Sclerostin, Osteocytes, and Wnt Signaling in Pediatric Renal Osteodystrophy

Abstract

The pathophysiology of chronic kidney disease-mineral and bone disorder (CKD-MBD) is not well understood. Specific factors secreted by osteocytes are elevated in the serum of adults and pediatric patients with CKD-MBD, including FGF-23 and sclerostin, a known inhibitor of the Wnt signaling pathway. The molecular mechanisms that promote bone disease during the progression of CKD are incompletely understood. In this study, we performed a cross-sectional analysis of 87 pediatric patients with pre-dialysis CKD and post-dialysis (CKD 5D). We assessed the associations between serum and bone sclerostin levels and biomarkers of bone turnover and bone histomorphometry. We report that serum sclerostin levels were elevated in both early and late CKD. Higher circulating and bone sclerostin levels were associated with histomorphometric parameters of bone turnover and mineralization. Immunofluorescence analyses of bone biopsies evaluated osteocyte staining of antibodies towards the canonical Wnt target, β-catenin, in the phosphorylated (inhibited) or unphosphorylated (active) forms. Bone sclerostin was found to be colocalized with phosphorylated β-catenin, which suggests that Wnt signaling was inhibited. In patients with low serum sclerostin levels, increased unphosphorylated "active" β-catenin staining was observed in osteocytes. These data provide new mechanistic insight into the pathogenesis of CKD-MBD and suggest that sclerostin may offer a potential biomarker or therapeutic target in pediatric renal osteodystrophy.

Keywords: CKD-MBD; Wnt signaling; bone biopsy; children; immunofluorescence; immunohistochemistry; sclerostin.

Figure 1

Distribution of Pediatric Renal Osteodystrophy Lesions in Pre-Dialysis CKD and CKD 5D Patients. Normal bone (Normal), M osteomalacia (OM), Mild bone lesion of secondary hyperparathyroidism (Mild), Mixed Uremic Osteodystrophy (MUO), osteitis fibrosa (OF), Adynamic Bone (ADB) and Osteomalacia (OM).

Figure 2

Representative Images of Sclerostin Immunofluorescence Analyses in CKD Bone. (A–C) Staining with an anti-sclerostin antibody (red) was assessed in both early-stage CKD (n = 6) and in dialysis CKD 5D patients (n = 6). As illustrated in the microscopy workflow diagram, patients were further designated into two groups with either high serum sclerostin (58–125 pmol/L) or low levels similar to healthy controls (18.6–36.0 pmol/L). DAPI was used as a nuclear marker (blue). (D,E) Bone stained from patients with high serum sclerostin. (F) Quantification of sclerostin-positive osteocytes normalized by total number of cells using bone unit area to normalize (n > 230 cells per condition). Dark grey bars indicate high serum sclerostin while light grey bars indicate low serum sclerostin. Trabecular (TB) and bone marrow (BM) are indicated. Arrows denote corresponding cells in red and blue channels.

Figure 3

Phosphorylated-β-Catenin and Sclerostin Immunofluorescence Analyses in CKD Bone. (A–D) Bone stained with anti-sclerostin antibody (red), phosphorylated (phos) β-catenin (green), and DAPI (blue). Arrows denote corresponding cells with colocalized sclerostin and phos-β-catenin. Bottom panel indicates insets from dotted white boxes. (E) Model of the monoclonal antibody used here to recognize the phosphorylated (Ser33/37/Thr41) “inhibited” β-catenin peptide, which denotes an inhibition of the canonical Wnt pathway. (F) Quantification of immunofluorescence by the % of osteocytes positive for both phos-β-catenin and sclerostin relative to the total number of cells using bone unit area to normalize (n > 230 cells per condition). Biopsies were stained and processed side-by-side. Trabecular (TB) bone and bone marrow (BM) are indicated.

Figure 4

Active β-catenin Immunofluorescence Analyses in CKD bone. (A–D) Bone stained with active β-catenin (green) and DAPI (blue). Arrows denote corresponding cells with colocalized sclerostin and phos-β-catenin. (E) Model of the monoclonal antibody used to recognize the non-phosphorylated, stabilized β-catenin that is not tri-phosphorylated and functionally active in the canonical Wnt signaling pathway. (F) Quantification of immunofluorescence by the % of osteocytes positive for active β-catenin relative to the total number of cells using bone unit area to normalize (n > 230 cells per condition). Trabecular (TB) bone and bone marrow (BM) are indicated.