Chromatin accessibility and epigenetic DNA modifications in CKD osteoblasts: a study of bone and osteoblasts from pediatric patients with chronic kidney disease
Aline Martin, R. Kawaguchi, Qing Wang, I. Salusky, Renata C Pereira, K. Wesseling-Perry
{"title":"Chromatin accessibility and epigenetic DNA modifications in CKD osteoblasts: a study of bone and osteoblasts from pediatric patients with chronic kidney disease","authors":"Aline Martin, R. Kawaguchi, Qing Wang, I. Salusky, Renata C Pereira, K. Wesseling-Perry","doi":"10.1093/jbmrpl/ziad015","DOIUrl":null,"url":null,"abstract":"\n Maturation defects are intrinsic features of osteoblast lineage cells in CKD patients. These defects persist ex vivo, suggesting that CKD induces epigenetic changes in bone cells. To gain insights into which signaling pathways contribute to CKD-mediated, epigenetically-driven, impairments in osteoblast maturation, we characterized RNA expression and DNA methylation patterns by RNA-Seq and Methylation Epic in primary osteoblasts from 9 adolescent and young adult dialysis patients with end-stage kidney disease and 3 healthy references. ATAC-Seq was also performed on a subset of osteoblasts. Bone matrix protein expression was extracted from iliac crest and evaluated by proteomics. GSEA analysis was used to establish signaling pathways consistently altered in chromatin accessibility, DNA methylation, and RNA expression patterns. Single genes were suppressed in primary osteoblasts using shRNA and mineralization characterized in vitro. The effect of NFAT signaling suppression was also assessed using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) incorporation. We found that signaling pathways critical for osteoblast differentiation were strongly downregulated in CKD osteoblasts. GSEA identified highly significant methylation changes, differential chromatin accessibility, and altered RNA expression in NFAT signaling targets. NFAT inhibition reduced osteoblast proliferation. Combined analysis of osteoblast RNA expression and whole bone matrix composition identified thirteen potential ligand-receptor pairs were identified. In summary, epigenetic changes in CKD osteoblasts associate with altered expression of multiple osteoblast genes and signaling pathways. An increase in NFAT signaling may play a role in impaired CKD osteoblast maturation. Epigenetic changes also associate with an altered bone matrix which may contribute to bone fragility. Further studies are necessary to elucidate the pathways affected by these genetic alterations since elucidating these pathways will be vital to correcting the underlying biology of bone disease in the CKD population.","PeriodicalId":14611,"journal":{"name":"JBMR Plus","volume":null,"pages":null},"PeriodicalIF":3.4000,"publicationDate":"2024-01-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"JBMR Plus","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.1093/jbmrpl/ziad015","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"ENDOCRINOLOGY & METABOLISM","Score":null,"Total":0}
引用次数: 0
Abstract
Maturation defects are intrinsic features of osteoblast lineage cells in CKD patients. These defects persist ex vivo, suggesting that CKD induces epigenetic changes in bone cells. To gain insights into which signaling pathways contribute to CKD-mediated, epigenetically-driven, impairments in osteoblast maturation, we characterized RNA expression and DNA methylation patterns by RNA-Seq and Methylation Epic in primary osteoblasts from 9 adolescent and young adult dialysis patients with end-stage kidney disease and 3 healthy references. ATAC-Seq was also performed on a subset of osteoblasts. Bone matrix protein expression was extracted from iliac crest and evaluated by proteomics. GSEA analysis was used to establish signaling pathways consistently altered in chromatin accessibility, DNA methylation, and RNA expression patterns. Single genes were suppressed in primary osteoblasts using shRNA and mineralization characterized in vitro. The effect of NFAT signaling suppression was also assessed using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) incorporation. We found that signaling pathways critical for osteoblast differentiation were strongly downregulated in CKD osteoblasts. GSEA identified highly significant methylation changes, differential chromatin accessibility, and altered RNA expression in NFAT signaling targets. NFAT inhibition reduced osteoblast proliferation. Combined analysis of osteoblast RNA expression and whole bone matrix composition identified thirteen potential ligand-receptor pairs were identified. In summary, epigenetic changes in CKD osteoblasts associate with altered expression of multiple osteoblast genes and signaling pathways. An increase in NFAT signaling may play a role in impaired CKD osteoblast maturation. Epigenetic changes also associate with an altered bone matrix which may contribute to bone fragility. Further studies are necessary to elucidate the pathways affected by these genetic alterations since elucidating these pathways will be vital to correcting the underlying biology of bone disease in the CKD population.