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Abstract: FR-OR050

Histone Demethylation near the Serum Amyloid A Promoter: Metabolic Memory Associated Inflammation

Session Information

Category: Diabetic Kidney Disease

  • 601 Diabetic Kidney Disease: Basic

Authors

  • Dieter, Brad, Providence Health Care, Spokane, Washington, United States
  • Tuttle, Katherine R., University of Washington School of Medicine, Spokane, Washington, United States
  • Meek, Rick L., Providence Sacred Heart Medical Center, Spokane, Washington, United States
Background

Episodic hyperglycemia increases risk of diabetic complications, also known as “metabolic memory.” The study aim was to determine if serum amyloid A (SAA) mediated inflammation is due to demethylation of trimethyl histone 3 lysine 27 (H3K27Me3) near the SAA promoter in podocytes and to correlate indicators of this demethylation with SAA expression in kidneys of humans and mice with diabetes.

Methods

SAA knockout (CRISPR-Cas9) and control mouse podocytes were exposed to advanced glycation end-products (AGE, 300 μg/mL) for 7 days, followed by 7 days without AGE. H3K27Me3 near the SAA promoter was measured via chromatin immunoprecipitation (ChIP). Levels of mRNA for SAA and inflammatory mediators: (CXCL5, CCL5, and CCL2) were measured by qt-PCR. H3K27Me3 demethylation was inhibited (GSK-1, 20 uM) in podocytes exposed to AGE for 1 day. ChIP measured H3K27Me3 near the SAA promoter in the kidney cortex of db/db and wild type mice (C57BLKS). Relationships between expression of JMDJ3 mRNA (H3K27Me3 specific demethylase) and SAA mRNA were determined in kidneys of diabetic and non-diabetic humans and mice (C57- BLKS, DBA/2, eNOS-deficient C57-BLKS, Nephroseq).

Results

H3K27Me3 was reduced (95%, n=6 pooled samples) after 7-day periods of AGE exposure and AGE withdrawal in podocytes. Levels of mRNA increased for SAA (17±6-fold, p=0.025) and inflammatory mediators: CXCL5 (18±12-fold, p=0.004), CCL5 (1.6±0.6-fold, p=0.026), and CCL2 (2.0±0.7-fold, p=0.001). SAA knockout reduced expression of these mediators by >60% (p<0.05 for all). GSK-1 reduced AGE-induced expression of SAA and these mediators by >60% (p<0.05 for all). H3K27Me3 was lower in the kidney cortex of db/db mice compared to wild type mice (25±6%, p=0.049, n=6). Expression of JMDJ3 mRNA correlated with SAA mRNA in diabetic and non-diabetic kidney tissue from humans (r=0.54, p<0.001) and mice (r=0.54, p<0.001).

Conclusion

Episodic exposure to AGE, a hyperglycemia-related perturbation, caused H3K27Me3 demethylation near the SAA promoter and increased expression of inflammatory mediators in podocytes. Correlative studies showed that indicators of this histone demethylation process and increased SAA expression carried over into kidney tissue of humans and mice.