1. Volume 95, Issue 5, Pages 1064-1078 (May 2019)
Calcimimetics maintain bone turnover in uremic rats despite the concomitant decrease in parathyroid hormone concentration 
Juan M. Díaz-Tocados, María E. Rodríguez-Ortiz, Yolanda Almadén, Carmen Pineda, Julio M. Martínez-Moreno, Carmen Herencia, Noemi Vergara, M. Victoria Pendón-Ruiz de Mier, Rafael Santamaría, Cristian Rodelo-Haad, Antonio Casado-Díaz, Víctor Lorenzo, Catarina Carvalho, João M. Frazão, Arnold J. Felsenfeld, William G. Richards, Escolástico Aguilera-Tejero, Mariano Rodríguez, Ignacio López, Juan R. Muñoz-Castañeda 
Kidney International 
Volume 95, Issue 5, Pages (May 2019)
DOI: /j.kint
Copyright © 2019 International Society of Nephrology Terms and Conditions

2. Kidney International 2019 95, 1064-1078DOI: (10.1016/j.kint.2018.12.015)
Copyright © 2019 International Society of Nephrology Terms and Conditions

3. Figure 1
Effects of a calcimimetic (CM) in the bone histomorphometric parameters of normal rats who underwent parathyroidectomy (PTX) to clamp parathyroid hormone (PTH). After 28 days, the bone volume (BV; a) remained similar in all groups. PTX decreased the osteoid volume (OV; b), osteoid surface (OS; c), osteoblast activity (d), and osteoclast activity (e) and did not modify the significantly eroded surface (f). PTH replacement reestablished bone remodeling similar to that in sham animals. Treatment with a CM produced a tendency to increase osteoblast activity. Bars show mean ± SD. A 1-way analysis of vairance with a Tukey post hoc test was conducted. *P < 0.05; **P < ●, Sham; ▪, PTX; ▴, PTx-PTH;▼, PTx-PTH-CM. BS, bone surface; ES, eroded surface; Ob.S, osteoblast surface; Oc.S, osteoclast surface; TV, tissue volume.
Kidney International  , DOI: ( /j.kint )
Copyright © 2019 International Society of Nephrology Terms and Conditions

4. Figure 2
Effects of a calcimimetic (CM) in bone histomorphometric parameters of nephrectomized (Nx) rats and Nx rats with parathyroidectomy (PTX) and parathyroid hormone (PTH) infusion. Nx induced a reduction in bone volume (BV; a), and increased bone turnover (b–f). (a) P < 0.05 versus sham (1-way analysis of variance [ANOVA] with a Tukey post hoc test). In Nx rats, treatment with CM prevented the decrease in bone turnover. In parathyroidectomized Nx rats, with PTH infusion in an amount 6 times the normal replacement dose (Nx-PTx-PTHx6), bone turnover decreased, and CM treatment (Nx-PTx-PTHx6-CM) increased bone cell activity accompanied by an increase in mineralization (g) and bone formation rate (h). *P < 0.05; **P < 0.01 (2-way ANOVA with Fisher’s least significant difference [LSD] post hoc test). The infusion of PTH at 9 times the normal replacement dose (PTHx9) significantly increased bone turnover compared with PTHx6. (b) P < 0.01 versus Nx-PTx-PTHx6 (2-way ANOVA with LSD post hoc test). In conditions of very high PTH, the administration of the CM (Nx-PTx-PTHx9-CM) did not produce a significant further increase in bone cell activity. Bars show mean ± SD. ●, Sham; ▲, Nx; ▲, Nx-CM; ▪, Nx-PTX-PTHx6; ▪, Nx-PTX-PTHx6-CM; ▼, Nx-PTX-PTHx9; ▼, Nx-PTX-PTHx9-CM. BFR, bone formation rate; BS, bone surface; ES, eroded surface; MS, mineralizing surface; n.s., not significant; Ob.S, osteoblast surface; Oc.S, osteoclast surface; OS, osteoid surface; OV, osteoid volume; TV, tissue volume.
Kidney International  , DOI: ( /j.kint )
Copyright © 2019 International Society of Nephrology Terms and Conditions

5. Figure 3
Effects of a calcimimetic (CM) on bone mineralization of nephrectomized (Nx) rats and Nx rats with parathyroidectomy (PTX) and parathyroid hormone (PTH) infusion. No significant differences were found between groups treated with vehicle or CM for mineral apposition rate (MAR; a), mineralization lag time (Mlt; b), mineralizing surface related to osteoid surface (MS/OS; c), adjusted apposition rate (Aj.AR; d), osteoid thickness (O.Th; e), or osteoid maturation time (Omt; f). (a) P < 0.05 versus sham (1-way analysis of variance [ANOVA] with Tukey post hoc test). (b) P < 0.05 versus rats given infusion of PTH at 6 times the normal replacement dose (Nx-PX-PTHx6; 2-way ANOVA with Fisher’s least significant difference [LSD] post hoc test). ●, Sham; ▲, Nx; ▲, Nx-CM; ▪, Nx-PTX-PTHx6; ▪, Nx-PTX-PTHx6-CM; ▼, Nx-PTX-PTHx9 (infusion of PTH at 9 times the normal replacement dose); ▼, Nx-PTX-PTHx9-CM. n.s: not significant.
Kidney International  , DOI: ( /j.kint )
Copyright © 2019 International Society of Nephrology Terms and Conditions

6. Figure 4
Representative photographs of Goldner’s trichrome staining and double calcein labeling in trabecular bone. In sham animals, an almost quiescent state of bone cells was observed (a), with normal mineralization (b). In nephrectomized (Nx) rats, an increase in bone cell activity was observed; note the high number of cuboidal cells (osteoblasts, arrowhead) and multinucleated cells (osteoclasts, asterisk) on the bone surface (c), accompanied by an increase in bone formation (d). Nx rats treated with the calcimimetic (CM) maintained bone cell activity (e) and mineralization (f) similar to that in Nx rats. In Nx animals who had undergone parathyroidectomy (PTX) and were given parathyroid hormone at 6 times the normal replacement dose (Nx-PTX-PTHx6), low bone turnover (g), and bone formation (h), compared with the Nx group, were detected. Treatment with CM increased the number of osteoblasts and osteoclasts (i) as well as mineralization and bone formation (j). In the Nx-PTX-PTHx9 (PTH at 9 times the normal replacement dose) group, rates of bone turnover (k) and mineralization (l) similar to those in the Nx group were observed. In Nx-PTX-PTHx9 + CM, bone cell activity (m) and bone formation (n) were also similar to those observed in their corresponding vehicle group, Nx-PTX-PTHx9. Mineralized tissue is stained in green, and the nonmineralized bone (osteoid) is stained in red. The distance between calcein labels (double-headed arrows) shows the bone being formed during the time interval (7 days), indicating the mineralization status. Original magnification ×200. Scale bar: 100 μm. To optimize viewing of this image, please see the online version of this article at
Kidney International  , DOI: ( /j.kint )
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7. Figure 5
The administration of a calcimimetic (CM) triggered Erk1/2 phosphorylation (P-ERK1/2) of osteoblasts. Osteoblasts with positive P-Erk1/2 staining (b,c) were counted in 3 random field samples and expressed as the percentage of total osteoblasts (a). Goldner’s staining of undecalcified bone sections showed cuboidal osteoblasts covering osteoid on the bone surface. Immunohistochemistry for P-Erk1/2 showed intense nuclear staining in osteoblasts in the nephrectomized (Nx)-CM (b), Nx-PTx-PTHx6-CM (Nx animals who had undergone parathyroidectomy [PTX] and were given parathyroid hormone [PTH] at 6 times the normal replacement dose), and Nx-PTx-PTHx9-CM (PTH at 9 times the normal replacement dose) groups (c), and scarce nuclear staining in the bone cells of the other groups. To determine that the target cells were osteoblasts, serial undecalcified bone sections were also stained for osteocalcin. Immunohistochemistry without primary antibody (Ab) in undecalcified bone samples showed no positive staining. Arrows indicate nuclear P-Erk1/2. Original magnification ×1000. Bar: 20 μm. To optimize viewing of this image, please see the online version of this article at
Kidney International  , DOI: ( /j.kint )
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8. Figure 6
Treatment with a calcimimetic (CM) triggered calcium-sensing receptor (CaSR) downstream signaling and upregulated osteogenic marker gene expression in low calcium conditions. Western blots of total protein extracts show positive expression of CaSR in the UMR106 cell line, and with the CM, there was a nonsignificant tendency to increase proliferating cell nuclear (PCNA) expression (a), and Erk1/2 phosphorylation (pERK1/2) was induced (b). The gene expression of osterix (c), Runx2 (d), and osteocalcin (e) was induced with the effective dose of CM. Bars show mean ± SD. A t-test was conducted. (a) P < 0.05 versus control; (b) P < 0.05 versus CM at 1 μM. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. To optimize viewing of this image, please see the online version of this article at
Kidney International  , DOI: ( /j.kint )
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9. Figure 7
Inhibition of CaSR signaling by calcilytics decreases osteogenesis in UMR-106 cells. Calhex administration at 10 μM reduced Erk1/2 phosphorylation (P-ERK1/2; a). In the presence of Calhex, there was a significant decrease of osterix (b), Runx2 (c), and osteocalcin (d) expression. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. To optimize viewing of this image, please see the online version of this article at
Kidney International  , DOI: ( /j.kint )
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10. Figure 8
Treatment with a calcimimetic (CM) during osteogenic differentiation of mesenchymal stem cells (MSC) in low calcium (Ca) medium increased mineralization and gene expression of osteogenic markers. Osteogenic differentiation induced matrix mineralization (a,b), and increased expression of osteogenic gene markers Runx2 (c), osterix (d), osteocalcin (e), and BMP2 (f). Both MSC and MSC-derived osteoblasts (OB) expressed calcium-sensing receptor (CaSR; g). Mineralization and osteogenic markers were further increased with CM addition. Low Ca was 0.5 mM. [CM] = 100 μM. Bars show mean ± SD. A t-test was conducted. (a) P < 0.05 versus MSC. (b) P < 0.05 versus OB. Original magnification ×200. Bar: 100 μm. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. To optimize viewing of this image, please see the online version of this article at
Kidney International  , DOI: ( /j.kint )
Copyright © 2019 International Society of Nephrology Terms and Conditions