1. Chapter 16 Chapter 16 Phosphatonins Copyright © 2013 Elsevier Inc. All rights reserved.

2. FIGURE 16.1 Opossum kidney (OK) cells expressing chimeric NaPi IIa-V5 were exposed to sFRP4 or parathyroid hormone (PTH). The presence of NaPi IIa-V5 was detected using an antibody directed against the V5 epitope. After exposure of the OK cells expressing NaPi IIa-V5 to sFRP4 or PTH for three hours, reduced NaPi IIa-V5 protein was detected compared to untreated cells. (A, original magnification, 200x; B, original magnification, 400x). Source: reprinted from Berndt etal. Pflugers Archives – European Journal of Physiology, 2006, with permission [47]. 2

3. Copyright © 2013 Elsevier Inc. All rights reserved. FIGURE 16.2 A similar mechansim of hypophosphatemia is found in patients with TIO, XLH, and ADHR. Increased production and/or decreased degradation of a phosphatonin leads to a phosphatonin mediated reduction in renal phosphate reabsorption. The phosphatonin also acts to reduce 1α-hydroxylase activity thereby reducing 1α,25-dihydroxyvitamin D concentrations which subsequently reduces gastrointestinal phosphate absorption. Abbreviations: FGF23: fibroblast growth factor-23; sFRP4: secreted frizzled-related protein 4; PHEX: phosphate-regulating gene with homologies to endopeptidases on the X chromosome. 3

4. Copyright © 2013 Elsevier Inc. All rights reserved. FIGURE 16.3 A. Fibroblast growth factor-23 (FGF23) is produced by bone cells, circulates in the blood, and acts on the kidney to inhibit the production of 1α,25(OH)2D3, and to induce phosphaturia. This has a negative feedback effect of decreasing bone FGF23 production. B. FGF23 acts at renal tubule cells to decrease the transcription, translation, and overall activity of the enzyme 25-hydroxyvitamin D3 1α-hydroxylase. Receptor binding also increases phosphate excretion due to effects on downregulation of NaPi IIa/c in the apical membrane. C. Osteocytes and osteoblasts in response to elevations in phosphorus and 1α,25(OH)2D3, increase FGF23 transcription, translation, and secretion. D. FGF23 processing is carried out by the enzymatic activity of polypeptide N-acetylgalactosaminyltransferase 3 (GALNT3) and a subtilisin-like proprotein convertase (SPC), probably furin. In the absence of glycosylation by GALNT3, FGF23 is cleaved by furin. Source: reprinted from Bhattacharyya etal. Trends in Endocrinology and Metabolism, 2012, with permission [76]. 4

5. Copyright © 2013 Elsevier Inc. All rights reserved. FIGURE 16.4 Chinese hamster ovary (CHO) cells expressing wild-type fibroblast growth factor-23 (FGF23) or mutant FGF23 (R176Q or R179Q) were implanted into mice. Serum phosphorus is reduced in the mice exposed to CHO cells expressing wild-type or mutant FGF23. (A, B) Radiographs of femurs (C, D) and ash content of femurs (E, F) demonstrate reduced mineral content in mice exposed to FGF23 (wild-type or mutant) compared to control animals. Source: reprinted from Shimada, etal. Endocrinology, 2002, with permission [90]. 5

6. Copyright © 2013 Elsevier Inc. All rights reserved. FIGURE 16.5 Effect of infusion of sFRP4 on solute excretion in intact rats. Intact rats were administered secreted frizzled-related protein-4 (sFRP-4) (black bars) at a dose of 0.3 μg/kg/hour or vehicle (white bars) by intravenous infusion over a period of 2 hours. C1 indicates equilibrium period prior to the infusion of sFRP4 or vehicle. C2 indicates the experimental period during which sFRP4 or vehicle was infused. Fractional excretion of inorganic phosphate increased significantly in the rats after infusion of sFRP4. Source: reprinted from Berndt etal. Journal of Clinical Investigation, 2003, with permission [52]. 6

7. Copyright © 2013 Elsevier Inc. All rights reserved. FIGURE 16.6 Exposure of mice to intraperitoneal injections of matrix extracellular phosphoglycoprotein (MEPE) or parathyroid hormone (PTH) cause a reduction in serum phosphorus concentrations after 31 hours compared to vehicle treated mice (A). Fractional excretion of phosphorus is increased in mice exposed to intraperitoneal injections of MEPE or PTH after 6 hours (B) or 31 hours (C). MEPE40, 40 μg/kg/30 hours; MEPE400, 400 μg/kg/30 hours; PTH, 80 μg/kg/30 hours. Source: reprinted from Rowe etal. Bone, 2004 [55]. 7

8. Copyright © 2013 Elsevier Inc. All rights reserved. FIGURE 16.7 Phosphate transport was measured in opossum kidney (OK) cells exposed to increasing concentrations of fibroblast growth factor (FGF)-7. A significant and dose-dependent decrease in OK cell phosphate transport was detected at FGF7 concentrations of 50 ng/mL or greater. Source: reprinted from Carpenter etal. Journal of Clinical Endocrinology and Metabolism, 2005, with permission [54]. 8