Epigenetic Histone β-Hydroxybutyrylation Contributes to Renoprotection by β-Hydroxybutyrate in the Dahl Rat

Abstract

Background: Previously, we demonstrated that the ketone body, β-hydroxybutyrate, is a potent antihypertensive and reno-protective metabolite in Dahl Salt-Sensitive rats. However, the mechanism by which β-hydroxybutyrate confers these beneficial effects is understudied. Here we focused on determining whether the reno-protective effect of β-hydroxybutyrate is due to its known ability to epigenetically remodel chromatin via histone β-hydroxybutyrylation.

Methods: We used the same animal protocol previously used for the discovery of the reno-protective effect of β-hydroxybutyrate. Briefly, postweaning, male and female Dahl Salt-Sensitive rats were split into 2 groups and supplemented with or without 1,3-butanediol for 6 weeks. At euthanasia, circulating β-hydroxybutyrate was quantitated. Renal homogenates were examined for histone 3 lysine 9 β-hydroxybutyrylation, chromatin occupancy, transcriptomic and proteomic profiles with validations.

Results: Rats supplemented with 1,3-butanediol had higher circulating β-hydroxybutyrate, renal histone β-hydroxybutyrylation, and significant remodeling of chromatin. Notably, regions of the genome associated with lipid catabolism were predominantly in an open chromatin configuration, leading to active transcription and translation. The most highly upregulated gene actively transcribed and translated was Hmgcs2 (3-hydroxy-3-methylglutaryl CoA synthase 2), a gene responsible for the biosynthesis of β-hydroxybutyrate in mitochondria. In contrast, regions with more compact chromatin structures contained immune function genes, Ptprc (protein tyrosine phosphatase receptor type C) and Lcp1 (lymphocyte cytosolic protein 1), which were suppressed.

Conclusions: These results reveal that renal epigenetic histone β-hydroxybutyrylation is a novel mechanism by which transcriptional regulation of both energy metabolism and immune function occur concomitantly and contribute to renoprotection in the hypertensive Dahl rat.

Keywords: chromatin remodeling; energy metabolism; histones; hypertension; ketones; kidney.

Figure 1:. Histone β-hydroxybutyrylation is elevated with 1,3-butanediol treatment.

Groups of S rats on a high salt diet (2% Nacl) were supplemented with or without 1,3-butanediol (20% v/v) in their drinking water as described in the methods section. (A-B) Systolic blood pressure of males (n= 5-6/group) and females (n= 5/group). (C-D) Serum levels of β-hydroxybutyrate in males (n=4-6/group) and females (n=6-8/group). (E-F) Immunoblotting for β-hydroxybutyrylation. (G-H) Quantification of the blots. (n=4-7/group), BHB: β-hydroxybutyrate, H3K9BHB: Histone 3 lysine 9 β-hydroxybutyrylation, H3: Histone 3. All data are mean ±SEM, *p<0.05, **p<0.01, ****p<0.0001.

Figure 2:. Chromatin accessibility, gene expression, and protein levels are altered with 1,3-butanediol treatment.

(A) Volcano plot for BHB vs. control groups. Each data point is a tested locus. The vertical lines correspond to the logFC cutoff. (B) Annotation summary of loci tested in BHB vs control groups. X-axis: genomic annotations; Y-axis proportion of loci in each category within each type of annotation. All Tested loci (red bars), serve as a background by which to compare the significant loci (blue and green bars). Loci or genomic regions were calculated based on differentially open if FDR < 0.05 and |logFC| > 0.585. (C) Pathways associated with the upregulated genes of BHB group. (D) Pathways associated with upregulated proteins in the BHB group. (E) Venn diagram showing common upregulated genes. Different shades of blue circles depict ATACseq, RNAseq, and Proteomics data and numbers within the circles are numbers of genes/proteins significantly upregulated. (F) Chromatin accessibility for genes of interest. The y-axis represents chromatin cut sites and thus open chromatin, and x-axis represents the chromatin location of genes of interest. Black peaks represent the control group, and green peaks represent BHB group. (n=3/group).

Figure 3:. 1,3-butanediol treatment reduces respiratory exchange ratio (RER) and inhibits mammalian target of rapamycin complex 1 (mTORC1).

(A-C) Metabolic CLAMS data showed reduced VO₂, VCO₂, and RER level in the BHB group compared to controls. Black line: control group and green line: BHB group. (n=5/group) (D-E) Reduced phospho-S6 ribosomal protein levels were found in male rats treated with BHB compared to control. See also Figure S5 for data from female rats. BHB: β-hydroxybutyrate. (n=5-6/group) All data are mean±SEM; *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001.

Figure 4:. Validation of common upregulated genes through CHIP-qPCR, Real time PCR, and proteomics.

(A) CHIP-qPCR data demonstrating enrichment of Hmgcs2, Acaa1b, Cyp2d4, and Cyp2e1 in the BHB group compared to control (n=3 replicates/group). Black bar: Control, Green bar: BHB. (B) Real time PCR showed higher expression of Hmgcs2, Acaa1b, Cyp2d4, and Cyp2e1 in BHB group compared to control (housekeeping gene is L36a) (n=5-6/group) (C) Normalized relative abundance of Hmgcs2, Acaa1b, Cyp2d4, Cyp2e1 proteins (n=3-6/group). See also Figure S2 for female data. Black open circle- control, green closed circle- BHB. All data are mean±SEM; *p< 0.05, **p< 0.01, ***p< 0.001, ****p <0.0001 and ^#p=0.0567.

Figure 5:. Kidney proximal tubule epithelial cell mitochondrial circulatory index was found to be higher in BHB group compared to control.

(A) Representative transmission electron microscopy images of renal mitochondria. Orange arrows point to mitochondria. (B-D) Bar graph shows quantification (B) Average mitochondrial area (C) Area/Perimeter, and (D) Circularity index. BHB: β-hydroxybutyrate. n=30 images per group. A total of 3 rats per group were used. All data are mean±SEM, ***p< 0.001, and ****p<0.0001.

Figure 6:. Chromatin accessibility, gene expression, protein levels, and CD3⁺ T cells proliferation were altered with 1,3-butanediol treatment.

(A) Venn diagrams showing common downregulated genes and proteins leading to downregulation of immune function pathways. Different shades of blue circles are used to indicate ATACseq, RNAseq, and Proteomics data. The gray box lists the names of common downregulated genes and proteins, beneath which are shown the associated downregulated pathways. (B) Chromatin accessibility at the promoter region of Ptprc and Lcp1 promoter. The y-axis represents chromatin cut sites and thus open chromatin, and x-axis represents the chromatin location of genes of interest. Black peaks represent the control group, and green peaks represent BHB group. (C) Real time PCR data showing reduced expression with Ptprc and Lcp1 in the 1,3-butanediol supplementation. (n=5-6/group) (D) Normalized relative abundances of Ptprc (Cd45) and Lcp1 detected in the quantitative proteomics study. Black open circle– control, green closed circle- BHB. (n=3/group). (E) Peripheral white blood cells and Lymphocytes in the control and BHB treated male S rats. (F) Representative histogram for percent CFSE-positive CD3+ T cells andquantification for percent CFSE-positive CD3⁺ T cells in control and BHB groups after 5 days. Data represented as Mean ± SEM and N was plotted for control and BHB group. N=7-8/group. WBC: white blood cells, Lym: Lymphocytes, CFSE: carboxyfluorescein diacetate succinimidyl ester, CD3: Cluster of differentiation 3, P= Parent population, D1= Daughter 1, and D2= Daughter 2 population All data are mean±SEM, *p<0.05, **p< 0.01, ***p< 0.001 and ****p<0.0001.