Valentin David

Regulation and Function of the FGF23/Klotho Endocrine Pathways

Calcium (Ca(2+)) and phosphate (PO(4)(3-)) homeostasis are coordinated by systemic and local factors that regulate intestinal absorption, influx and efflux from bone, and kidney excretion and reabsorption of these ions through a complex hormonal network. Traditionally, the parathyroid hormone (PTH)/vitamin D axis provided the conceptual framework to understand mineral metabolism. PTH secreted by the parathyroid gland in response to hypocalcemia functions to maintain serum Ca(2+) levels by increasing Ca(2+) reabsorption and 1,25-dihydroxyvitamin D [1,25(OH)(2)D] production by the kidney, enhancing Ca(2+) and PO(4)(3-) intestinal absorption and increasing Ca(2+) and PO(4)(3-) efflux from bone, while maintaining neutral phosphate balance through phosphaturic effects. FGF23 is a recently discovered hormone, predominately produced by osteoblasts/osteocytes, whose major functions are to inhibit renal tubular phosphate reabsorption and suppress circulating 1,25(OH)(2)D levels by decreasing Cyp27b1-mediated formation and stimulating Cyp24-mediated catabolism of 1,25(OH)(2)D. FGF23 participates in a new bone/kidney axis that protects the organism from excess vitamin D and coordinates renal PO(4)(3-) handling with bone mineralization/turnover. Abnormalities of FGF23 production underlie many inherited and acquired disorders of phosphate homeostasis. This review discusses the known and emerging functions of FGF23, its regulation in response to systemic and local signals, as well as the implications of FGF23 in different pathological and physiological contexts.

Bone proteins PHEX and DMP1 regulate fibroblastic growth factor Fgf23 expression in osteocytes through a common pathway involving FGF receptor (FGFR) signaling

Fibroblastic growth factor 23 (FGF23) is a circulating phosphaturic hormone. Inactivating mutations of the endopeptidase PHEX or the SIBLING protein DMP1 result in equivalent intrinsic bone mineralization defects and increased Fgf23 expression in osteocytes. The mechanisms whereby PHEX and DMP1 regulate Fgf23 expression are unknown. We examined the possibility that PHEX and DMP1 regulate Fgf23 through a common pathway by analyzing the phenotype of compound Phex and Dmp1 mutant mice (Hyp/Dmp1–/–). Compared to single‐mutant littermates, compound‐mutant Hyp/Dmp1–/– mice displayed nonadditive elevations of serum FGF23 (1912 ± 183, 1715 ± 178, and 1799 ± 181 pg/ml), hypophosphatemia (Pi: 6.0 ± 0.3, 5.8 ± 0.2, and 5.4 ±0.1 mg/dl), and severity of rickets/osteomalacia (bone mineral density: –36, –36, and –30%). Microarray analysis of long bones identified gene expression profiles implicating common activation of the FGFR pathway in all the mutant groups. Furthermore, inhibiting FGFR signaling using SU5402 in Hyp‐ and Dmp1–/– ‐derived bone marrow stromal cells prevented the increase in Fgf23 mRNA expression (129‐ and 124‐fold increase in Hyp and Dmp1–/– vs. 1.3‐fold in Hyp+SU5402 and 2.5‐fold in Dmp1–/– +SU5402, P<0.05). For all analyses, samples collected from nonmutant wild‐type littermates served as controls. These findings indicate that PHEX and DMP1 control a common pathway regulating bone mineralization and FGF23 production, the latter involving activation of the FGFR signaling in osteocytes.—Martin, A., Liu, S., David, V., Li, H., Karydis, A., Feng, J. Q., Quarles, L. D. Bone proteins PHEX and DMP1 regulate fibroblastic growth factor Fgf23 expression in osteocytes through a common pathway. FASEB J. 25, 2551–2562 (2011). www.fasebj.org

Noninvasive in vivo monitoring of bone architecture alterations in hindlimb-unloaded female rats using novel three-dimensional microcomputed tomography.

We tested a novel microcomputed tomograph designed to longitudinally and noninvasively monitor bone alterations in hindlimb‐unloaded female rats at a resolution of 26 μm over a period of 3 weeks. This prototype has a potential to detect three‐dimensional trabecular microarchitectural changes induced by growth and unloading.

Inflammation and functional iron deficiency regulate fibroblast growth factor 23 production

Circulating levels of fibroblast growth factor 23 (FGF23) are elevated in patients with chronic kidney disease (CKD), but the mechanisms are poorly understood. Here we tested whether inflammation and iron deficiency regulate FGF23. In wild-type mice, acute inflammation induced by single injections of heat-killed Brucella abortus or interleukin-1β (IL-1β) decreased serum iron within 6 hours, and was accompanied by significant increases in osseous Fgf23 mRNA expression and serum levels of C-terminal FGF23, but no changes in intact FGF23. Chronic inflammation induced by repeated bacteria or IL-1β injections decreased serum iron, increased osseous Fgf23 mRNA and serum C-terminal FGF23, but modestly increased biologically active, intact FGF23 serum levels. Chronic iron deficiency mimicked chronic inflammation. Increased osseous FGF23 cleavage rather than a prolonged half-life of C-terminal FGF23 fragments accounted for the elevated C-terminal FGF23 but near-normal intact FGF23 levels in inflammation. IL-1β injection increased Fgf23 mRNA and C-terminal FGF23 levels similarly in wild-type and Col4a3KO mice with CKD, but markedly increased intact FGF23 levels only in the CKD mice. Inflammation increased Fgf23 transcription by activating Hif1α signaling. Thus, inflammation and iron deficiency stimulate FGF23 production. Simultaneous upregulation of FGF23 cleavage in osteocytes maintains near-normal levels of biologically active, intact circulating FGF23, whereas downregulated or impaired FGF23 cleavage may contribute to elevated intact serum FGF23 in CKD.

A Comparative Transcriptome Analysis Identifying FGF23 Regulated Genes in the Kidney of a Mouse CKD Model

Elevations of circulating Fibroblast growth factor 23 (FGF23) are associated with adverse cardiovascular outcomes and progression of renal failure in chronic kidney disease (CKD). Efforts to identify gene products whose transcription is directly regulated by FGF23 stimulation of fibroblast growth factor receptors (FGFR)/α-Klotho complexes in the kidney is confounded by both systemic alterations in calcium, phosphorus and vitamin D metabolism and intrinsic alterations caused by the underlying renal pathology in CKD. To identify FGF23 responsive genes in the kidney that might explain the association between FGF23 and adverse outcomes in CKD, we performed comparative genome wide analysis of gene expression profiles in the kidney of the Collagen 4 alpha 3 null mice (Col4a3−/−) model of progressive kidney disease with kidney expression profiles of Hypophosphatemic (Hyp) and FGF23 transgenic mouse models of elevated FGF23. The different complement of potentially confounding factors in these models allowed us to identify genes that are directly targeted by FGF23. This analysis found that α-Klotho, an anti-aging hormone and FGF23 co-receptor, was decreased by FGF23. We also identified additional FGF23-responsive transcripts and activation of networks associated with renal damage and chronic inflammation, including lipocalin 2 (Lcn2), transforming growth factor beta (TGF-β) and tumor necrosis factor-alpha (TNF-α) signaling pathways. Finally, we found that FGF23 suppresses angiotensin-converting enzyme 2 (ACE2) expression in the kidney, thereby providing a pathway for FGF23 regulation of the renin-angiotensin system. These gene products provide a possible mechanistic links between elevated FGF23 and pathways responsible for renal failure progression and cardiovascular diseases.

Compound deletion of Fgfr3 and Fgfr4 partially rescues the Hyp mouse phenotype.

Uncertainty exists regarding the physiologically relevant fibroblast growth factor (FGF) receptor (FGFR) for FGF23 in the kidney and the precise tubular segments that are targeted by FGF23. Current data suggest that FGF23 targets the FGFR1c-Klotho complex to coordinately regulate phosphate transport and 1,25-dihydroxyvitamin D [1,25(OH)(2)D] production in the proximal tubule. In studies using the Hyp mouse model, which displays FGF23-mediated hypophosphatemia and aberrant vitamin D, deletion of Fgfr3 or Fgfr4 alone failed to correct the Hyp phenotype. To determine whether FGFR1 is sufficient to mediate the renal effects of FGF23, we deleted Fgfr3 and Fgfr4 in Hyp mice, leaving intact the FGFR1 pathway by transferring compound Fgfr3/Fgfr4-null mice on the Hyp background to create wild-type (WT), Hyp, Fgfr3(-/-)/Fgfr4(-/-), and Hyp/Fgfr3(-/-)/Fgfr4(-/-) mice. We found that deletion of Fgfr3 and Fgfr4 in Fgfr3(-/-)/Fgfr4(-/-) and Hyp/Fgfr3(-/-)/Fgfr4(-/-) mice induced an increase in 1,25(OH)(2)D. In Hyp/Fgfr3(-/-)/Fgfr4(-/-) mice, it partially corrected the hypophosphatemia (P(i) = 9.4 ± 0.9, 6.1 ± 0.2, 9.1 ± 0.4, and 8.0 ± 0.5 mg/dl in WT, Hyp, Fgfr3(-/-)/Fgfr4(-/-), and Hyp/Fgfr3(-/-)/Fgfr4(-/-) mice, respectively), increased Na-phosphate cotransporter Napi2a and Napi2c and Klotho mRNA expression in the kidney, and markedly increased serum FGF23 levels (107 ± 20, 3,680 ± 284, 167 ± 22, and 18,492 ± 1,547 pg/ml in WT, Hyp, Fgfr3(-/-)/Fgfr4(-/-), and Hyp/Fgfr3(-/-)/Fgfr4(-/-) mice, respectively), consistent with a compensatory response to the induction of end-organ resistance. Fgfr1 expression was unchanged in Hyp/Fgfr3(-/-)/Fgfr4(-/-) mice and was not sufficient to transduce the full effects of FGF23 in Hyp/Fgfr3(-/-)/Fgfr4(-/-) mice. These studies suggest that FGFR1, FGFR3, and FGFR4 act in concert to mediate FGF23 effects on the kidney and that loss of FGFR function leads to feedback stimulation of Fgf23 expression in bone.

Matrix Extracellular Phosphoglycoprotein (MEPE) Is a New Bone Renal Hormone and Vascularization Modulator

Increased matrix extracellular phosphoglycoprotein (MEPE) expression occurs in several phosphate and bone-mineral metabolic disorders. To resolve whether MEPE plays a role, we created a murine model overexpressing MEPE protein (MEPE tgn) in bone. MEPE tgn mice displayed a growth and mineralization defect with altered bone-renal vascularization that persisted to adulthood. The growth mineralization defect was due to a decrease in bone remodeling, and MEPE tgn mice were resistant to diet-induced renal calcification. MEPE protein-derived urinary ASARM peptides and reduced urinary Ca X PO4 product mediated the suppressed renal calcification. Osteoblastic cells displayed reduced activity but normal differentiation. Osteoclastic precursors were unable to differentiate in the presence of osteoblasts. In the kidney, NPT2a up-regulation induced an increase in phosphate renal reabsorption, leading to hyperphosphatemia. We conclude MEPE and MEPE-phosphate-regulating gene with homologies to endopeptidases on the X chromosome (MEPE-PHEX) interactions are components to an age-diet-dependent pathway that regulates bone turnover and mineralization and suppresses renal calcification. This novel pathway also modulates bone-renal vascularization and bone turnover.

Calcium Regulates FGF-23 Expression in Bone

Calcium has recently been shown to regulate fibroblast growth factor 23 (FGF-23), a bone-derived phosphate and vitamin D-regulating hormone. To better understand the regulation of FGF-23 by calcium, phosphorus, 1,25 dihydroxyvitamin D3 [1,25(OH)2D], and PTH, we examined FGF-23 expression under basal conditions and in response to PTH, doxercalciferol, or high-calcium diet treatment in Gcm2(-/-) and Cyp27b1(-/-) mutant mice. Gcm2(-/-) mice exhibited low serum PTH and 1,25(OH)2D concentrations, hypocalcemia, and hyperphosphatemia, whereas Cyp27b1(-/-) mice had high PTH, undetectable 1,25(OH)2D, hypocalcemia, and hypophosphatemia. Serum FGF-23 levels were decreased in both mutant models. Doxercalciferol administration increased serum FGF-23 levels in both mutant models. PTH administration to Gcm2(-/-) mice also increased serum FGF-23 levels, in association with an increase in both 1,25(OH)2D and calcium concentrations. Multiple regression analysis of pooled data indicated that changes in FGF-23 were positively correlated with serum calcium and 1,25(OH)2D but not related to changes in serum phosphate concentrations. A high-calcium diet also increased serum FGF-23 concentrations in Cyp27b1(-/-) mice in the absence of 1,25(OH)2D and in Gcm2(-/-) mice with low PTH. The addition of calcium to the culture media also stimulated FGF-23 message expression in MC3T3-E1 osteoblasts. In addition, FGF-23 promoter activity in cultured osteoblasts was inhibited by the L-calcium-channel inhibitor nifedipine and stimulated by calcium ionophores. The effects of chronic low calcium to prevent 1,25(OH)2D and PTH stimulation of FGF-23 in these mutant mouse models suggest that suppression of FGF-23 plays an important physiological adaptive response to hypocalcemia.

Longitudinal evaluation of FGF23 changes and mineral metabolism abnormalities in a mouse model of chronic kidney disease.

Fibroblast growth factor 23 (FGF23) is a phosphaturic and vitamin D‐regulatory hormone of putative bone origin that is elevated in patients with chronic kidney disease (CKD). The mechanisms responsible for elevations of FGF23 and its role in the pathogenesis of chronic kidney disease‐mineral bone disorder (CKD‐MBD) remain uncertain. We investigated the association between FGF23 serum levels and kidney disease progression, as well as the phenotypic features of CKD‐MBD in a Col4a3 null mouse model of human autosomal‐recessive Alport syndrome. These mice exhibited progressive renal failure, declining 1,25(OH)2D levels, increments in parathyroid hormone (PTH) and FGF23, late‐onset hypocalcemia and hyperphosphatemia, high‐turnover bone disease, and increased mortality. Serum levels of FGF23 increased in the earliest stages of renal damage, before elevations in blood urea nitrogen (BUN) and creatinine. FGF23 gene transcription in bone, however, did not increase until late‐stage kidney disease, when serum FGF23 levels were exponentially elevated. Further evaluation of bone revealed trabecular osteocytes to be the primary cell source for FGF23 production in late‐stage disease. Changes in FGF23 mirrored the rise in serum PTH and the decline in circulating 1,25(OH)2D. The rise in PTH and FGF23 in Col4a3 null mice coincided with an increase in the urinary fractional excretion of phosphorus and a progressive decline in sodium‐phosphate cotransporter gene expression in the kidney. Our findings suggest elevations of FGF23 in CKD to be an early marker of renal injury that increases before BUN and serum creatinine. An increased production of FGF23 by bone may not be responsible for early increments in FGF23 in CKD but does appear to contribute to FGF23 levels in late‐stage disease. Elevations in FGF23 and PTH coincide with an increase in urinary phosphate excretion that likely prevents the early onset of hyperphosphatemia in the face of increased bone turnover and a progressive decline in functional renal mass.

Ex Vivo bone formation in bovine trabecular bone cultured in a dynamic 3D bioreactor is enhanced by compressive mechanical strain.

Our aim was to test cell and trabecular responses to mechanical loading in vitro in a tissue bone explant culture model. We used a new three-dimensional culture model, the ZetOS system, which provides the ability to exert cyclic compression on cancellous bone cylinders (bovine sternum) cultured in forced flow circumfusion chambers, and allows to assess mechanical parameters of the cultivated samples. We evaluated bone cellular parameters through osteocyte viability test, gene and protein expression, and histomorphometric bone formation rate, in nonloaded versus loaded samples. The microarchitecture of bone cores was appraised by in vivo micro-CT imaging. After 3 weeks, the samples receiving daily cyclic compression exhibited increased osteoblast differentiation and activity associated with thicker, more plate-like-shaped trabeculae and higher Youngs modulus and ultimate force as compared to unloaded samples. Osteoclast activity was not affected by mechanical strain, although it was responsive to drug treatments (retinoic acid and bisphosphonate) during the first 2 weeks of culture. Thus, in the ZetOS apparatus, we reproduce in vitro the osteogenic effects of mechanical strain known in vivo, making this system a unique and an essential laboratory aid for ex vivo testing of lamellar bone remodeling.