Loss of Histone H3 K79 Methyltransferase Dot1l Facilitates Kidney Fibrosis by Upregulating Endothelin 1 through Histone Deacetylase 2

Abstract

BackgroundThe progression rate of CKD varies substantially among patients. The genetic and epigenetic contributions that modify how individual patients respond to kidney injury are largely unknown. Emerging evidence has suggested that histone H3 K79 methyltransferase Dot1l has an antifibrotic effect by repressing Edn1, which encodes endothelin 1 in the connecting tubule/collecting duct.MethodsTo determine if deletion of the Dot1l gene is a genetic and epigenetic risk factor through regulating Edn1, we studied four groups of mice: wild-type mice, connecting tubule/collecting duct–specific Dot1l conditional knockout mice (Dot1lAC), Dot1l and Edn1 double-knockout mice (DEAC), and Edn1 connecting tubule/collecting duct–specific conditional knockout mice (Edn1AC), under three experimental conditions (streptozotocin-induced diabetes, during normal aging, and after unilateral ureteral obstruction). We used several approaches (colocalization, glutathione S-transferase pulldown, coimmunoprecipitation, yeast two-hybrid, gel shift, and chromatin immunoprecipitation assays) to identify and confirm interaction of Dot1a (the major Dot1l splicing variant in the mouse kidney) with histone deacetylase 2 (HDAC2), as well as the function of the Dot1a-HDAC2 complex in regulating Edn1 transcription.ResultsIn each case, Dot1lAC mice developed more pronounced kidney fibrosis and kidney malfunction compared with wild-type mice. These Dot1lAC phenotypes were ameliorated in the double-knockout DEAC mice. The interaction between Dot1a and HDAC2 prevents the Dot1a-HDAC2 complex from association with DNA, providing a counterbalancing mechanism governing Edn1 transcription by modulating H3 K79 dimethylation and H3 acetylation at the Edn1 promoter.ConclusionsOur study confirms Dot1l to be a genetic and epigenetic modifier of kidney fibrosis, reveals a new mechanism regulating Edn1 transcription by Dot1a and HDAC2, and reinforces endothelin 1 as a therapeutic target of kidney fibrosis.

HDAC2 targeting stabilizes the CoREST complex in renal tubular cells and protects against renal ischemia/reperfusion injury

“…Given microarray data showing changes in endothelin levels confirmed by direct testing and in light of very recent data linking HDAC2 to regulation of Edn1 expression 27 , we hypothesized that Edn1 could be an important downstream mediator of HDAC2′s effects during renal IRI. Edn1 is produced by endothelial cells as well as renal tubular epithelial cells and is well known as a vasoconstrictor via its paracrine effects on vascular smooth muscle ET-A receptors.…”

“…Edn1 is produced by endothelial cells as well as renal tubular epithelial cells and is well known as a vasoconstrictor via its paracrine effects on vascular smooth muscle ET-A receptors. These effects play a role in hypertension, as well as mediating kidney damage independently of hypertension by increasing inflammation and kidney fibrosis 27 , 31 , 53 . Other groups have shown that endothelin is increased in response to renal IRI via a TNF-α mediated pathway 54 .…”

“…Other groups have shown that endothelin is increased in response to renal IRI via a TNF-α mediated pathway 54 . Of particular interest, Zhang et al showed, using a model of chronic kidney disease, that HDAC2 forms a complex with Dot1l, inhibiting its association with DNA, independent of HDAC2 catalytic activity 27 . Dot1l is a histone H3K79 methyltransferase that acts on the Edn1 promotor to repress endothelin in renal tubular cells.…”

“…In contrast, HDAC3 forms a canonical, deacetylase-dependent complex with NCoR1/NCoR2 and can be recruited to the nucleus by various transcription factors 25 , or can function via a non-canonical deacetylase-independent manner independently of NCoR1/NCoR2 26 . One relevant non-histone protein whose functions are regulated by class I HDACs is endothelin-1 (Edn1) 27 . Edn1 is a potent vasoconstrictor produced by endothelial cells and renal tubular epithelial cells and is known to mediate kidney injury and fibrosis 28 – 31 .…”

Epigenetic Histone Modifications in the Pathogenesis of Diabetic Kidney Disease

“…Considerable evidence has suggested that injured kidneys increased the expression of DOT1L and H3K79 dimethylation, particularly in renal tubular epithelial cells and myofibroblasts, eventually aggravating renal fibrosis, even developing end-stage renal disease. However, emerging evidence by Zhang et al 135 showed that Dot1l has an antifibrotic effect. Using several approaches in groups of mice, Dot1a-HDAC2 complex regulated H3K79me2 and H3 acetylation at the endothelin 1 (Edn1) promoter, ensuring the balance of endothelin transcription.…”