-2, NF-kappaB, c-Fos, and c-Jun), and heat shock proteins (HSP-70 and heme oxygenase-1) to the HMGCR promoter and transcription region (start/end exons) were assessed by Matrix ChIP assay. HMGCR mRNA, protein, and cholesterol levels were determined. Finally, histone modifications at HMGCR were assessed. Ischemia/reperfusion (I/R) induced marked cholesterol loading, which corresponded with elevated Pol II recruitment to HMGCR and increased expression levels of both HMGCR protein and mRNA. I/R also induced the binding of multiple transcription factors (SREBP-1, SREBP-2, c-Fos, c-Jun, NF-kappaB) and heat shock proteins to the HMGCR promoter and transcription regions. Significant histone modifications (increased H3K4m3, H3K19Ac, and H2A.Z variant) at these loci were also observed but were not identified at either the 5' and 3' HMGCR flanking regions (+/-5000 bps) or at negative control genes (beta-actin and beta-globin). In conclusion, I/R activates the HMGCR gene via multiple stress-activated transcriptional and epigenetic pathways, contributing to renal cholesterol loading.

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Figures

Figure 1
Figure 1
Renal histological injury, as assessed at 3 days after unilateral ischemic injury. As a frame of reference for interpreting the biochemical data, 4-μm kidney sections were cut from 10% formalin-fixed tissues and stained with H&E. Extensive proximal tubular necrosis and cast formation was observed in the renal cortex (A) and in the outer medullary stripe (B). C: The contralateral kidney manifested normal histology. Thus, extensive renal injury was present in the kidney samples that were used to study the HMG CoA reductase pathway.
Figure 2
Figure 2
Free cholesterol (left), cholesterol esters (middle), and HMG CoA reductase (HMGCR) protein levels (right, by Western blot) were assessed 3 days after unilateral I/R and in contralateral (CL) control kidneys. An ∼35% increase in free cholesterol levels was observed. This was accompanied by a sixfold increase in cholesterol esters, a cholesterol storage form. Increases in HMGCR protein were also observed, the magnitude of which paralleled the extent of the free cholesterol accumulation (each ∼35%).
Figure 3
Figure 3
A: HMGCR mRNA levels in control and post-I/R cortical kidney samples obtained at 4 or 72 hours after surgery were quantified by PCR. In both instances, an ∼30% increase in mRNA levels was observed (expressed as a ratio to GAPDH). B: RNA polymerase II (Pol II) densities were assessed at the HMGCR promoter, and at the start and end exons of the HMGCR gene (exons 1 and 19). I/R caused a small, albeit statistically significant, Pol II increase at the HMGCR promoter. Conversely, I/R induced an approximately threefold increase in Pol II at both exons 1 and 19. As expected, the absolute Pol II amounts in both the control and post-I/R kidneys were greater at the start versus the end exon.
Figure 4
Figure 4
The HMGCR promoter region was probed for: i) sterol-responsive element binding proteins (SREBP 1, 2); ii) general transcription factors (NF-κB, c-FOS, c-Jun); and iii) two heat shock proteins (HSP-70 and HSP-32, ie, HO-1). I/R caused twofold to threefold increases in the densities of both SREBP 1 and 2 at the HMGCR promoter. Significantly greater amounts of NF-κB, c-Fos, c-Jun, HSP-70, and HO-1 were also observed in the I/R versus control kidneys. Thus, multiple factors appear to activate HMGCR transcription as part of the stress response.
Figure 5
Figure 5
Localization of general transcription factors (NF-κB, c-Fos, c-Jun), HSP-70, and HO-1, at the start and end HMGCR exons. I/R induced significant increases of each protein at both HMGCR exons 1 and 19, compared to control kidneys. The absolute levels were consistently greater at the start versus the end exon (as would be expected with gene transcription).
Figure 6
Figure 6
Assessments performed at a control gene (β-actin, exon 1). I/R did not significantly alter Pol II levels at β-actin, exon 1, serving as a negative control for the data shown at the right of Figure 2. Furthermore, I/R did not impact NF-κB, c-Fos, or c-Jun binding to β-actin, (negative control for the data in Figure 4). Finally, no difference in the extent of trimethylation of histone 3 lysine 4 (H3K4m3) was observed, indicating the relative specificity for data presented in Figure 8. [Note: H3K9AC and H2A.Z variants at β-actin were not assessed.]
Figure 7
Figure 7
Assessments performed at the β-globin gene. As additional controls for the experiments depicted in Figures 1 to 5 and Figure 7, the SREBPs, general transcription factors, heat shock proteins, and histone modifications/variants were assessed at the normally silent β-globin gene (assessed between exon 1 and intron 1). In no instance was a statistically significant difference observed between control and the I/R kidney samples. Not shown (because of a different y axis scale), the amount of H3K4m3 was also not increased by I/R (2.2% versus 2.5% input for control and I/R samples; NS).
Figure 8
Figure 8
Histone variant H2A.Z levels and H3 histone modifications at the HMGCR promoter, and along the gene. I/R caused a twofold to fourfold increase in the amounts of H2A.Z variant and of H3K4m3 at all assessed sites. H3 lysine 19 acetylation was also increased at exons 1 and 19 (but not at the promoter).
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References

    1. Honda N, Hishida A, Ikuma K, Yonemura K. Acquired resistance to acute renal failure. Kidney Int. 1987;31:1233–1238. - PubMed
    1. Zager RA, Baltes LA, Sharma HM, Jurkowitz MS. Responses of the ischemic acute renal failure kidney to additional ischemic events. Kidney Int. 1984;26:689–700. - PubMed
    1. Reimer KA, Murry CE, Jennings RB. Cardiac adaptation to ischemia. Ischemic preconditioning increases myocardial tolerance to subsequent ischemic episodes. Circulation. 1990;82:2266–2268. - PubMed
    1. Zhao H. The protective effect of ischemic postconditioning against ischemic injury: from the heart to the brain. J Neuroimmune Pharmacol. 2007;2:313–318. - PubMed
    1. Hausenloy DJ, Yellon DM. Remote ischaemic preconditioning: underlying mechanisms and clinical application. Cardiovasc Res. 2008;79:377–386. - PubMed

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