Osteoclast-Released Wnt-10b Underlies Cinacalcet Related Bone Improvement in Chronic Kidney Disease

Abstract

Secondary hyperparathyroidism (SHPT) relates to high turnover bone loss and is responsible for most bone fractures among chronic kidney disease (CKD) patients. Changes in the Wingless/beta-catenin signaling (Wnt/β-catenin) pathway and Wnt inhibitors have been found to play a critical role in CKD related bone loss. A calcimimetic agent, cinacalcet, is widely used for SHPT and found to be similarly effective for parathyroidectomy clinically. A significant decrease in hip fracture rates is noted among US hemodialysis Medicare patients since 2004, which is probably related to the cinacalcet era. In our previous clinical study, it was proven that cinacalcet improved the bone mineral density (BMD) even among severe SHPT patients. In this study, the influence of cinacalcet use on bone mass among CKD mice was determined. Cinacalcet significantly reduced the cortical porosity in femoral bones of treated CKD mice. It also improved the whole-bone structural properties through increased stiffness and maximum load. Cinacalcet increased femoral bone wingless 10b (Wnt10b) expression in CKD mice. In vitro studies revealed that cinacalcet decreased osteoclast bone resorption and increased Wnt 10b release from osteoclasts. Cinacalcet increased bone mineralization when culturing the osteoblasts with cinacalcet treated osteoclast supernatant. In conclusion, cinacalcet increased bone quantity and quality in CKD mice, probably through increased bone mineralization related with osteoclast Wnt 10b secretion.

Keywords: Wnt 10b; chronic kidney disease; cinacalcet; osteoclast; renal osteodystrophy.

Figure 1

Serum (parathyroid hormone) PTH and Wnt 10b levels before and after 4 weeks in control, non-treated and cinacalcet-treated (chronic kidney disease) CKD mice. (A) PTH levels significantly increased in CKD mice compared with controls. The cinacalcet treatment significantly decreased PTH levels in CKD mice. (B) Wnt 10b levels increased significantly in both CKD with or without treatment. (C) P1NP levels significantly decreased in all groups. (D) CTX-1 levels are significantly decreased in CKD+Cin group after 4 weeks treated compared with CKD group. n = 6 in each group, * p < 0.05, ** p < 0.01.

Figure 2

Cinacalcet improves both trabecular and cortical bone microarchitecture. (A) Histomorphometric parameters of trabecular and cortical bones of femur in sham-operated (Control) mice, CKD mice treated with saline (CKD), CKD mice treated with cinacalcet 10mg/kg/day (CKD+cin) Scale bar 500 μm. (B,C) Quantitative results of the experiment shown in A. The cortical porosity significantly improved in cinacalcet treatment group. Cortical bone: Bone mineral density (BMD); cortical thickness (Cr. Th); polar moment of inertia (MMI); total porosity (Po(tot)); bone volume/tissue volume ratio (BV/TV); trabecular thickness (Tb. Th); trabecular number (Tb. N); trabecular separation (Tb. Sp). n = 6 in each group, * p < 0.05, ** p < 0.01.

Figure 3

Cinacalcet increases femoral bone structural properties in 5/6 nephrectomy CKD mice. (A) Biomechanical three-point bending test of the femoral bone in sham-operated (Control) mice, CKD mice treated with saline (CKD), CKD mice treated with cinacalcet 10 mg/kg/day (CKD+cin) for 4 weeks (left = before; right = after; Scale bar 5 mm). (B) Quantitative results of the experiment shown in A. The femoral bone stiffness is significantly increased in the CKD+cin group compared to the CKD group. The maximum load is significantly decreased in the CKD group compared to the control and CKD+cin group. n = 6 in each group, * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 4

Cinacalcet increases Wnt 10b expression in femoral cortical bones of treated mice. Confocal microscopic analysis of femoral bone cells in the control, CKD, and CKD with cinacalcet treated mice. Blue, nuclei; red, calcitonin receptors; green, Wnt 10b. Scale bar 20 μm.

Figure 5

Cinacalcet inhibits osteoclastic bone resorption, increases osteoclast Wnt10b expression and improves mineralization. (A) TRAP staining of osteoclasts treated with culture medium alone (control) or culture medium plus Cinacalcet (Cin, 400 nM). Red intracellular staining in the presence of multiple nuclei indicates positive labeling of osteoclasts. Scale bar 20 μm. (B) Quantitative results of the experiment shown in A. (C) Confocal analysis of osteoclasts treated with culture medium alone (Con) or culture medium plus Cinacalcet (400 nM Cinacalcet). Osteoclasts were labeled with rhodamine-phalloidin (red) to visualize F-actin and TOTO3 (blue) to visualize nuclei. Blue, nuclei; red, actin; green, Wnt10b. Scale bar 20 μm. Each figure represents at least three replicate experiments with a total of at least 500 osteoclasts. (D,E) Western blot analyses of Wnt 10b expression in osteoclasts. (D) Wnt10b expression is significantly increased in osteoclasts treated with Cinacalcet 400 nM. (E) Pretreatment with C-59, a Wnt10b secretion inhibitor, further increases the Wnt10b expression by cinacalcet. (F) When calvarial osteoblastic cells were cultured with the supernatant derived from cinacalcet-treated osteoclasts, mineralization is increased as expressed by increased Alizarin Red Staining, Scale bar 20 μm, and expressed quantitatively as in (G). * p < 0.05, ** p < 0.01.

Figure 5

Cinacalcet inhibits osteoclastic bone resorption, increases osteoclast Wnt10b expression and improves mineralization. (A) TRAP staining of osteoclasts treated with culture medium alone (control) or culture medium plus Cinacalcet (Cin, 400 nM). Red intracellular staining in the presence of multiple nuclei indicates positive labeling of osteoclasts. Scale bar 20 μm. (B) Quantitative results of the experiment shown in A. (C) Confocal analysis of osteoclasts treated with culture medium alone (Con) or culture medium plus Cinacalcet (400 nM Cinacalcet). Osteoclasts were labeled with rhodamine-phalloidin (red) to visualize F-actin and TOTO3 (blue) to visualize nuclei. Blue, nuclei; red, actin; green, Wnt10b. Scale bar 20 μm. Each figure represents at least three replicate experiments with a total of at least 500 osteoclasts. (D,E) Western blot analyses of Wnt 10b expression in osteoclasts. (D) Wnt10b expression is significantly increased in osteoclasts treated with Cinacalcet 400 nM. (E) Pretreatment with C-59, a Wnt10b secretion inhibitor, further increases the Wnt10b expression by cinacalcet. (F) When calvarial osteoblastic cells were cultured with the supernatant derived from cinacalcet-treated osteoclasts, mineralization is increased as expressed by increased Alizarin Red Staining, Scale bar 20 μm, and expressed quantitatively as in (G). * p < 0.05, ** p < 0.01.

Figure 6

Summary of possible effects of cinacalcet on bones and bone cells. Decreased; Increased.