Losartan reverses permissive epigenetic changes in renal glomeruli of diabetic db/db mice

Abstract

Epigenetic mechanisms such as chromatin histone H3 lysine methylation and acetylation have been implicated in diabetic vascular complications. However, histone modification profiles at pathologic genes associated with diabetic nephropathy in vivo and their regulation by the angiotensin II type 1 receptor (AT1R) are not clear. Here we tested whether treatment of type 2 diabetic db/db mice with the AT1R blocker losartan not only ameliorates diabetic nephropathy, but also reverses epigenetic changes. As expected, the db/db mice had increased blood pressure, mesangial hypertrophy, proteinuria, and glomerular expression of RAGE and PAI-1 vs. control db/+ mice. This was associated with increased RNA polymerase II recruitment and permissive histone marks as well as decreased repressive histone marks at these genes, and altered expression of relevant histone modification enzymes. Increased MCP-1 mRNA levels were not associated with such epigenetic changes, suggesting post-transcriptional regulation. Losartan attenuated key parameters of diabetic nephropathy and gene expression, and reversed some but not all the epigenetic changes in db/db mice. Losartan also attenuated increased H3K9/14Ac at RAGE, PAI-1, and MCP-1 promoters in mesangial cells cultured under diabetic conditions. Our results provide novel information about the chromatin state at key pathologic genes in vivo in diabetic nephropathy mediated in part by AT1R. Thus, combination therapies targeting epigenetic regulators and AT1R could be evaluated for more effective treatment of diabetic nephropathy.

Fig. 1

Schematic diagram showing the study design.

Fig. 2. Effect of Losartan treatment on blood pressure and parameters of diabetic nephropathy in DB/DB mice

A. Systolic blood pressure (mmHg) was determined by tail cuff method in the three groups: db/+ mice treated with water (db/+H2O), db/db mice treated with water (db/dbH2O) and db/db mice treated with Losartan (db/dbLOS) for 10 weeks (n=14). B. Bar graph showing total urine protein levels in 24 h urine samples from the indicated mice (n=11 to 14). C. Bar graph showing albuminuria assayed using ELISA as described in the methods section. D. Images of PAS staining in kidney sections from indicated mice were collected using an Olympus BX51 microscope (40X lens). E–F. Mesangial expansion (E) and glomerular area (F) in PAS stained kidney sections determined using Image-Pro Plus software were expressed as “fold over db/+H2O” (n=8). A–C and E–F: Data represents Mean±SEM; ***, p<0.0001; **, p<0.001; *, p<0.05. Scale bar-50 μm.

Fig. 3. Losartan treatment can inhibit the expression of key inflammatory and fibrotic genes in renal glomeruli isolated from db/db mice

A–C. RT-qPCR results showing the expression of indicated genes in glomeruli from db/+H2O, db/dbH2O and db/dbLOS mice. Kidneys from three mice were pooled to prepare glomeruli from each group of mice and total RNA was extracted to analyze mRNA expression by RT-qPCR. Gene expression was normalized to the β-actin gene and expressed as % of db/+H2O. Data represents Mean±SEM; *, p<0.05 (n=9–17). D. IHC staining of kidney sections from indicated mice using RAGE, PAI-1 and MCP-1 antibodies. IHC was performed as described in the Methods section and images were collected using an Olympus DP-72 microscope (40X lens). E. Intensities of RAGE, PAI-1 and MCP-1staining were quantified using Smart segmentation method in Image-Pro-Premier software (Media Cybernetics, Rockville, MD). Results were expressed as “% of db/+H2O”. Data represents Mean±SEM; *, p<0.05 (n=10–13). Scale bar-50 μm.

Fig. 4. Matrix ChIP analysis of RNA polymerase II and histone H3 levels at MCP-1, PAI-1 and RAGE genes in renal glomeruli from three groups of mice

A. Schematic of the ChIP primer locations. Large black bold arrows indicate location of ChIP primers. Exons are shown as rectangles and the small arrows indicate the direction of transcription. B. Matrix ChIP assays with RNA polymerase II C-terminal domain (Pol II CTD) and histone H3 (H3) antibodies. Kidneys from 3 mice were pooled to prepare glomeruli for eight chromatin preparations from each group of mice. Cross-linked and sheared renal glomeruli chromatin was assayed using Pol II CTD (row1), and histone H3 (row2) antibodies. ChIP DNA was analyzed using primers at or near the promoters and the last exon using real-time PCR. Data were acquired, analyzed and graphed using in-house generated PCRCrunch and GraphGrid software. Statistical significance is indicated in the grid below the graphs (see Fig. S1). Each vertical line and its attached horizontal component is associated with the bar above it. The Bonferroni corrected p-value of the t-test between any two groups is indicated by the size of the solid circle at the intersection of their respective lines. Data represents Mean±SEM expressed as fraction of input (n=14 glomeruli preparations for each group). The circles below each bar indicate statistical significance (p<0.05 and p<0.01 are represented by small and larger circles, respectively).

Fig. 5. Matrix ChIP analysis of transcription permissive histone covalent modifications at MCP-1, PAI-1 and RAGE genes in renal glomeruli from three groups of mice

Cross-linked and sheared renal glomeruli chromatin was assayed using H3K9/14Ac (row1), H3K36me3 (row 2), H3K4me1 (row3), H3K4me2 (row4) and H3K4me3 (row5) antibodies. Matrix ChIP assays were performed as described in the legend of Fig. 4.

Fig. 6. Matrix ChIP analysis of repressive histone covalent modifications at MCP-1, PAI-1 and RAGE genes in renal glomeruli from three groups of mice

Cross-linked and sheared renal glomeruli chromatin was assayed using, H3K9me2 (row1), H3K9me3 (row2) and H3K27me3 (row3), antibodies. ChIP assays were performed as described in the legend of Fig 4.

Fig. 7. Reversal of glomerular Epigenetic factors expression by Losartan

A–H. Total RNA from glomeruli was used to analyze expression of indicated genes by RT-qPCR as described in the Methods section. Results were expressed as % of db/+H2O (*, p<0.05, n=6–9).

Fig. 8. Effect of Losartan on promoter H3K9/14Ac in cultured mesangial cells

A. Schematic of RMC treatment. RMC were cultured in normal (5.5 mM) glucose (NG), or high (25 mM) glucose (HG) for 48 h and then pre-treated for 1 h without (No LOS) or with 1 μM Losartan (LOS) followed by treatment with HG or HG + 0.1 μM Angiotensin II (HG/A) for an additional 24 h. B–E. ChIP-qPCR results showing H3K9/14Ac at the RAGE (B), PAI-1 (C), MCP-1 (D) and CypA (E) gene promoters in rat MC. Chromatin preparations from RMC treated as described in A were subjected to ChIP assays using H3K9/14Ac antibodies and ChIP-enriched DNA samples were analyzed by qPCR using primers for indicated gene promoters. Results were expressed as % of NG. Data represents Mean±SEM; *, p<0.05 (n=3–6).