Primary osteoblast-like cells from patients with end-stage kidney disease reflect gene expression, proliferation, and mineralization characteristics ex vivo

Abstract

Osteocytes regulate bone turnover and mineralization in chronic kidney disease. As osteocytes are derived from osteoblasts, alterations in osteoblast function may regulate osteoblast maturation, osteocytic transition, bone turnover, and skeletal mineralization. Thus, primary osteoblast-like cells were cultured from bone chips obtained from 24 pediatric ESKD patients. RNA expression in cultured cells was compared with RNA expression in cells from healthy individuals, to RNA expression in the bone core itself, and to parameters of bone histomorphometry. Proliferation and mineralization rates of patient cells were compared with rates in healthy control cells. Associations were observed between bone osteoid accumulation, as assessed by bone histomorphometry, and bone core RNA expression of osterix, matrix gla protein, parathyroid hormone receptor 1, and RANKL. Gene expression of osteoblast markers was increased in cells from ESKD patients and signaling genes including Cyp24A1, Cyp27B1, VDR, and NHERF1 correlated between cells and bone cores. Cells from patients with high turnover renal osteodystrophy proliferated more rapidly and mineralized more slowly than did cells from healthy controls. Thus, primary osteoblasts obtained from patients with ESKD retain changes in gene expression ex vivo that are also observed in bone core specimens. Evaluation of these cells in vitro may provide further insights into the abnormal bone biology that persists, despite current therapies, in patients with ESKD.

Figure 1

Lesions of renal osteodystophy on bone biopsy represented in the cohort of ESKD patients evaluated for osteoblast-like cell and bone core RNA. Adynamic bone (low bone turnover) (grey): n=6; normal bone turnover (striped): n=3; normal bone turnover with fibrosis (white): n=9; osteitis fibrosa (high bone turnover) (black): n=5; mixed uremic osteodystrophy (high bone turnover with defective mineralization) (checkered): n=1.

Figure 2

Relationship between FGF23 RNA expression in bone cores and osteoid thickness (O.Th), as determined by bone histomorphometry.

Figure 3

Gene expression of osteoblast markers in primary osteoblasts from ESKD patients. Values are expressed as a multiple of (fold of) the expression found in healthy control cells. Box plots indicate the medians (interquartile ranges); the 5^th and 95^th percentiles are denoted by the lower and upper whiskers, respectively. The asterisks denote significant (p<0.001) differences from healthy controls.

Figure 4

Gene expression of cell signaling genes in primary osteoblasts from CKD patients. Values are expressed as a multiple of (fold of) the expression found in normal control cells. Box plots indicate the medians (interquartile ranges); the 5^th and 95^th percentiles are denoted by the lower and upper whiskers, respectively. The asterisks denote significant (p<0.001) differences from healthy controls.

Figure 5

Mineralization rates in cells from dialysis patients with high turnover renal osteodystrophy as compared to those from healthy controls. A) Alizarin Red S staining of primary human osteoblast-like cells from healthy controls and dialysis patients with high turnover renal osteodystrophy grown under mineralizing conditions (10 mM β-glycerolphosphate and 100 µg/ml ascorbic acid) for 7, 14, 21 and 28 days. B) Alizarin red stained mineralized nodules under light microscopy (40x) at 14, 21, and 28 days. C) Mineralization of primary osteoblast-like cells, as quantified by the ratio of absorbance of Alizarin Red S to Crystal violet (i.e. ratio of mineral content to cells), occurs more rapidly in cells from healthy controls (black bars) than in dialysis patients with high turnover renal osteodystrophy (open bars). The asterisk denotes significant (p<0.01) difference in Alizarin Red S to Crystal Violet absorbance ratios between dialysis patients with high turnover renal osteodystrophy and healthy controls. D) Primary osteoblast-like cells from healthy controls after 28 days in mineralizing conditions a) display mineralized nodules under light microscopy and b) express osteocalcin (green color) as detected by immunofluorescence. DAPI staining (c) confirms that osteocalcin expression localizes to living cells at the site of the mineralized nodule formation.

Figure 6

Proliferation of primary human osteoblast-like cells as assessed by MTS incorporation. Data are expressed as percentage increase in absorbance from baseline. A higher proliferation rate was observed in dialysis patients with high turnover renal osteodystrophy (open symbols) than in controls (closed symbols) (p<0.01). The asterisk denotes a significant (p<0.01) difference in proliferation rates between groups.

Figure 7

Flowchart of the study.