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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 α Klotho 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. Synopsis FGF23 serum levels are elevated in chronic kidney disease patients. FGF23 is here shown to be a regulator of the sodium‐chloride channel NCC in distal renal tubules, thus affecting renal sodium retention, plasma expansion, hypertension, and heart hypertrophy. FGF23 regulates membrane abundance and activity of the renal sodium‐chloride channel NCC through the ERK1/2‐SGK1‐WNK4 signaling pathway. FGF23 is a sodium‐conserving hormone. Elevated circulating FGF23 leads to volume expansion, hypertension, and cardiac hypertrophy in a Klotho‐dependent fashion. The NCC inhibitor chlorothiazide blunts FGF23‐induced hypertension. A low sodium diet aggravates the hypertensive action of FGF23 through crosstalk with aldosterone signaling at the level of SGK1. Graphical Abstract FGF23 serum levels are elevated in chronic kidney disease patients. FGF23 is here shown to be a regulator of the sodium‐chloride channel NCC in distal renal tubules, thus affecting renal sodium retention, plasma expansion, hypertension, and heart hypertrophy.

OPG is a new member of the tumor necrosis factor (TNF) receptor family which plays a key role in the physiological regulation of osteoclastic bone resorption. The protein, which is produced by osteoblasts and marrow stromal cells, lacks a transmembrane domain and acts as a secreted decoy receptor which has no direct signaling capacity. OPG acts by binding to its natural ligand OPGL, which is also known as RANKL (receptor activator of NF-kB ligand). This binding prevents OPGL from activating its cognate receptor RANK, which is the osteoclast receptor vital for osteoclast differentiation, activation and survival. Overexpression of OPG in transgenic mice leads to profound osteopetrosis secondary to a near total lack of osteoclasts. Conversely, ablation of the OPG gene causes severe osteoporosis in mice. Ablation of OPGL or RANK also produces profound osteopetrosis, indicating the important physiological role of these proteins in regulating bone resorption. The secretion of OPG and OPGL from osteoblasts and stromal cells is regulated by numerous hormones and cytokines, often in a reciprocal manner. The relative levels of OPG and OPGL production are thought to ultimately dictate the extent of bone resorption. Excess OPGL increases bone resorption, whereas excess OPG inhibits resorption. Recombinant OPG blocks the effects of virtually all factors which stimulate osteoclasts, in vitro and in vivo. OPG also inhibits bone resorption in a variety of animal disease models, including ovariectomy-induced osteoporosis, humoral hypercalcemia of malignancy, and experimental bone metastasis. OPG might represent an effective therapeutic option for diseases associated with excessive osteoclast activity.

Vitamin D status in different populations relies on accurate measurement of total serum 25-hydroxyvitamin D [25(OH)D] concentrations [i.e., 25(OH)D3 and 25(OH)D2]. This study evaluated agreement between the ADVIA Centaur Vitamin D Total assay for 25(OH)D testing (traceable to the NIST-Ghent reference method procedure) and a liquid chromatography tandem mass spectrometry (LC-MS/MS) method for various populations with different levels of vitamin D binding protein (DBP). Total serum 25(OH)D concentrations were measured for 36 pregnant women, 40 hemodialysis patients, and 30 samples (DBP-spiked or not) from healthy subjects. ELISA measured DBP levels. The mean serum DBP concentrations were higher for pregnancy (415 μg/mL) and lower for hemodialysis subjects (198 μg/mL) than for healthy subjects and were highest for spiked serum (545 μg/mL). The average bias between the ADVIA Centaur assay and the LC-MS/MS method was −1.4% (healthy), −6.1% (pregnancy), and 4.4% (hemodialysis). The slightly greater bias for samples from some pregnancy and hemodialysis subjects with serum DBP levels outside of the normal healthy range fell within a clinically acceptable range—reflected by analysis of their low-range (≤136 μg/mL), medium-range (137–559 μg/mL), and high-range (≥560 μg/mL) DBP groups. Thus, the ADVIA Centaur Vitamin D Total assay demonstrates acceptable performance compared with an LC-MS/MS method for populations containing different amounts of DBP.

The graphs in Figure 4(b) appear as duplicates of graphs in Figure 4(a) and are corrected as follows.

Chronic kidney disease-mineral and bone disorder (CKD-MBD) is associated with secondary hyperparathyroidism (HPT) and serum elevations in the phosphaturic hormone FGF23, which may be maladaptive and lead to increased morbidity and mortality. To determine the role of FGF23 in the pathogenesis of CKD-MBD and development of secondary HPT, we developed a monoclonal FGF23 antibody to evaluate the impact of chronic FGF23 neutralization on CKD-MBD, secondary HPT, and associated comorbidities in a rat model of CKD-MBD. CKD-MBD rats fed a high-phosphate diet were treated with low or high doses of FGF23-Ab or an isotype control antibody. Neutralization of FGF23 led to sustained reductions in secondary HPT, including decreased parathyroid hormone, increased vitamin D, increased serum calcium, and normalization of bone markers such as cancellous bone volume, trabecular number, osteoblast surface, osteoid surface, and bone-formation rate. In addition, we observed dose-dependent increases in serum phosphate and aortic calcification associated with increased risk of mortality in CKD-MBD rats treated with FGF23-Ab. Thus, mineral disturbances caused by neutralization of FGF23 limited the efficacy of FGF23-Ab and likely contributed to the increased mortality observed in this CKD-MBD rat model.

Chronic kidney disease (CKD) is a worldwide health problem. Serum levels of FGF23, a phosphaturic hormone, increase at the earliest stages of CKD and have been found to be independently associated with the mortality and morbidity of CKD patients. The purpose of this study was to evaluate whether FGF23 neutralization was able to improve bone quality and osseointegration of titanium implants. Uremia was induced by 5/6 nephrectomy in adult female mice. Postsurgery, the mice were injected with vehicle or FGF23 neutralizing antibody (5 mg/kg body weight) 3 times a week. Experimental titanium implants were inserted in the distal end of the femurs. FGF23 neutralization significantly increased serum phosphate, 1,25(OH) 2 D and BUN and decreased serum PTH and FGF23, relative to vehicle-treated CKD mice. Histomorphometric analysis of the tibiae indicated that FGF23 neutralization normalized the osteoidosis observed in vehicle-treated CKD mice. Although bone-implant contact ratio remained unchanged by anti-FGF23 antibody treatment, the strength of osseointegration, as evidenced by a biomechanical push-in test, was significantly improved by FGF23 neutralization. Our findings revealed that FGF23 neutralization effectively improves bone quality and osseointegration of titanium implants in CKD mice, suggesting FGF23 as a key factor of CKD related bone diseases.

Osteocalcin, a bone-specific protein and marker of the mature osteoblast, is expressed only in nonproliferating osteoblasts in a mineralizing extracellular matrix, while type I collagen is expressed in proliferating cells. The nuclear proteins encoded by the c-fos and c-jun protooncogenes are expressed during the proliferation period of osteoblast phenotype development. We present evidence that AP-1 (HeLa cell-activating protein 1) sites residing within two promoter elements of the osteocalcin gene bind the Fos-Jun protein complex: the osteocalcin box (OC box; nucleotides -99 to -76), which contains a CCAAT motif as a central element and influences tissue-specific basal levels of osteocalcin gene transcription, and the vitamin D-responsive element (VDRE; nucleotides -462 to -440), which mediates enhancement of osteocalcin gene transcription. Gel electrophoretic mobility-shift analysis demonstrated high AP-1 binding activity in proliferating osteoblasts and dramatic changes in this activity after the down-regulation of proliferation and the initiation of extracellular-matrix mineralization in primary cultures of normal diploid osteoblasts. Methylation interference analysis established at single nucleotide resolution that purified recombinant Fos and Jun proteins bind in a sequence-specific manner to the AP-1 sites within the VDRE and OC box. Similarly, an AP-1 motif within a putative VDRE of the alkaline phosphatase gene, which is also expressed after the completion of proliferation, binds the Fos-Jun complex. These results support a model in which coordinate occupancy of the AP-1 sites in the VDRE and OC box in proliferating osteoblasts may suppress both basal level and vitamin D-enhanced osteocalcin gene transcription as well as transcription of other genes associated with osteoblast differentiation--a phenomenon we describe as phenotype suppression. This model is further supported by binding of the Fos-Jun complex at an AP-1 site in the type alpha I collagen promoter that is contiguous with, but not overlapping, the VDRE. Such a sequence organization in the collagen VDRE motif is compatible with vitamin D modulation of collagen but not with osteocalcin and alkaline phosphatase expression in proliferating osteoblasts.

In the osteocalcin (OC) gene promoter, both independent positive and negative regulatory elements, as well as others with contiguous [kTATA/glucocorticoid-responsive elements (GRE)] or overlapping [TATA/GRE, vitamin D-responsive enhancer elements (VDRE)/AP-1, and OC box/AP-1] domains, are sites for modifications in protein-DNA interactions. In the present studies, we have examined nuclear protein extracts from fetal rat calvarial cells that undergo a developmental sequence of bone cell differentiation. Our results demonstrate modifications in protein-DNA interactions that relate to the developmental stages of the osteoblast and support developmental regulation of OC gene transcription. Basal expression of the OC gene is associated with sequence-specific protein-DNA interactions at the OC box, VDRE, and TATA/GRE box. Distinct differences are observed in proliferating osteoblasts, where the OC gene is not transcribed compared to postproliferative, differentiated osteoblasts that transcribe the OC gene. Furthermore, the protein-DNA complexes that reflect hormonal control are also developmentally regulated, mediating both the transcriptionally active and repressed states of the OC gene. For example, in proliferating osteoblasts, a vitamin D receptor-antibody-sensitive complex is formed that is different from the DNA binding complex induced by vitamin D postproliferatively when the OC gene is transcribed. Mutational analysis of the steroid hormone binding domain and the overlapping AP-1 site at the VDRE supports mutually exclusive occupancy by Fos-Jun heterodimers and vitamin D receptor. Such protein-DNA interactions at the VDRE are consistent with repression of competency for vitamin D-mediated transcriptional enhancement in proliferating osteoblasts expressing high levels of Fos and Jun.