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

<b><i>Background:</i></b> 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. <b><i>Methods:</i></b> 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 <i>collagen I</i> and <i>tgfβ</i> gene expression and histological quantification of collagen I staining. In addition, the effect of decitabine treatment on tubular injury (<i>Kim-1</i>, <i>Ngal</i>), inflammation (<i>TNFa</i>, <i>IL6</i>), DNA-methyltransferases (<i>Dnmt1, 3a, and 3b</i>), and global methylation status was determined. <b><i>Results:</i></b> Following ischemia there was a significant increase in fibrotic, injury, and inflammatory markers as well as an increase of the various <i>dnmts</i>. 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. <b><i>Conclusion:</i></b> Administration of untargeted nucleoside analogues seems unsuitable as a first-line treatment option in developing or established CKD.

**<i>背景：</i>** 当前慢性肾脏病（chronic kidney disease, CKD）的治疗选择十分有限，临床主要通过干预合并症以延缓疾病进展。纤维化（fibrosis）作为慢性肾脏病常见的组织病理学进程，是当前治疗研究的核心靶点。在慢性肾脏病中，成纤维细胞（fibroblasts）因DNA甲基化（DNA methylation）模式改变，尤其是高甲基化（hypermethylation），而发生终末活化。阻断增殖性成纤维细胞中病理性DNA甲基化模式的传递，或可成为治疗慢性肾脏病的新型有效策略。
**<i>方法：</i>** 为评估DNA甲基转移酶（DNA-methyltransferase, DNMT）抑制剂地西他滨（decitabine）短期治疗对进展期或已确诊纤维化的干预效果，本研究对雄性C57Bl/6小鼠实施单侧热缺血再灌注损伤造模。分别于造模后3天、3周及6周启动地西他滨干预（剂量为0.25 mg/kg），连续给药10天，随后将小鼠随访至缺血造模后12周。通过检测I型胶原（collagen I）、转化生长因子β（tgfβ）的基因表达水平，以及I型胶原染色的组织学定量分析，评估干预对纤维化的治疗效果。此外，本研究还检测了地西他滨干预对肾小管损伤标志物（Kim-1、Ngal）、炎症因子（TNF-α、IL-6）、DNA甲基转移酶家族（Dnmt1、3a、3b）表达及全基因组甲基化状态（global methylation status）的影响。
**<i>结果：</i>** 缺血造模后，小鼠的纤维化、损伤及炎症标志物水平，以及各类DNA甲基转移酶的表达均显著升高。尽管地西他滨干预可一过性加重肾损伤，且在即刻治疗时对DNA甲基转移酶表达及全基因组甲基化水平具有轻度下调作用，但所有干预方案均未能在长期随访中实现纤维化的预防、减轻或缓解。
**<i>结论：</i>** 非靶向核苷类似物（untargeted nucleoside analogues）似乎不适用于进展期或已确诊慢性肾脏病的一线治疗方案。