Volume 92, Issue 3, Pages 599-611 (September 2017) Klotho expression in osteocytes regulates bone metabolism and controls bone formation  Hirotaka Komaba, Jovana Kaludjerovic, Dorothy Z. Hu, Kenichi Nagano, Katsuhiko Amano, Noriko Ide, Tadatoshi Sato, Michael J. Densmore, Jun-ichi Hanai, Hannes Olauson, Teresita Bellido, Tobias E. Larsson, Roland Baron, Beate Lanske  Kidney International  Volume 92, Issue 3, Pages 599-611 (September 2017) DOI: 10.1016/j.kint.2017.02.014 Copyright © 2017 International Society of Nephrology Terms and Conditions

Figure 1 Generation of mice with osteocyte-specific Klotho deletion and tdTomato expression. (a) Schematic representation of wild-type (WT) allele (top) and floxed Klotho allele before (middle) and after Cre-mediated deletion (bottom). (b) Schematic representation of floxed STOP tdTomato allele before (top) and after Cre-mediated recombination (bottom). (c) Localization of tdTomato fluorescence in frozen sections from 5-week-old control mice (Dmp1-tdTomato). tdTomato is expressed at high levels in osteocytes and is also present in the kidney medulla but is not expressed in the renal proximal and distal tubular cells and parathyroid cells (arrowheads). Bar = 300 μm. (d) Immunohistochemical staining of Klotho in tibial cortical bone and kidney from control mice and Dmp1-Klotho−/− mice: arrowheads indicate positive Klotho staining in osteocytes. Bar = 100 μm. (e) Quantitative real-time polymerase chain reaction analysis of Klotho transcript in the femur, calvarium, and kidney from control mice and Dmp1-Klotho−/− mice. Data are represented as mean ± SD; *P < 0.05 by t test, n = 5–7 in each group. (f) Immunofluorescence staining of Klotho in osteocytes isolated from the long bones of control mice and Dmp1-Klotho−/− mice. Bar = 20 μm (also refer Figure S1). DAPI, 4′,6-diamidino-2-phenylindole; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org. Kidney International 2017 92, 599-611DOI: (10.1016/j.kint.2017.02.014) Copyright © 2017 International Society of Nephrology Terms and Conditions

Figure 2 Osteocyte-specific deletion of Klotho increases bone formation and bone mass. (a) von Kossa staining of distal femur sections from 5-week-old control mice and Dmp1-Klotho−/− mice. Bar = 500 μm. (b) Histomorphometric analysis of tibiae from control mice and Dmp1-Klotho−/− mice. Data are represented as mean ± SD; *P < 0.05 by t test, n = 6 in each group. (c) Serum N-terminal propeptide of type I procollagen (PINP) and C-terminal telopeptide fragments of the type I collagen (CTX) in control mice and Dmp1-Klotho−/− mice. Data are represented as mean ± SD; *P < 0.05 by t test, n = 7 in each group. (d) Quantitative real-time polymerase chain reaction analysis of bone metabolism-related genes in femur from control mice and Dmp1-Klotho−/− mice. Data are represented as mean ± SD; *P < 0.05 by t test, n = 7 in each group. ALP, alkaline phosphatase; BFR/BS, bone formation rate; BV/TV, bone volume fraction; Col1a1, collagen type 1; Dkk1, Dickkopf-1; FGF23, fibroblast growth factor 23; MAR, mineral apposition rate; MCSF, macrophage colony-stimulating factor; MS/BS, mineralizing surface; Ob.S/BS, osteoblast surface; OCN, osteocalcin; Oc.S/BS, osteoclast surface; OPN, osteopontin; RANKL/OPG, receptor activator of nuclear factor-kappaB ligand-osteoprotegerin; Runx2, runt-domain transcription factor 2; Tb.N, trabecular number; Tb.Sp, trabecular separation; Tb.Th, trabecular thickness. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org. Kidney International 2017 92, 599-611DOI: (10.1016/j.kint.2017.02.014) Copyright © 2017 International Society of Nephrology Terms and Conditions

Figure 3 Klotho overexpression inhibits mineralization and osteogenic activity in MC3T3.E1 cells during osteocyte differentiation. (a) Alizarin red staining of differentiated MC3T3.E1 cells with lentivirus-mediated overexpression of green fluorescent protein (GFP) or Klotho. (b) Quantification of Alizarin red staining. Data are represented as mean ± SD; *P < 0.05 by t test, n = 6 in each group. (c) Quantitative real-time polymerase chain reaction analysis of bone metabolism-related genes in differentiated MC3T3.E1 cells overexpressing GFP or Klotho. Data are represented as mean ± SD; *P < 0.05 by t test, n = 6 in each group. ALP, alkaline phosphatase; Col1a1, collagen type 1; FGF23, fibroblast growth factor 23; LV, lentivirus; OCN, osteocalcin; OPN, osteopontin; Runx2, runt-domain transcription factor 2. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org. Kidney International 2017 92, 599-611DOI: (10.1016/j.kint.2017.02.014) Copyright © 2017 International Society of Nephrology Terms and Conditions

Figure 4 Quantitative real-time polymerase chain reaction analysis of renal transcripts from control mice and Dmp1-Klotho−/− mice with sham operation or 5/6 nephrectomy (NTx) under normal or high-phosphate diet (HPD) feeding. Data are represented as mean ± SD; P values are from 2-way analysis of variance, n = 8 in each group. FGFR1, fibroblast growth factor receptor 1. Kidney International 2017 92, 599-611DOI: (10.1016/j.kint.2017.02.014) Copyright © 2017 International Society of Nephrology Terms and Conditions

Figure 5 Skeletal effect of osteocyte-specific Klotho deletion is attenuated in a model of high-turnover renal osteodystrophy. (a) Histomorphometric analysis of tibiae from control mice and Dmp1-Klotho−/− mice with sham operation or 5/6 nephrectomy (NTx) under normal or high-phosphate diet (HPD) feeding. Data are represented as mean ± SD; P values are from 2-way analysis of variance (ANOVA), n = 6 in each group. (b) Serum N-terminal propeptide of type I procollagen (PINP) and C-terminal telopeptide fragments of the type I collagen (CTX) in control mice and Dmp1-Klotho−/− mice with sham operation or NTx under normal or HPD feeding. Data are represented as mean ± SD; P values are from 2-way ANOVA, n = 8 in each group. BFR/BS, bone formation rate; BV/TV, bone volume fraction; MAR, mineral apposition rate; MS/BS, mineralizing surface; N.Ot/B.Ar, number of osteoblast per bone area; Ob.S/BS, osteoblast surface; Oc.S/BS, osteoclast surface; OS/BS, osteoid surface; Tb.N, trabecular number; Tb.Sp, trabecular separation; Tb.Th, trabecular thickness. Kidney International 2017 92, 599-611DOI: (10.1016/j.kint.2017.02.014) Copyright © 2017 International Society of Nephrology Terms and Conditions

Figure 6 Quantitative real-time polymerase chain reaction analysis of bone metabolism-related genes in femurs from control mice and Dmp1-Klotho−/− mice with sham operation or 5/6 nephrectomy (NTx) under normal or high-phosphate diet (HPD) feeding. Data are represented as mean ± SD; P values are from 2-way analysis of variance, n = 6–8 in each group. ALP, alkaline phosphatase; Col1a1, collagen type 1; FGF23, fibroblast growth factor 23; FGFR1, fibroblast growth factor receptor 1; OCN, osteocalcin; OPN, osteopontin; MCSF, macrophage colony-stimulating factor; RANKL/OPG, receptor activator of nuclear factor-kappaB ligand-osteoprotegerin; Runx2, runt-domain transcription factor 2. Kidney International 2017 92, 599-611DOI: (10.1016/j.kint.2017.02.014) Copyright © 2017 International Society of Nephrology Terms and Conditions