Volume 90, Issue 1, Pages 77-89 (July 2016) Direct inhibition of osteoblastic Wnt pathway by fibroblast growth factor 23 contributes to bone loss in chronic kidney disease  Natalia Carrillo-López, Sara Panizo, Cristina Alonso-Montes, Pablo Román-García, Isabel Rodríguez, Carlos Martínez-Salgado, Adriana S. Dusso, Manuel Naves, Jorge B. Cannata-Andía  Kidney International  Volume 90, Issue 1, Pages 77-89 (July 2016) DOI: 10.1016/j.kint.2016.01.024 Copyright © 2016 International Society of Nephrology Terms and Conditions

Figure 1 In vivo effects of CRF and dietary phosphorus intake on bone turnover and components of the Wnt pathway. (a) Osteocalcin, Runx2, and cathepsin K, and (b) Sfrp1, Sfrp4, Dkk1, and Sost mRNA expression measured by qPCR in the tibia of CRF rats fed a normal-phosphorus diet (NPD) or a high-phosphorus diet (HPD). All values are expressed relative to the reference group (rats with normal renal function fed an NPD), assigned a value of 0.0. Mean ± SD values are shown. #P < 0.05 compared to time-matched NPD groups. *P < 0.05 compared to reference group. CRF, chronic renal failure; qPCR, quantitative real-time polymerase chain reaction; R.U., relative units versus reference group. Kidney International 2016 90, 77-89DOI: (10.1016/j.kint.2016.01.024) Copyright © 2016 International Society of Nephrology Terms and Conditions

Figure 2 In vivo effects of CRF and dietary phosphorus intake on bone turnover and components of the Wnt pathway. (a) Representative image of Western blot analysis and (b) relative quantification of Sfrp1 and Dkk1 proteins in the tibia of CRF rats fed a normal-phosphorus diet (NPD) or a high-phosphorus diet (HPD). Gapdh was used as a loading control. All values are expressed relative to the reference group (rats with normal renal function were fed an NPD) and were assigned a value of 0.0. Mean ± SD values are shown. #P < 0.05 compared to time-matched NPD groups. *P < 0.05 compared to reference group. (c) Representative staining for osteoblasts and osteoclast detection (toluidine blue and Tartrate-resistant acid phosphatase [TRAP] stainnings, respectively) and immunohistochemical analysis of osteocalcin and Dkk1 expression in tibias from sham-operated controls (Sham) and 7/8 nephrectomized rats fed HPD after 12 weeks of CRF induction (HPD). Each bar indicates the relative scale. CRF; chronic renal failure; Gapdh, glyceraldehyde-3-phosphate dehydrogenase; R.U., relative units versus reference group. Kidney International 2016 90, 77-89DOI: (10.1016/j.kint.2016.01.024) Copyright © 2016 International Society of Nephrology Terms and Conditions

Figure 3 Uremic serum regulation of Wnt pathway components in UMR106-01 cells. (a) Sfrp1, Sfrp4, and Dkk1 gene expression measured by qPCR from UMR106-01 cells exposed to control or uremic serum for 24 and 48 hours. (b) Representative image of Western blot analysis and (c) relative quantification of phospho-β-catenin (p-β-cat), β-catenin (β-cat), Sfrp1, Sfrp4, and Dkk1 proteins and p-β-cat/β-cat ratio in UMR106-01 cells exposed to control or uremic serum for 24 or 48 hours. Gapdh was used as loading control. Horizontal bar represents the control group (cells exposed to control serum). Mean ± SD of 6 independent experiments is shown. *P < 0.05 compared to the control group. qPCR, quantitative real-time polymerase chain reaction; R.U., relative units versus control group; Gapdh, glyceraldehyde-3-phosphate dehydrogenase. Kidney International 2016 90, 77-89DOI: (10.1016/j.kint.2016.01.024) Copyright © 2016 International Society of Nephrology Terms and Conditions

Figure 4 Combined PTH and Fgf23/rsKlotho regulation of the Wnt pathway in vitro. (a) Sfrp1, Sfrp4, and Dkk1 gene expression measured by qPCR from UMR106-01 cells exposed to parathyroid hormone (PTH) 10–7 M, Fgf23 1000 ng/ml, and rsKlotho 50 ng/ml for 48 hours. (b) Representative image of Western blot analysis and (c) relative quantification of phospho-β-catenin (p-β-cat), β-catenin (β-cat), Sfrp1, Sfrp4, and Dkk1 proteins and p-β-cat/β-cat ratio in UMR106-01 cells exposed to PTH, Fgf23, and rsKlotho for 48 hours. Gapdh was used as loading control. The horizontal broken line represents the control group (cells cultured with vehicle). Mean ± SD of 6 independent experiments is shown. *P < 0.005 compared to the control group. Gapdh, glyceraldehyde-3-phosphate dehydrogenase; qPCR, quantitative real-time polymerase chain reaction; R.U., relative units versus control group. Kidney International 2016 90, 77-89DOI: (10.1016/j.kint.2016.01.024) Copyright © 2016 International Society of Nephrology Terms and Conditions

Figure 5 PTH regulation of the Wnt pathway in vitro. (a) Sfrp1, Sfrp4, and Dkk1 mRNA levels measured by qPCR from UMR106-01 cells exposed to vehicle (control) or parathyroid hormone (PTH) 10–7 M for 48 hours. (b) Representative image of Western blot analyses and (c) relative quantification of phospho-β-catenin (p-β-cat), β-catenin (β-cat), Sfrp1, Sfrp4, and Dkk1 proteins and p-β-cat/β-cat ratio in UMR106-01 cells exposed to PTH 10–7 M. Gapdh was used as loading control. The horizontal broken line represents the control group (cells cultured with vehicle). Mean ± SD of 6 independent experiments is shown. *P < 0.05 compared to the control group. Gapdh, glyceraldehyde-3-phosphate dehydrogenase; PTH, parathyroid hormone; qPCR, quantitative real-time polymerase chain reaction; R.U., relative units referred to the control group. Kidney International 2016 90, 77-89DOI: (10.1016/j.kint.2016.01.024) Copyright © 2016 International Society of Nephrology Terms and Conditions

Figure 6 Fgf23/rsKlotho regulation of the Wnt and MAPK pathways in vitro. (a) Sfrp1, Sfrp4, and Dkk1 mRNA levels measured by qPCR from UMR106-01 cells exposed to vehicle (control), rsKlotho (50 ng/ml), or Fgf23 (1000 ng/ml) alone or combined with rsKlotho (50 ng/ml) for 48 hours. (b) Representative image of Western blot analysis. (c) Relative quantification of phospho-β-catenin (p-β-cat), β-catenin (β-cat), Sfrp1, Sfrp4, and Dkk1 proteins and p-β-cat/β-cat ratio in UMR106-01 cells exposed to Fgf23 (1000 ng/ml) and rsKlotho (50 ng/ml). (d) Representative image of Western blot analysis and (e) relative quantification of phospho-ERK (p-ERK), ERK, Sfrp1, and Dkk1 proteins and p-ERK/ERK ratio in UMR106-01 cells exposed to Fgf23 (1000 ng/ml), rsKlotho (50 ng/ml), U0126 (5 μM), or some combination of these. Gapdh was used as loading control. The horizontal broken line represents the control group (cells exposed to vehicle). Mean ± SD of 6 independent experiments is shown. *P < 0.05 compared to the control group. R.U., relative units referred to the control group. ERK, extracellular regulated MAP kinase; Gapdh, glyceraldehyde-3-phosphate dehydrogenase; MAPK, mitogen-activated protein kinase. Kidney International 2016 90, 77-89DOI: (10.1016/j.kint.2016.01.024) Copyright © 2016 International Society of Nephrology Terms and Conditions

Figure 7 Dkk1 is required for Fgf23/rsKlotho downregulation of the osteoblastic Wnt pathway. (a) Representative image of Western blot analysis and (b) relative quantification of Sfrp1, phospho-β-catenin (p-β-cat), β-catenin (β-cat), and Dkk1 proteins and p-β-cat/β-cat ratio in Sfrp1 small, interfering RNA (siRNA) UMR106-01 cells exposed to Fgf23 (1000 ng/ml) and rsKlotho (50 ng/ml). (c) Representative image of Western blot analysis and (d) relative quantification of Dkk1, p-β-cat, β-cat, and Sfrp1 proteins and p-β-cat/β-cat ratio in Dkk1 siRNA UMR106-01 cells exposed to Fgf23 (1000 ng/ml) and rsKlotho (50 ng/ml). Gapdh was used as loading control. Mean ± SD of 6 independent experiments is shown. *P < 0.05 compared to the scrambled group (cells transfected with control siRNA). R.U., relative units referred to the scramble group. Gapdh, glyceraldehyde-3-phosphate dehydrogenase. Kidney International 2016 90, 77-89DOI: (10.1016/j.kint.2016.01.024) Copyright © 2016 International Society of Nephrology Terms and Conditions

Figure 8 Proposed Fgf23/sKlotho regulation of the osteoblastic Wnt pathway. Fgf23/sKlotho upregulates Sfrp1 and Dkk1 through the activation of the MAPK pathway. Dkk1, secreted to the extracellular space, would bind lipoprotein receptor–related peptides 5 and 6 (Lrp5/6) at the cell membrane of the same osteoblast, adjacent osteoblasts, or both, favoring the phosphorylation of β-catenin and, finally, autocrine/paracrine inactivation of the Wnt pathway. β-cat, β-catenin; ERK, extracellular regulated MAP kinase; MAPK, mitogen-activated protein kinase; p-β-cat, phospho-β-catenin; Frz, frizzled; p-ERK, phospho-ERK. Kidney International 2016 90, 77-89DOI: (10.1016/j.kint.2016.01.024) Copyright © 2016 International Society of Nephrology Terms and Conditions