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Comparative Study
. 2015 Oct;88(4):734-44.
doi: 10.1038/ki.2015.164. Epub 2015 Jun 10.

Heterogeneity of epigenetic changes at ischemia/reperfusion- and endotoxin-induced acute kidney injury genes

Daniel Mar  1 , Sina A Gharib  1   2 , Richard A Zager  3 , Ali Johnson  3 , Oleg Denisenko  1 , Karol Bomsztyk  1
Affiliations
Comparative Study

Heterogeneity of epigenetic changes at ischemia/reperfusion- and endotoxin-induced acute kidney injury genes

Daniel Mar et al. Kidney Int. 2015 Oct.

Abstract

Aberrant gene expression is a molecular hallmark of acute kidney injury (AKI). As epigenetic processes control gene expression in a cell- and environment-defined manner, understanding the epigenetic pathways that regulate genes altered by AKI may open vital new insights into the complexities of disease pathogenesis and identify possible therapeutic targets. Here we used matrix chromatin immunoprecipitation and integrative analysis to study 20 key permissive and repressive epigenetic histone marks at transcriptionally induced Tnf, Ngal, Kim-1, and Icam-1 genes in mouse models of AKI; unilateral renal ischemia/reperfusion, lipopolysaccharide (LPS), and their synergistically injurious combination. Results revealed unexpected heterogeneity of transcriptional and epigenetic responses. Tnf and Ngal were transcriptionally upregulated in response to both treatments individually, and to combination treatment. Kim-1 was induced by ischemia/reperfusion and Icam-1 by LPS only. Epigenetic alterations at these genes exhibited distinct time-dependent changes that shared some similarities, such as reduction in repressive histone modifications, and also had major ischemia/reperfusion versus endotoxin differences. Thus, diversity of changes at AKI genes in response to different insults indicates involvement of several epigenetic pathways. This could be exploited pharmacologically through rational-drug design to alter the course and improve clinical outcomes of this syndrome.

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Figures

Fig.1
Fig.1. Cluster analysis of temporal gene expression profiles in AKI
The timing of harvesting the kidneys post I/R injury was 3, 26 and 76 hrs, and 2 hrs after either LPS or saline treatment (Methods). Total RNA from mice renal cortex was used in RT reactions with oligo dT. cDNA was used in real time PCR (qPCR) with gene specific primers (Table S1, supplement). mRNA level of a given gene in each sample was normalized to β-actin transcript. The mRNA levels of 56 AKI-associated genes were measured using and clustered based on expression pattern differences across time and injury model. Note the diverse transcriptional responses to injury of co-clustered genes as highlighted in the figure. Data are represented as mean, n=6 mice from each group. Four AKI candidate genes—Tnf, Ngal, Icam-1, and Kim-1—were selected for further in-depth epigenetic analysis.
Fig.2
Fig.2. Renal cortical Tnf, Ngal, Kim-1 and Icam-1 expression following unilateral kidney I/R and LPS injection
Total RNA from mice renal cortex was used in RT reactions with oligo dT. cDNA was used in real time PCR with gene specific primers (Table S1, supplement). mRNA level of a given gene in each sample was normalized to β-actin transcript. Data are represented as mean± SEM, n= 6 mice in each group. Statistical differences between two means (p value) are shown by the size of the solid circle under the x-axis: p<0.05 by small circle, p<0.01 by large circle, and no circle indicating the differences are not statistically significant (Methods and Fig.S1, supplement).
Fig.3
Fig.3. RNA polymerase II (Pol II) Tnf, Ngal, Kim-1 and Icam-1 genes following unilateral kidney I/R and LPS injection
Sheared cross-linked renal cortex chromatin from mice were assayed using a monoclonal antibody to (4H8) that detects both unphosphorylated and phosphorylated C-terminal domain repeats YSPTSPS. ChIP DNA were analyzed at Tnf, Ngal, Kim-1 and Icam-1 genes in qPCR. Data represent mean ± SEM (6 animals from each group), expressed as fraction of input.
Fig.4
Fig.4. Permissive histone H3 lysine 9 acetylation (H3K9Ac) at Tnf, Ngal, Kim-1 and Icam-1 genes following unilateral kidney I/R and LPS injection
Sheared cross-linked renal cortex chromatin from mice were assayed using H3K9Ac antibody. Data represent mean ± SEM (6 animals from each group), expressed as fraction of input.
Fig.5
Fig.5. Permissive histone H3 lysine 4 tri-methylation (H3K4m3) at Tnf, Kim-1, Ngal, and Icam-1 genes following unilateral kidney I/R and LPS injection
Sheared cross-linked renal cortex chromatin from mice were assayed using H3K4m3 antibody. Data represent mean ± SEM (6 animals from each group), expressed as fraction of input.
Fig.6
Fig.6. Transcription elongation histone H3 lysine 36 tri-methylation (H4K36m3) at Tnf, Kim-1, Ngal, and Icam-1 genes following unilateral kidney I/R and LPS injection
Sheared cross-linked renal cortex chromatin from mice were assayed using H3K36m3 antibody. Data represent mean ± SEM (6 animals from each group), expressed as fraction of input.
Fig.7
Fig.7. Transcription repressive histone H4 serine 1 phosphorylation (H4pSer1) at Tnf, Kim-1, Ngal, and Icam-1 genes following unilateral kidney I/R and LPS injection
Sheared cross-linked renal cortex chromatin from mice were assayed using H4pSer1 antibody. Data represent mean ± SEM (6 animals from each group), expressed as fraction of input.
Fig.8
Fig.8. Integrated transcriptional and epigenetic analysis of Tnf, Kim-1, Ngal, and Icam-1 portraits following unilateral kidney injury
Log-transformed values for mRNA and Pol II levels, as well as several epigenetic modifications during temporal progression of AKI due to I/R, LPS and I/R+LPS are depicted as a heatmap. While there is a general increase in permissive marks and reduction of repressive marks in a time-dependent manner, note the heterogeneous response pattern of individual AKI-induced genes.
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