Supplementary Material for: Untargeted DNA-Demethylation Therapy Neither Prevents Nor Attenuates Ischemia-Reperfusion-Induced Renal Fibrosis

Background: Current treatment options for chronic
kidney disease (CKD) are limited and their focus is on slowing its
progression by addressing comorbidities. Fibrosis, the common
histopathological process in CKD, is a major therapeutic research
target. In CKD, fibroblasts are terminally activated due to alterations
in their DNA-methylation pattern, particularly hypermethylation.
Preventing the copying of pathological DNA-methylation patterns in
proliferating fibroblasts could be a new effective therapeutic strategy
for treating CKD. Methods: To evaluate the therapeutic
effect of short-term treatment with the DNA-methyltransferase
(DNMT)-inhibitor decitabine on fibrosis (either developing or already
established), male C57Bl/6 mice underwent warm unilateral
ischemia-reperfusion injury. Respectively 3 days, 3 and 6 weeks after
surgery, decitabine treatment (0.25 mg/kg) was initiated for 10 days
after which animals were followed up to 12 weeks after ischemia. The
efficacy of therapy on fibrosis was evaluated by collagen I and tgfβ
gene expression and histological quantification of collagen I staining.
In addition, the effect of decitabine treatment on tubular injury (Kim-1, Ngal), inflammation (TNFa, IL6), DNA-methyltransferases (Dnmt1, 3a, and 3b), and global methylation status was determined. Results:
Following ischemia there was a significant increase in fibrotic,
injury, and inflammatory markers as well as an increase of the various dnmts.
Although decitabine treatment transiently increased renal injury and
had a moderately decreasing effect on dnmt expression and on global
DNA-methylation upon immediate treatment, none of the treatment regimens
succeeded in preventing, attenuating, or diminishing fibrosis in the
long run. Conclusion: Administration of untargeted
nucleoside analogues seems unsuitable as a first-line treatment option
in developing or established CKD.