Abstract
Progression of chronic kidney disease remains a principal problem in clinical nephrology and there is a pressing need for novel therapeutics and biomarkers. Aberrant promoter CpG island methylation and subsequent transcriptional silencing of specific genes have emerged as contributors to progression of chronic kidney disease. Here, we report that transcriptional silencing of the Ras-GTP suppressor RASAL1 contributes causally to progression of kidney fibrosis and we identified that circulating methylated RASAL1 promoter DNA fragments in peripheral blood correspond with levels of intrarenal levels of RASAL1 promoter methylation and degree of fibrosis in kidney biopsies, enabling non-invasive longitudinal analysis of intrarenal CpG island methylation.Retrospective analysis of patients with hypertensive nephrosclerosis revealed that circulating methylated RASAL1 promoter DNA fragments in peripheral blood decrease with Dihydralazine treatment in patients with hypertensive nephrosclerosis, and provided evidence that low-dose Dihydralazine delays decline of excretory kidney function, whereas Dihydralazine at standard doses had no protective effect. We demonstrate that the protective effect of Dihydralazine is due to induction of endogenous Tet3/Tdg-mediated DNA-de-methylation activity reversing aberrant promoter CpG island methylation, while HIF1α induction at standard doses counterbalances its protective activity. We conclude that RASAL1 promoter methylation is a therapeutic target and a biomarker of renal fibrosis. Our study suggests that therapeutic use of low-dose Dihydralazine in patients with chronic kidney disease and fibrosis deserves further consideration.
Highlights
Based on encouraging studies demonstrating the de-methylating activity of Hydralazine with regard to cancer cells, we first aimed to determine if this de-methylating activity could be utilized to inhibit renal fibrogenesis, as was previously shown for the prototypical de-methylating drug 5′-Azacytidine (Bechtel et al, 2010)
We challenged CD1 mice with a single injection of folic acid (FA, which results in severe renal fibrosis within 5 months) and administered 5′-Azacytidine or Hydralazine at 10 mg/kg/day and 5 mg/kg/day, respectively, from day 3 to day 21 after the folic acid injection
We focused on Rasal1 methylation because we previously identified RASAL1 to be selectively methylated in fibrotic kidney biopsies utilizing a genomewide methylation screen and we verified that Rasal1 promoter CpG island methylation correlated with degree of fibrosis in murine models of renal fibrosis as well as in renal biopsies (Bechtel et al, 2010; Tampe et al, 2014)
Summary
Due to the imminent clinical need for effective anti-fibrotic therapies, identification of additional, already approved drugs with reno-protective potential would be highly desirable. In this regard, aberrant methylation of genomic DNA has emerged as a novel therapeutic target. While methylation of promoter CpG islands is a physiological mechanism to determine cell lineage and differentiation, aberrant promoter methylation (typically referred to as hypermethylation) and subsequent transcriptional silencing of affected genes are potent contributors to carcinogenesis. The importance of aberrant promoter CpG island methylation is highlighted by the effectiveness of 5′Azacytidine, the first de-methylating agent which was approved for clinical use for patients with high-risk myelodysplastic syndrome (MDS) and acute myeloid leukemia (Zeisberg and Zeisberg, 2013). More specific normalization of aberrant hypermethylation without incorporation of potentially toxic nucleotide analogs would be much more attractive
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
