Epiphyseal growth plate growth hormone receptor signaling is decreased in chronic kidney disease–related growth retardation  Ariel Troib, Daniel Landau, Leonid Kachko, Ralph Rabkin, Yael Segev  Kidney International  Volume 84, Issue 5, Pages 940-949 (November 2013) DOI: 10.1038/ki.2013.196 Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 1 Growth hormone receptor (GHR) expression. (a) Epiphyseal growth plate GH receptor (GHR) mRNA. n=12 per group. Results are presented as fold of the control (C) group. (b) Growth plate GHR/tubulin protein ratio. GHR was detected in 120kD, determination by western blotting, n=6 per group. (c) GHR distribution in the growth plate tested by immunohistochemistry. Bar=100μm. Groups with common letters are similar. CKD, chronic kidney disease; EGP, epiphyseal growth plate; H, hypertrophic zone; NC, negative C without primary antibody; P, proliferative zone. Kidney International 2013 84, 940-949DOI: (10.1038/ki.2013.196) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 2 Defects in Janus kinase 2/signal transducer and activator of transcription (JAK/STAT) signal transduction. (a) Epiphyseal growth plate STAT5 detected in 94kD, determination by western blotting, n=7 per group. (b) Growth plate p-STAT5 detected in 91kD, determination by western blotting, n=7 per group. (c) Growth plate p-STAT5/STAT5 ratio, n=7 per group. (d) Growth plate total JAK2 detected in 120kD, determination by western blotting, n=6 per group. (e) Growth plate p-STAT3/STAT3 ratio, detected in 80kD, determination by western blotting, n=9 per group. (f) Growth plate SOCS2 mRNA, n=12 per group. Results are presented as fold of control (C). Different letters above bars indicate a significant difference between groups (P<0.05); groups with common letters are similar. CKD, chronic kidney disease; EGP, epiphyseal growth plate. Kidney International 2013 84, 940-949DOI: (10.1038/ki.2013.196) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 3 Insulin-like growth factor-I (IGF-I) expression in the epiphyseal growth plate. (a) Immunohistochemical staining for IGF-I expression in the growth plate. P, proliferative zone; H, hypertrophic zone; POC, primary ossification center. Black arrows point to IGF-I-positive-stained hypertrophic chondrocytes. NC, negative control without primary antibody. Bar=100μm. (b) Growth plate IGF-I mRNA, n=16 per group. Results are presented as fold of the control (C) group. C fed ad lib (gray bar), C animals that were allowed free access to food; C pair-fed, C animals pair-fed with chronic kidney disease (CKD). Different letters above bars indicate a significant difference between groups (P<0.05). (c) Growth plate IGFBP2 mRNA, n=16 per group. Results are presented as fold of the C group. *P<0.05 vs. C. Different letters above bars indicate a significant difference between groups (P<0.05); groups with common letters are similar. Kidney International 2013 84, 940-949DOI: (10.1038/ki.2013.196) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 4 Insulin-like growth factor-1 receptor (IGF1R) expression and signaling in the epiphyseal growth plate. (a) Growth plate IGF1R mRNA, n=16 per group. Results are presented as fold of control (C) group. *P<0.05 vs. C. (b) Growth plate IGF1R detected in 97kD, determination by western blotting, n=8 per group. (c) Growth plate IRS-1 detected in 170–185kD, determination by western blotting, n=8 per group. (d) Growth plate p-AKT/AKT ratio, n=8 per group. Different letters above bars indicate a significant difference between groups (P<0.05); groups with common letters are similar. CKD, chronic kidney disease; EGP, epiphyseal growth plate. Kidney International 2013 84, 940-949DOI: (10.1038/ki.2013.196) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 5 Vascularization distal to the epiphyseal growth plate. (a) Movat’s pentachrome staining of tibial growth plate and primary ossification center (POC). Black arrow points to decrease in vascularization in the POC. Bar=100μm. (b) Immunohistochemical staining for vascular endothelial growth factor (VEGF) in the growth plate. Upper figures, × 100 magnification; lower figures, × 200 magnification; CKD, chronic kidney disease; H, hypertrophic zone; NC, negative control (C) without primary antibody. Bar=100μm. (c) Growth plate VEGF mRNA, n=14 per group. Results are presented as fold of C. *P<0.05 vs. C. EGP, epiphyseal growth plate. Kidney International 2013 84, 940-949DOI: (10.1038/ki.2013.196) Copyright © 2013 International Society of Nephrology Terms and Conditions