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

<p><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.</p

Conditional mouse models support the role of SLC39A14 (ZIP14) in Hyperostosis Cranialis Interna and in bone homeostasis

Hyperostosis Cranialis Interna (HCI) is a rare bone disorder characterized by progressive intracranial bone overgrowth at the skull. Here we identified by whole-exome sequencing a dominant mutation (L441R) in SLC39A14 (ZIP14). We show that L441R ZIP14 is no longer trafficked towards the plasma membrane and excessively accumulates intracellular zinc, resulting in hyper-activation of cAMP-CREB and NFAT signaling. Conditional knock-in mice overexpressing L438R Zip14 in osteoblasts have a severe skeletal phenotype marked by a drastic increase in cortical thickness due to an enhanced endosteal bone formation, resembling the underlying pathology in HCI patients. Remarkably, L438R Zip14 also generates an osteoporotic trabecular bone phenotype. The effects of osteoblastic overexpression of L438R Zip14 therefore mimic the disparate actions of estrogen on cortical and trabecular bone through osteoblasts. Collectively, we reveal ZIP14 as a novel regulator of bone homeostasis, and that manipulating ZIP14 might be a therapeutic strategy for bone diseases