In this regard Hydralazine syn

In this regard, Hydralazine (syn. 1-Hydrazinylphthalazine) was primarily synthesized by Ciba as an anti-hypertensive dhpg and has long been known to possess de-methylating activity (Arce et al., 2006; Druey, 1949). In clinical studies, Hydralazine and its derivate Dihydralazine were effective in de-methylating and re-activating expression of aberrantly silenced genes in cancer patients and a recent phase III clinical study demonstrated delayed cancer progression in patients receiving Hydralazine (Coronel et al., 2011). After its introduction to the clinic in 1952 as an anti-hypertensive drug, Hydralazine became part of the standard triple-regimen (Hydrochlorothiazide, Propranolol and Hydralazine) (Heagerty et al., 1982). With the emergence of ACEIs and ARBs as anti-hypertensive drugs with cardio-protective activity, Hydralazine today is mainly used as a third-line drug in patients with therapy-resistant hypertension (Leonetti et al., 1990). Due to it being highly safe, it is still a first-line therapy for hypertension during pregnancy (James et al., 2014). To our knowledge, its effectiveness to decrease the prevalence of ESRD has not yet been assessed. The aim of this study was to elucidate the potential of Hydralazine to correct aberrant DNA methylation in fibrotic kidneys and subsequently inhibit renal fibrogenesis.
Materials and Methods
Results
Discussion This study adds further evidence for a contribution of aberrant promoter CpG island methylation to the progression of renal fibrogenesis and its potential as a biomarker and therapeutic target. While there are likely additional genes affected by CpG island promoter methylation, this study provides mechanistic evidence for the first time that aberrant transcriptional silencing of Rasal1 causally contributes to progression of renal fibrogenesis and that Rasal1 methylation is not just part of an unspecific methylation signature in renal fibrosis. In this regard, normalization of aberrant promoter methylation through administration of 5′-Azacytidine or Hydralazine was associated with attenuated fibroblast activation and fibrogenesis in experimental fibrosis. Due to DNA incorporation, 5′-Azacytidine and its derivate 5′-Aza-2′-Deoxycytidine have considerable cytotoxicity, in addition to their unspecific de-methylating activity. Hydralazine is not incorporated into DNA and recruits crucial steps in dynamic DNA de-methylation involving Tet3-mediated formation of 5-hydroxymethyl-cytosine (5hmC), 5-formyl-cytosine (5fC), and 5-carboxyl-cytosine (5caC) with subsequent Tdg-mediated excision and replacement with naked cytosine (Fig. 12A–C). Due to the CXXC motif target selectivity of Tet proteins, which directs Tet3 directly to the CpG islands within the RASAL1 promoter, one can speculate that the de-methylating activity is more specific for aberrantly methylated genes as compared to 5′-Azacytidine, possibly accounting to its benign toxicity (Bechtel et al., 2010; Xu et al., 2012). Due to the prominent functional contribution of RASAL1 promoter CpG island methylation to progression of chronic kidney disease, tools to specifically de-methylate RASAL1 should be developed and explored in the future. Our study adds further evidence for the utility of RASAL1 methylation as a biomarker of renal fibrosis, which is not surprising as several transcriptional profiling studies revealed that RASAL1 expression is consistently decreased in kidney biopsies from patients with chronic kidney disease (Tampe et al., 2014). Importantly, we demonstrate for the first time that circulating methylated RASAL1 CpG island promoter fragments correlate with degree of intrarenal RASAL1 methylation and degree of fibrosis, similar to increased levels of methylated CpG fragments which can be detected in cancer patients. While the mechanism of how methylated promoter CpG island fragments are released into the circulation is not clear, we suspect that they are unspecifically liberated from injured cells, similar to what is observed in solid tumors. Because levels of methylated Rasal1 CpG island promoter fragments decreased in mice which were treated with de-methylating and anti-fibrotic agent 5′-Azacytidine or Hydralazine, we speculate that circulating methylated DNA fragment levels dynamically reflect RASAL1 CpG island promoter methylation within the kidney and may have utility to monitor effectiveness of de-methylating and anti-fibrotic therapies. As such decrease in circulating methylated Rasal1 CpG island promoter fragments in mice correlated with blunted kidney fibrosis upon de-methylating therapy, it is attractive to speculate that decreased levels of methylated RASAL1 DNA fragments and blood pressure-independent attenuation of CKD progression which were observed in patients which had received low-dose Dihydralazine are also reflective of its potential reno-protection.