Chromatin accessibility and epigenetic deoxyribose nucleic acid (DNA) modifications in chronic kidney disease (CKD) osteoblasts: a study of bone and osteoblasts from pediatric patients with CKD

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 MethylationEpic in primary osteoblasts from nine adolescent and young adult dialysis patients with end-stage kidney disease and three healthy references. ATAC-Seq was also performed on a subset of osteoblasts. Bone matrix protein expression was extracted from the iliac crest and evaluated by proteomics. Gene set enrichment 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 nuclear factor of activated T cells (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. Gene set enrichment analysis 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 13 potential ligand-receptor pairs. 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.

Keywords: CKD; NFAT; bone matrix; epigenetics; osteoblast maturation.

Graphical Abstract

Figure 1

Proportional area chart of differentially regulated genes. The 674 differentially expressed (out of 25 343 total expressed) genes in confluent CKD osteoblasts relative to reference osteoblasts are grouped according to functional categories in confluent CKD osteoblasts relative to healthy reference osteoblasts.

Figure 2

Gene expression patterns in CKD and healthy reference osteoblasts segregate uniformly. (A) Heatmap and (B) multidimensional scale representation of gene expression patterns in CKD (n = 9) and healthy reference (n = 3) osteoblasts. Reference cells: left side; CKD cells:right side; open circles:high bone turnover; closed triangles: low bone turnover: circles: normal bone turnover.

Figure 3

GSEA using transcription factor (TFT) molecular signatures database (MSigDB). (A) Significant methylation differences in CKD relative to reference osteoblasts vs significant changes in RNA expression in reference relative to CKD osteoblasts. The shaded box indicates the area of the graph where the NES is greater than 2.5 (indicating strong statistical significance) for changes in both methylation and RNA expression. (B) Enlargement of shaded area shown in (A). NES: normalized enrichment score (>2.5 considered significant). TGGAAA_NFAT_Q4_01: NFATc1.

Figure 4

Analysis of differentially accessible chromatin regions by ATAC-Seq. Normalized enrichment scores for the 20 transcription factors whose expression is most significantly differentially accessible in CKD osteoblasts relative to references. The negative value for the NES indicates decreased chromatin accessibility in reference, relative to CKD, osteoblasts.

Figure 5

(A) NFAT inhibition (NFATi) decreases proliferation in primary osteoblasts. Ctrl: control; NFATi: NFAT inhibitor (100 mM). *P < .01 from control (no NFATi) condition.(B) Gene expression in HOXA2 and HOXA3 knockdown osteoblasts. All expression is expressed relative to nontargeting (NT) shRNA transduced cells at confluence. A2kd: HOXA2 knockdown; A3kd: HOXA3 knockdown; BGLAP: osteocalcin; asterisk: P < .05 from NT at the same time point.