Aldosterone Is Positively Associated With Circulating FGF23 Levels in Chronic Kidney Disease Across Four Species, and May Drive FGF23 Secretion Directly.

BACKGROUND: Chronic kidney disease (CKD) is accompanied by increases in circulating fibroblast growth factor 23 (FGF23) and aldosterone levels. Here, we tested the hypothesis that aldosterone may be one of the driving forces behind increased FGF23 secretion in CKD. METHODS: Using data from a prospective study in humans, a retrospective study in dogs and cats, and an experimental study in 5/6-nephrectomized mice, we analyzed the relationship between circulating FGF23 and serum aldosterone levels in CKD across four species. To assess the effects of acute inhibition of aldosterone signaling on circulating FGF23, we acutely treated mice with established CKD with the mineralocorticoid receptor blocker canrenone (50 mg/kg iv/sc), and measured intact FGF23 before and 24 h as well as 72 h after start of administration of the drug. RESULTS: We found a tight positive association between circulating intact FGF23 and serum aldosterone in human, canine, and feline CKD patients, as well as in experimental murine CKD (humans: r S = 0.57, p = 0.0368; dogs: r S = 0.66, p = 0.0019; cats: r S = 0.75, p = 0.0003; mice: r S = 0.49, p = 0.0004). Injection of canrenone in mice with established CKD did not lead to changes in FGF23 levels within 24 h, but reduced FGF23 in all mice at 72 h. CONCLUSION: Aldosterone may drive enhanced FGF23 secretion in CKD, possibly explaining the tight positive association between circulating intact FGF23 and aldosterone in human, canine, and feline CKD patients as well as in experimental CKD models.

The bone is the major source of high circulating intact fibroblast growth factor-23 in acute murine polymicrobial sepsis induced by cecum ligation puncture.

Fibroblast growth factor-23 (FGF23), a bone-produced hormone, plays a critical role in mineral homeostasis. Human diseases associated with excessive intact circulating FGF23 (iFGF23) result in hypophosphatemia and low vitamin D hormone in patients with normal kidney function. In addition, there is accumulating evidence linking FGF23 with inflammation. Based on these studies and the frequent observation of hypophosphatemia among septic patients, we sought to elucidate further the relationship between FGF23 and mineral homeostasis in a clinically relevant murine polymicrobial sepsis model. Medium-severity sepsis was induced by cecum ligation puncture (CLP) in adult CD-1 mice of both sexes. Healthy CD-1 mice (without CLP) were used as controls. Forty-eight hours post-CLP, spontaneous urine was collected, and serum, organs and bones were sampled at necropsy. Serum iFGF23 increased ~20-fold in CLP compared to control mice. FGF23 protein concentration was increased in the bones, but not in spleen or liver of CLP mice. Despite the ~20-fold iFGF23 increase, we did not observe any significant changes in mineral homeostasis or parathyroid hormone levels in the blood of CLP animals. Urinary excretion of phosphate, calcium, and sodium remained unchanged in male CLP mice, whereas female CLP mice exhibited lower urinary calcium excretion, relative to healthy controls. In line with renal FGF23 resistance, expression of phosphate-, calcium- and sodium-transporting proteins did not show consistent changes in the kidneys of male and female CLP mice. Renal expression of the co-receptor αKlotho was downregulated in female, but not in male CLP mice. In conclusion, our data demonstrate that the dramatic, sex-independent rise in serum iFGF23 post-CLP was mainly caused by an upregulation of FGF23 secretion in the bone. Surprisingly, the upsurge in circulating iFGF23 did not alter humoral mineral homeostasis in the acutely septic mice. Hence, the biological function of elevated FGF23 in sepsis remains unclear and warrants further studies.

Role of KLHL3 and dietary K+ in regulating KS-WNK1 expression.

The physiological role of the shorter isoform of with no lysine kinase (WNK)1 that is exclusively expressed in the kidney (KS-WNK1), with particular abundance in the distal convoluted tubule, remains elusive. KS-WNK1, despite lacking the kinase domain, is nevertheless capable of stimulating the NaCl cotransporter, apparently through activation of WNK4. It has recently been shown that a less severe form of familial hyperkalemic hypertension featuring only hyperkalemia is caused by missense mutations in the WNK1 acidic domain that preferentially affect cullin 3 (CUL3)-Kelch-like protein 3 (KLHL3) E3-induced degradation of KS-WNK1 rather than that of full-length WNK1. Here, we show that full-length WNK1 is indeed less impacted by the CUL3-KLHL3 E3 ligase complex compared with KS-WNK1. We demonstrated that the unique 30-amino acid NH2-terminal fragment of KS-WNK1 is essential for its activating effect on the NaCl cotransporter and recognition by KLHL3. We identified specific amino acid residues in this region critical for the functional effect of KS-WNK1 and KLHL3 sensitivity. To further explore this, we generated KLHL3-R528H knockin mice that mimic human mutations causing familial hyperkalemic hypertension. These mice revealed that the KLHL3 mutation specifically increased expression of KS-WNK1 in the kidney. We also observed that in wild-type mice, the expression of KS-WNK1 was only detectable after exposure to a low-K+ diet. These findings provide new insights into the regulation and function of KS-WNK1 by the CUL3-KLHL3 complex in the distal convoluted tubule and indicate that this pathway is regulated by dietary K+ levels.NEW & NOTEWORTHY In this work, we demonstrated that the kidney-specific isoform of with no lysine kinase 1 (KS-WNK1) in the kidney is modulated by dietary K+ and activity of the ubiquitin ligase protein Kelch-like protein 3. We analyzed the role of different amino acid residues of KS-WNK1 in its activity against the NaCl cotransporter and sensitivity to Kelch-like protein 3.

Lack of vitamin D signalling per se does not aggravate cardiac functional impairment induced by myocardial infarction in mice.

Epidemiological studies have linked vitamin D deficiency to an increased incidence of myocardial infarction and support a role for vitamin D signalling in the pathophysiology of myocardial infarction. Vitamin D deficiency results in the development of secondary hyperparathyroidism, however, the role of secondary hyperparathyroidism in the pathophysiology of myocardial infarction is not known. Here, we aimed to explore further the secondary hyperparathyroidism independent role of vitamin D signalling in the pathophysiology of myocardial infarction by inducing experimental myocardial infarction in 3-month-old, male, wild-type mice and in mice lacking a functioning vitamin D receptor. In order to prevent secondary hyperparathyroidism in vitamin D receptor mutant mice, all mice were maintained on a rescue diet enriched with calcium, phosphorus, and lactose. Surprisingly, survival rate, cardiac function as measured by echocardiography and intra-cardiac catheterisation and cardiomyocyte size were indistinguishable between normocalcaemic vitamin D receptor mutant mice and wild-type controls, 2 and 8 weeks post-myocardial infarction. In addition, the myocardial infarction-induced inflammatory response was similar in vitamin D receptor mutants and wild-type mice, as evidenced by a comparable upregulation in cardiac interleukin-1-ß and tumor-necrosis-factor-α mRNA abundance and similar elevations in circulating interleukin-1-ß and tumor-necrosis-factor-α. Our data suggest that the lack of vitamin D signalling in normocalcaemic vitamin D receptor mutants has no major detrimental effect on cardiac function and outcome post-myocardial infarction. Our study may have important clinical implications because it suggests that the secondary hyperparathyroidism induced by vitamin D deficiency, rather than the lack of vitamin D signalling per se, may negatively impact cardiac function post-myocardial infarction.

Augmented Fibroblast Growth Factor-23 Secretion in Bone Locally Contributes to Impaired Bone Mineralization in Chronic Kidney Disease in Mice.

Chronic kidney disease-mineral and bone disorder (CKD-MBD) is a systemic disorder of mineral and bone metabolism caused by CKD. Impaired bone mineralization together with increased bony secretion of fibroblast growth factor-23 (FGF23) are hallmarks of CKD-MBD. We recently showed that FGF23 suppresses the expression of tissue nonspecific alkaline phosphatase (TNAP) in bone cells by a Klotho-independent, FGF receptor-3-mediated signaling axis, leading to the accumulation of the mineralization inhibitor pyrophosphate. Therefore, we hypothesized that excessive FGF23 secretion may locally impair bone mineralization in CKD-MBD. To test this hypothesis, we induced CKD by 5/6 nephrectomy in 3-month-old wild-type (WT) mice and Fgf23-/-/VDRΔ/Δ (Fgf23/VDR) compound mutant mice maintained on a diet enriched with calcium, phosphate, and lactose. Eight weeks postsurgery, WT CKD mice were characterized by reduced bone mineral density at the axial and appendicular skeleton, hyperphosphatemia, secondary hyperparathyroidism, increased serum intact Fgf23, and impaired bone mineralization as evidenced by bone histomorphometry. Laser capture microdissection in bone cryosections showed that both osteoblasts and osteocytes contributed to the CKD-induced increase in Fgf23 mRNA abundance. In line with our hypothesis, osteoblastic and osteocytic activity of alkaline phosphatase was reduced, and bone pyrophosphate concentration was ~2.5-fold higher in CKD mice, relative to Sham controls. In Fgf23/VDR compound mice lacking Fgf23, 5/6-Nx induced secondary hyperparathyroidism and bone loss. However, 5/6-Nx failed to suppress TNAP activity, and bone pyrophosphate concentrations remained unchanged in Fgf23/VDR CKD mice. Collectively, our data suggest that elevated Fgf23 production in bone contributes to the mineralization defect in CKD-MBD by auto-/paracrine suppression of TNAP and subsequent accumulation of pyrophosphate in bone. Hence, our study has identified a novel mechanism involved in the pathogenesis of CKD-MBD.

Selective inhibition of receptor activator of NF-κB ligand (RANKL) in hematopoietic cells improves outcome after experimental myocardial infarction.

The RANK (receptor activator of nuclear factor κB)/RANKL (RANK ligand)/OPG (osteoprotegerin) axis is activated after myocardial infarction (MI), but its pathophysiological role is not well understood. Here, we investigated how global and cell compartment-selective inhibition of RANKL affects cardiac function and remodeling after MI in mice. Global RANKL inhibition was achieved by treatment of human RANKL knock-in (huRANKL-KI) mice with the monoclonal antibody AMG161. huRANKL-KI mice express a chimeric RANKL protein wherein part of the RANKL molecule is humanized. AMG161 inhibits human and chimeric but not murine RANKL. To dissect the pathophysiological role of RANKL derived from hematopoietic and mesenchymal cells, we selectively exchanged the hematopoietic cell compartment by lethal irradiation and across-genotype bone marrow transplantation between wild-type and huRANKL-KI mice, exploiting the specificity of AMG161. After permanent coronary artery ligation, mice were injected with AMG161 or an isotype control antibody over 4 weeks post-MI. MI increased RANKL expression mainly in cardiomyocytes and scar-infiltrating cells 4 weeks after MI. Only inhibition of RANKL derived from hematopoietic cellular sources, but not global or mesenchymal RANKL inhibition, improved post-infarct survival and cardiac function. Mechanistically, hematopoietic RANKL inhibition reduced expression of the pro-inflammatory cytokine IL-1ß in the cardiac cellular infiltrate. In conclusion, inhibition of RANKL derived from hematopoietic cellular sources is beneficial to maintain post-ischemic cardiac function by reduction of pro-inflammatory cytokine production. KEY MESSAGES: Experimental myocardial infarction (MI) augments cardiac RANKL expression in mice. RANKL expression is increased in cardiomyocytes and scar-infiltrating cells after MI. Global or mesenchymal cell RANKL inhibition has no influence on cardiac function after MI. Inhibition of RANKL derived from hematopoietic cells improves heart function post-MI. Hematopoietic RANKL inhibition reduces pro-inflammatory cytokines in scar-infiltrating cells.

Response of human periodontal ligament stem cells to IFN-γ and TLR-agonists.

Periodontal ligament stem cells similarly to the mesenchymal stem cells of other tissues possess immunomodulatory properties, which are regulated by different cytokines, particularly by interferon-γ (IFN-γ). In contrast, less information is provided about the effect of toll-like receptors ligand on immunomodulatory properties of these cells. In the present study we investigated the response of human periodontal ligament stem cells (hPDLSCs) in response to simultaneous stimulation with IFN-γ and toll-like receptor (TLR) agonists. The resulting expression of indoleamine-2,3-dioxygenase-1 (IDO-1), interleukin (IL)-6, IL-8 and monocyte chemotactic protein 1 (MCP-1) was investigated. The expression of IDO-1 was upregulated by IFN-γ in both gene and protein levels. TLR2 agonists Pam3CSK4 induced gene expression of IDO-1, but had no effect on protein expression. IFN-γ induced IDO-1 protein expression was further enhanced by Pam3CSK4. TLR-4 agonist E. coli LPS has no significant effect on neither basal nor IFN-γ induced IDO-1 protein expression. The production of IL-6, IL-8, and MCP-1 was induced by TLR agonists. Neither basal nor TLR agonists induced production of these proteins was affected by IFN-γ. Our data shows potential interaction between IFN-γ and TLR2 responses in hPDLSCs, which might be involved in regulation of immune response in inflammatory diseases, and particularly periodontitis.

Genetic Ablation of Fgf23 or Klotho Does not Modulate Experimental Heart Hypertrophy Induced by Pressure Overload.

Left ventricular hypertrophy (LVH) ultimately leads to heart failure in conditions of increased cardiac pre- or afterload. The bone-derived phosphaturic and sodium-conserving hormone fibroblast growth factor-23 (FGF23) and its co-receptor Klotho have been implicated in the development of uremic LVH. Using transverse aortic constriction (TAC) in gene-targeted mouse models, we examine the role of Fgf23 and Klotho in cardiac hypertrophy and dysfunction induced by pressure overload. TAC profoundly increases serum intact Fgf23 due to increased cardiac and bony Fgf23 transcription and downregulation of Fgf23 cleavage. Aldosterone receptor blocker spironolactone normalizes serum intact Fgf23 levels after TAC by reducing bony Fgf23 transcription. Notably, genetic Fgf23 or Klotho deficiency does not influence TAC-induced hypertrophic remodelling, LV functional impairment, or LV fibrosis. Despite the profound, aldosterone-mediated increase in circulating intact Fgf23 after TAC, our data do not support an essential role of Fgf23 or Klotho in the pathophysiology of pressure overload-induced cardiac hypertrophy.

Estrogen Regulates Bone Turnover by Targeting RANKL Expression in Bone Lining Cells.

Estrogen is critical for skeletal homeostasis and regulates bone remodeling, in part, by modulating the expression of receptor activator of NF-κB ligand (RANKL), an essential cytokine for bone resorption by osteoclasts. RANKL can be produced by a variety of hematopoietic (e.g. T and B-cell) and mesenchymal (osteoblast lineage, chondrocyte) cell types. The cellular mechanisms by which estrogen acts on bone are still a matter of controversy. By using murine reconstitution models that allow for selective deletion of estrogen receptor-alpha (ERα) or selective inhibition of RANKL in hematopoietic vs. mesenchymal cells, in conjunction with in situ expression profiling in bone cells, we identified bone lining cells as important gatekeepers of estrogen-controlled bone resorption. Our data indicate that the increase in bone resorption observed in states of estrogen deficiency in mice is mainly caused by lack of ERα-mediated suppression of RANKL expression in bone lining cells.

Klotho Lacks an FGF23-Independent Role in Mineral Homeostasis.

Fibroblast growth factor-23 (FGF23) is a bone-derived hormone regulating vitamin D hormone production and renal handling of minerals by signaling through an FGF receptor/αKlotho (Klotho) receptor complex. Whether Klotho has FGF23-independent effects on mineral homeostasis is a controversial issue. Here, we aimed to shed more light on this controversy by comparing male and female triple knockout mice with simultaneous deficiency in Fgf23 and Klotho and a nonfunctioning vitamin D receptor (VDR) (Fgf23/Klotho/VDR) with double (Fgf23/VDR, Klotho/VDR, and Fgf23/Klotho) and single Fgf23, Klotho, and VDR mutants. As expected, 4-week-old Fgf23, Klotho, and Fgf23/Klotho knockout mice were hypercalcemic and hyperphosphatemic, whereas VDR, Fgf23/VDR, and Klotho/VDR mice on rescue diet were normocalcemic and normophosphatemic. Serum levels of calcium, phosphate, and sodium did not differ between 4-week-old triple Fgf23/Klotho/VDR and double Fgf23/VDR or Klotho/VDR knockout mice. Notably, 3-month-old Fgf23/Klotho/VDR triple knockout mice were indistinguishable from double Fgf23/VDR and Klotho/VDR compound mutants in terms of serum calcium, serum phosphate, serum sodium, and serum PTH, as well as urinary calcium and sodium excretion. Protein expression analysis revealed increased membrane abundance of sodium-phosphate co-transporter 2a (NaPi-2a), and decreased expression of sodium-chloride co-transporter (NCC) and transient receptor potential cation channel subfamily V member 5 (TRPV5) in Fgf23/Klotho/VDR, Fgf23/VDR, and Klotho/VDR mice, relative to wild-type and VDR mice, but no differences between triple and double knockouts. Further, ex vivo treatment of live kidney slices isolated from wild-type and Klotho/VDR mice with soluble Klotho did not induce changes in intracellular phosphate, calcium or sodium accumulation assessed by two-photon microscopy. In conclusion, our data suggest that the main physiological function of Klotho for mineral homeostasis in vivo is its role as co-receptor mediating Fgf23 action. © 2017 American Society for Bone and Mineral Research.

Acute Parathyroid Hormone Injection Increases C-Terminal but Not Intact Fibroblast Growth Factor 23 Levels.

The acute effects of parathyroid hormone (PTH) on fibroblast growth factor 23 (FGF23) in vivo are not well understood. After a single subcutaneous PTH (1-34) injection (50 nmol/kg) in mice, FGF23 levels were assessed in plasma using assays that measure either intact alone (iFGF23) or intact/C-terminal FGF23 (cFGF23). Furthermore, FGF23 messenger RNA (mRNA) and protein levels were assessed in bone. In addition, we examined the effects of PTH treatment on FGF23 production in vitro using differentiated calvarial osteocyte-like cells. cFGF23 levels increased by three- to fivefold within 2 hours following PTH injection, which returned to baseline by 4 hours. In contrast, iFGF23 levels remained unchanged for the first 2 hours, yet declined to ∼60% by 6 hours and remained suppressed before returning to baseline after 24 hours. Using homozygous mice for an autosomal dominant hypophosphatemic rickets-FGF23 mutation or animals treated with a furin inhibitor, we showed that cFGF23 and iFGF23 levels increased equivalently after PTH injection. These findings are consistent with increased FGF23 production in bone, yet rapid cleavage of the secreted intact protein. Using primary osteocyte-like cell cultures, we showed that PTH increased FGF23 mRNA expression through cyclic adenosine monophosphate/protein kinase A, but not inositol triphosphate/protein kinase C signaling; PTH also increased furin protein levels. In conclusion, PTH injection rapidly increases FGF23 production in bone in vivo and in vitro. However, iFGF23 is rapidly degraded. At later time points through an unidentified mechanism, a sustained decrease in FGF23 production occurs.

FGF23-Klotho signaling axis in the kidney.

Fibroblast growth factor-23 (FGF23) is a bone-derived hormone protecting against the potentially deleterious effects of hyperphosphatemia by suppression of phosphate reabsorption and of active vitamin D hormone synthesis in the kidney. The kidney is one of the main target organs of FGF23 signaling. The purpose of this review is to highlight the recent advances in the area of FGF23-Klotho signaling in the kidney. During recent years, it has become clear that FGF23 acts independently on proximal and distal tubular epithelium. In proximal renal tubules, FGF23 suppresses phosphate reabsorption by a Klotho dependent activation of extracellular signal-regulated kinase-1/2 (ERK1/2) and of serum/glucocorticoid-regulated kinase-1 (SGK1), leading to phosphorylation of the scaffolding protein Na+/H+ exchange regulatory cofactor (NHERF)-1 and subsequent internalization and degradation of sodium-phosphate cotransporters. In distal renal tubules, FGF23 augments calcium and sodium reabsorption by increasing the apical membrane expression of the epithelial calcium channel TRPV5 and of the sodium-chloride cotransporter NCC through a Klotho dependent activation of with-no-lysine kinase-4 (WNK4). In proximal and distal renal tubules, FGF receptor-1 is probably the dominant FGF receptor mediating the effects of FGF23 by forming a complex with membrane-bound Klotho in the basolateral membrane. The newly described sodium- and calcium-conserving functions of FGF23 may have major implications for the pathophysiology of diseases characterized by chronically increased circulating FGF23 concentrations such as chronic kidney disease.

Soluble CD14 Enhances the Response of Periodontal Ligament Stem Cells to P. gingivalis Lipopolysaccharide.

Periodontal ligament stem cells (PDLSCs) are lacking membrane CD14, which is an important component of lipopolysaccharide (LPS) signaling through toll-like receptor (TLR) 4. In the present study we investigated the effect of soluble CD14 on the response of human PDLSCs to LPS of Porphyromonas (P.) gingivalis. Human PDLSCs (hPDLSCs) were stimulated with P. gingivalis LPS in the presence or in the absence of soluble CD14 (sCD14) and the production of interleukin (IL)-6, chemokine C-X-C motif ligand 8 (CXCL8), and chemokine C-C motif ligand 2 (CCL2) was measured. The response to P. gingivalis LPS was compared with that to TLR4 agonist Escherichia coli LPS and TLR2-agonist Pam3CSK4. The response of hPDLSCs to both P. gingivalis LPS and E. coli LPS was significantly enhanced by sCD14. In the absence of sCD14, no significant difference in the hPDLSCs response to two kinds of LPS was observed. These responses were significantly lower compared to that to Pam3CSK4. In the presence of sCD14, the response of hPdLSCs to P. gingivalis LPS was markedly higher than that to E. coli LPS and comparable with that to Pam3CSK4. The response of hPdLSCs to bacterial LPS is strongly augmented by sCD14. Local levels of sCD14 could be an important factor for modulation of the host response against periodontal pathogens.

Fgf23 and parathyroid hormone signaling interact in kidney and bone.

Fibroblast growth factor-23 (FGF23) is a bone-derived hormone, suppressing renal phosphate reabsorption and vitamin D hormone synthesis in proximal tubules, and stimulating calcium reabsorption in distal tubules of the kidney. Here, we analyzed the long term sequelae of deficient Fgf23 signaling on bone and mineral metabolism in 9-month-old mice lacking both Fgf23 or Klotho and a functioning vitamin D receptor (VDR). To prevent hypocalcemia in VDR deficient mice, all mice were kept on a rescue diet enriched with calcium, phosphate, and lactose. VDR mutants were normocalcemic and normophosphatemic, and had normal tibial bone mineral density. Relative to VDR mutants, Fgf23/VDR and Klotho/VDR compound mutants were characterized by hypocalcemia, hyperphosphatemia, and very high serum parathyroid hormone (PTH). Despite ∼10-fold higher serum PTH levels in compound mutants, urinary excretion of phosphate and calcium as well as osteoclast numbers in bone remained unchanged relative to VDR mutants. The increase in plasma cAMP after hPTH(1-34) injection was similar in all genotypes. However, a 5-day infusion of hPTH(1-34) via osmotic minipumps resulted in reduced phosphorylation of extracellular signal-regulated kinase 1 and 2 (ERK1/2) in bone and kidney of Fgf23/VDR and Klotho/VDR compound mutants, relative to VDR and WT controls. Similarly, the PTH-mediated ERK1/2 phosphorylation was reduced in primary osteoblasts isolated from Fgf23 and Klotho deficient mice, but was restored by concomitant treatment with recombinant FGF23. Collectively, our data indicate that the phosphaturic, calcium-conserving, and bone resorption-stimulating actions of PTH are blunted by Fgf23 or Klotho deficiency. Hence, FGF23 may be an important modulator of PTH signaling in bone and kidney.

Excessive Osteocytic Fgf23 Secretion Contributes to Pyrophosphate Accumulation and Mineralization Defect in Hyp Mice.

X-linked hypophosphatemia (XLH) is the most frequent form of inherited rickets in humans caused by mutations in the phosphate-regulating gene with homologies to endopeptidases on the X-chromosome (PHEX). Hyp mice, a murine homologue of XLH, are characterized by hypophosphatemia, inappropriately low serum vitamin D levels, increased serum fibroblast growth factor-23 (Fgf23), and osteomalacia. Although Fgf23 is known to be responsible for hypophosphatemia and reduced vitamin D hormone levels in Hyp mice, its putative role as an auto-/paracrine osteomalacia-causing factor has not been explored. We recently reported that Fgf23 is a suppressor of tissue nonspecific alkaline phosphatase (Tnap) transcription via FGF receptor-3 (FGFR3) signaling, leading to inhibition of mineralization through accumulation of the TNAP substrate pyrophosphate. Here, we report that the pyrophosphate concentration is increased in Hyp bones, and that Tnap expression is decreased in Hyp-derived osteocyte-like cells but not in Hyp-derived osteoblasts ex vivo and in vitro. In situ mRNA expression profiling in bone cryosections revealed a ~70-fold up-regulation of Fgfr3 mRNA in osteocytes versus osteoblasts of Hyp mice. In addition, we show that blocking of increased Fgf23-FGFR3 signaling with anti-Fgf23 antibodies or an FGFR3 inhibitor partially restored the suppression of Tnap expression, phosphate production, and mineralization, and decreased pyrophosphate concentration in Hyp-derived osteocyte-like cells in vitro. In vivo, bone-specific deletion of Fgf23 in Hyp mice rescued the suppressed TNAP activity in osteocytes of Hyp mice. Moreover, treatment of wild-type osteoblasts or mice with recombinant FGF23 suppressed Tnap mRNA expression and increased pyrophosphate concentrations in the culture medium and in bone, respectively. In conclusion, we found that the cell autonomous increase in Fgf23 secretion in Hyp osteocytes drives the accumulation of pyrophosphate through auto-/paracrine suppression of TNAP. Hence, we have identified a novel mechanism contributing to the mineralization defect in Hyp mice.

FGF23 Regulates Bone Mineralization in a 1,25(OH)2 D3 and Klotho-Independent Manner.

Fibroblast growth factor-23 (Fgf23) is a bone-derived hormone, suppressing phosphate reabsorption and vitamin D hormone (1,25(OH)2 D3 ) production in the kidney. It has long been an enigma why lack of Fgf23 or of Klotho, the coreceptor for Fgf23, leads to severe impairment in bone mineralization despite the presence of hypercalcemia and hyperphosphatemia. Using Fgf23(-/-) or Klotho(-/-) mice together with compound mutant mice lacking both Fgf23 or Klotho and a functioning vitamin D receptor, we show that in Klotho(-/-) mice the mineralization defect is solely driven by 1,25(OH)2 D3 -induced upregulation of the mineralization-inhibiting molecules osteopontin and pyrophosphate in bone. In Fgf23(-/-) mice, the mineralization defect has two components, a 1,25(OH)2 D3 -driven component similar to Klotho(-/-) mice and a component driven by lack of Fgf23, causing additional accumulation of osteopontin. We found that FGF23 regulates osteopontin secretion indirectly by suppressing alkaline phosphatase transcription and phosphate production in osteoblastic cells, acting through FGF receptor-3 in a Klotho-independent manner. Hence, FGF23 secreted from osteocytes may form an autocrine/paracrine feedback loop for the local fine-tuning of bone mineralization.

Amphiregulin lacks an essential role for the bone anabolic action of parathyroid hormone.

Although parathyroid hormone (PTH) has long been known to act as a bone anabolic agent when administered intermittently, the exact underlying mechanisms remain largely unknown. Amphiregulin (AREG), a ligand of the epidermal growth factor receptor, has been identified to be a PTH target gene in vitro and in vivo. Here, we used female global AREG knockout (AREG-KO) mice to explore the role of AREG in mediating the bone anabolic effects of PTH. AREG-KO mice were characterized by unchanged distal femoral cancellous bone mass and only subtle decreases in bone mineral density (BMD) and cortical thickness at the femoral midshaft at 3 and 8 months of age, relative to wildtype controls. AREG deficiency was associated with complex changes in the mRNA expression of other EGFR ligands in femoral cancellous bone osteoblasts in situ in 3-week-old mice. To examine the bone anabolic effects of PTH in the absence and presence of AREG, we injected 3-month-old AREG-KO females and wildtype control littermates with 80 µg/kg PTH or vehicle 5 times per week over 4 weeks. Intermittent PTH treatment of AREG-KO mice led to increases in femoral trabecular and cortical BMD, cortical thickness, endocortical and periosteal bone formation, cancellous bone formation rate, and serum osteocalcin, comparable to those observed in wildtype control mice. In conclusion, our data indicate that the bone anabolic effects of PTH do not require AREG, at least in 3-month-old female mice.

FGF23 regulation of renal tubular solute transport.

PURPOSE OF REVIEW: Fibroblast growth factor-23 (FGF23) is a bone-derived hormone known to suppress phosphate reabsorption in the kidney. The purpose of this review was to highlight the recent advances in the area of FGF23-regulated solute transport in the kidney. RECENT FINDINGS: Recent evidence suggests that FGF23 suppresses phosphate reabsorption in renal proximal tubular epithelium by a Klotho-dependent, FGF receptor (FGFR)-1 and FGFR4-mediated signaling mechanism that may also involve Janus kinase 3. Moreover, it was recently established that FGF23 signaling in the distal renal tubule targets with-no-lysine kinase-4 (WNK4), a key molecule in the regulation of solute transport in the distal nephron. By targeting WNK4, FGF23 has been shown to increase the membrane abundance of the epithelial calcium channel TRPV5 and of the sodium-chloride cotransporter NCC, resulting in augmented renal calcium and sodium reabsorption. SUMMARY: Significant progress has been made in the further characterization of the signaling pathways involved in the FGF23-induced inhibition of phosphate transport in proximal tubular epithelium, and major new functions of FGF23 in solute transport have been discovered in distal renal tubules. The calcium- and sodium-conserving functions of FGF23 may have major implications for the pathophysiology of cardiovascular diseases. VIDEO ABSTRACT.

Experimental Myocardial Infarction Upregulates Circulating Fibroblast Growth Factor-23.

Myocardial infarction (MI) is a major cause of death worldwide. Epidemiological studies have linked vitamin D deficiency to MI incidence. Because fibroblast growth factor-23 (FGF23) is a master regulator of vitamin D hormone production and has been shown to be associated with cardiac hypertrophy per se, we explored the hypothesis that FGF23 may be a previously unrecognized pathophysiological factor causally linked to progression of cardiac dysfunction post-MI. Here, we show that circulating intact Fgf23 was profoundly elevated, whereas serum vitamin D hormone levels were suppressed, after induction of experimental MI in rat and mouse models, independent of changes in serum soluble Klotho or serum parathyroid hormone. Both skeletal and cardiac expression of Fgf23 was increased after MI. Although the molecular link between the cardiac lesion and circulating Fgf23 concentrations remains to be identified, our study has uncovered a novel heart-bone-kidney axis that may have important clinical implications and may inaugurate the new field of cardio-osteology.

FGF23 regulates renal sodium handling and blood pressure.

Fibroblast growth factor-23 (FGF23) is a bone-derived hormone regulating renal phosphate reabsorption and vitamin D synthesis in renal proximal tubules. Here, we show that FGF23 directly regulates the membrane abundance of the Na(+):Cl(-) co-transporter NCC in distal renal tubules by a signaling mechanism involving the FGF receptor/αKlotho complex, extracellular signal-regulated kinase 1/2 (ERK1/2), serum/glucocorticoid-regulated kinase 1 (SGK1), and with-no lysine kinase-4 (WNK4). Renal sodium (Na(+)) reabsorption and distal tubular membrane expression of NCC are reduced in mouse models of Fgf23 and αKlotho deficiency. Conversely, gain of FGF23 function by injection of wild-type mice with recombinant FGF23 or by elevated circulating levels of endogenous Fgf23 in Hyp mice increases distal tubular Na(+) uptake and membrane abundance of NCC, leading to volume expansion, hypertension, and heart hypertrophy in a αKlotho and dietary Na(+)-dependent fashion. The NCC inhibitor chlorothiazide abrogates FGF23-induced volume expansion and heart hypertrophy. Our findings suggest that FGF23 is a key regulator of renal Na(+) reabsorption and plasma volume, and may explain the association of FGF23 with cardiovascular risk in chronic kidney disease patients.