Abstract
Introduction: Epigenetic regulation of fibrogenesis through post-translational histone modifications (marks) may be a key determinant of progression in renal disease. In this study, we examined the distribution and acquisition of histone 3 Lysine 9 (H3K9) marks after injury and stimulation with the pro-fibrotic cytokine TGF-β1. Our focus was on their presence in activated fibroblasts (myofibroblasts) and epithelial cells (epithelial-mesenchymal transition).Methods and Results: Immunofluorescent microscopy was used to examine global H3K9 acetylation (H3K9Ac) and tri-methylation (H3K9Me3) after unilateral ureteric obstruction (UUO) in mice. Confocal, super resolution microscopy and flow cytometry were used to determine the in vitro effect of TGF-β1 on structural arrangement of these marks, and their relationship with α-smooth muscle actin (αSMA) expression, a marker of myofibroblasts and early EMT. The number of individual histone marks was increased 10 days after UUO (p < 0.05 vs. control), with both marks clearly seen in various cell types including proximal tubules and myofibroblasts. Sub-nuclear microscopy in primary rat renal fibroblasts and a proximal tubule cell line (NRK-52e) showed that H3K9Ac was co-localized with phosphorylated-Ser2 RNA polymerase II (pRNAPol II), while H3K9Me3 was not, consistent with permissive and repressive effects on gene expression respectively. In both cell types H3K9Ac was diffusely distributed throughout the nucleus, while H3K9Me3 was found in compartments resembling the nucleolus, and in the case of the fibroblast, also juxtapositioned with the nuclear membrane. TGF-β1 had no effect on H3K9Ac marks in either cell, but resulted in a redistribution of H3K9Me3 within the fibroblast nucleus. This was unrelated to any change in mitogenesis, but was associated with increased αSMA expression.Conclusion: These findings highlight why it is important to consider the epigenetics of each cell individually, because whilst no overall enrichment occurred, renal myofibroblast differentiation was accompanied by distinct changes in histone mark arrangements.
Highlights
Epigenetic regulation of fibrogenesis through post-translational histone modifications may be a key determinant of progression in renal disease
Representative micrographs are shown from normal kidneys (D0) and kidneys 3 days (D3) and 10 days (D10) post-ureteric obstruction (UUO) (Figure 1)
ΑSMA staining was confined to the vasculature
Summary
Epigenetic regulation of fibrogenesis through post-translational histone modifications (marks) may be a key determinant of progression in renal disease. After acute renal injury occurs, normal tissue repair mechanisms are often able to restore kidney function If such mechanisms are disrupted, the injury is severe, or the cause of the injury persists, acute kidney injury can progress into a chronic disorder characterized by non-recoverable organ remodeling and scarring (fibrosis) (Hewitson, 2009). Renal parenchymal fibrosis represents the final common pathway in all progressive chronic kidney disease (CKD). This process manifests itself as glomerular and vascular sclerosis and tubulointerstitial fibrosis, with the last of these the best predictor of disease progression (Hewitson, 2009). In each case activation can be recognized by de novo expression of α-smooth muscle actin (αSMA), a marker of myofibroblast differentiation in fibroblasts, and epithelialmesenchymal transition (EMT) in renal tubule epithelial cells (Hewitson, 2009)
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