Extracellular sodium regulates fibroblast growth factor 23 (FGF23) formation.

The bone-derived hormone fibroblast growth factor-23 (FGF23) has recently received much attention due to its association with chronic kidney disease and cardiovascular disease progression. Extracellular sodium concentration ([Na+]) plays a significant role in bone metabolism. Hyponatremia (lower serum [Na+]) has recently been shown to be independently associated with FGF23 levels in patients with chronic systolic heart failure. However, nothing is known about the direct impact of [Na+] on FGF23 production. Here, we show that an elevated [Na+] (+20 mM) suppressed FGF23 formation, whereas low [Na+] (-20 mM) increased FGF23 synthesis in the osteoblast-like cell lines UMR-106 and MC3T3-E1. Similar bidirectional changes in FGF23 abundance were observed when osmolality was altered by mannitol but not by urea, suggesting a role of tonicity in FGF23 formation. Moreover, these changes in FGF23 were inversely proportional to the expression of NFAT5 (nuclear factor of activated T cells-5), a transcription factor responsible for tonicity-mediated cellular adaptations. Furthermore, arginine vasopressin, which is often responsible for hyponatremia, did not affect FGF23 production. Next, we performed a comprehensive and unbiased RNA-seq analysis of UMR-106 cells exposed to low versus high [Na+], which revealed several novel genes involved in cellular adaptation to altered tonicity. Additional analysis of cells with Crisp-Cas9-mediated NFAT5 deletion indicated that NFAT5 controls numerous genes associated with FGF23 synthesis, thereby confirming its role in [Na+]-mediated FGF23 regulation. In line with these in vitro observations, we found that hyponatremia patients have higher FGF23 levels. Our results suggest that [Na+] is a critical regulator of FGF23 synthesis.

FGF23 promotes proliferation, migration and invasion by regulating miR-340-5p in osteosarcoma.

BACKGROUND: Increasing evidences have been indicated that FGF23 is associated with the biological behavior of malignant tumors, but its role in osteosarcoma and the specific mechanism need to be elucidated. The purpose of this study is to investigate the effects of FGF23 on the proliferation, migration and invasion of osteosarcoma cells, and the possible molecular mechanisms. METHODS: Western blot was used to detect differences in FGF23 expression in osteosarcoma cells MG-63 and U2-OS and osteoblasts hFOB1.19. FGF23-overexpressing adenoviruses and FGF-silencing plasmids were transfected into osteosarcoma cells, and transfection efficiency was verified using Western blot. MTT and colony formation assays were performed to detect osteosarcoma cell proliferation. Cell cycle was measured by flow cytometry. Scratch assay, holographic imaging cell analyzer Holomonitor ® M4 and transwell were applied to detect cell migration and invasion. Dual-luciferase reporter assay was performed to validate the interaction between FGF23 and miR-340-5p. Changes in miR-340-5p mRNA levels were measured by QRT-PCR. RESULTS: FGF23 is highly expressed in osteosarcoma cells compared to hFOB1.19. Overexpression of FGF23 significantly promoted the proliferation, migration and invasion of MG-63 and U2-OS cells. MiR-340-5p is a target of FGF23. Transfection of miR-340-5p mimics reversed the promoting effects of FGF23 on proliferation, migration and invasion of MG-63 and U2-OS cells. CONCLUSION: FGF23 promotes osteosarcoma cell proliferation, migration and invasion by targeting miR-340-5p gene expression.

Association of Genetically Predicted Fibroblast Growth Factor-23 with Heart Failure: A Mendelian Randomization Study.

BACKGROUND AND OBJECTIVES: Elevated fibroblast growth factor-23 (FGF23) has been consistently associated with heart failure, particularly heart failure with preserved ejection fraction, among patients with CKD and in the general population. FGF23 may directly induce cardiac remodeling and heart failure. However, biases affecting observational studies impede robust causal inferences. Mendelian randomization leverages genetic determinants of a risk factor to examine causality. We performed a two-sample Mendelian randomization to assess causal associations between FGF23 and heart failure. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: Genetic instruments were genome-wide significant genetic variants associated with FGF23, including variants near PIP5K1B, RGS14, LINC01229, and CYP24A1. We analyzed data from the Heart Failure Molecular Epidemiology for Therapeutic Targets and BioVU biobanks to examine associations of the four variants with overall heart failure, heart failure with preserved ejection fraction, and heart failure with reduced and mid-range ejection fraction. We developed an eGFR polygenic risk score using summary statistics from the Chronic Kidney Disease Genetics Consortium (CKDGen) genome-wide association study of eGFR in >1 million individuals and performed stratified analyses across eGFR polygenic risk score strata. RESULTS: Genetically determined FGF23 was not associated with overall heart failure in the Heart Failure Molecular Epidemiology for Therapeutic Targets consortium (odds ratio, 1.13; 95% confidence interval, 0.89 to 1.42 per unit higher genetically predicted log FGF23) and the full BioVU sample (odds ratio, 1.32; 95% confidence interval, 0.95 to 1.84). In stratified analyses in BioVU, higher FGF23 was associated with overall heart failure (odds ratio, 3.09; 95% confidence interval, 1.38 to 6.91) among individuals with low eGFR-polygenic risk score (<1 SD below the mean), but not those with high eGFR-polygenic risk score (P interaction = 0.02). Higher FGF23 was also associated with heart failure with preserved ejection fraction among all BioVU participants (odds ratio, 1.47; 95% confidence interval, 1.01 to 2.14) and individuals with low eGFR-polygenic risk score (odds ratio, 7.20; 95% confidence interval, 2.80 to 18.49), but not those high eGFR-polygenic risk score (P interaction = 2.25 × 10-4). No significant associations were observed with heart failure with reduced and midrange ejection fraction. CONCLUSION: We found no association between genetically predicted FGF23 and heart failure in the Heart Failure Molecular Epidemiology for Therapeutic Targets consortium. In BioVU, genetically elevated FGF23 was associated with higher heart failure risk, specifically heart failure with preserved ejection fraction, particularly among individuals with low genetically predicted eGFR. PODCAST: This article contains a podcast at https://www.asn-online.org/media/podcast/CJASN/2022_07_28_CJN00960122.mp3.

FGF23-Klotho Axis and Fractures in Patients Without and With Early CKD: A Case-Cohort Analysis of CARTaGENE.

CONTEXT: Whether fibroblast growth factor-23 (FGF23) and α-Klotho are associated with fractures, especially in chronic kidney disease (CKD), remains controversial. OBJECTIVE: We evaluated how FGF23, α-Klotho, and traditional mineral parameters predict fractures in individuals with and without early CKD. METHODS: We conducted a stratified case-cohort analysis using CARTaGENE, a population-based survey from Quebec, Canada. Individuals aged 40 to 69 years were selected according to outcome and CKD status (non-CKD: eGFR > 60 mL/min/1.73 m2; CKD stage 3: eGFR 30-60 mL/min/1.73 m2]). Baseline levels of c-terminal FGF23 (cFGF23), α-Klotho, parathyroid hormone (PTH), phosphate, and calcium were analyzed for associations with osteoporotic fracture incidence from recruitment (2009-2010) through March 2016. Adjusted Cox models were used, and predictors were treated linearly or flexibly using splines. RESULTS: A total of 312 patients (159 non-CKD; 153 CKD) were included; 98 had ≥ 1 fracture at any site during a median follow up of 70 months. Compared with non-CKD, CKD patients had increased levels of cFGF23 but similar levels of α-Klotho. cFGF23 was linearly associated with increased fracture incidence (adjusted HR = 1.81 [1.71, 1.93] per doubling for all participants). The association of α-Klotho with fracture followed a U-curve (overall P = 0.019) but was attenuated by adjustment for potential mediators (bone mineral density, phosphate, PTH). PTH and phosphate also had U-shaped associations with fracture. Associations were mostly similar between non-CKD and CKD. Adjustment for cFGF23 strongly attenuated the association between CKD status and fractures. CONCLUSION: cFGF23 is associated linearly with fracture incidence while α-Klotho, PTH, and phosphate levels have a U-shaped association.

The effect of vitamin D supplementation on serum levels of fibroblast growth factor- 23: A systematic review and meta-analysis of randomized controlled trials.

Previous studies of the effect of vtamin D on serum levels of fibroblast growth factor- 23 (FGF-23) have yeilded an inconsistent findings. This systematic review and meta-analysis of randomized controlled trials (RCTs) sought to investigate the effect of vitamin D supplementation on serum levels of FGF-23. PubMed, Scopus, ISI Web of Science, and the Cochrane Library were searched, from database inception to November 2020, for RCTs that evaluated the effects of native or active vitamin D supplementation on serum levels of FGF-23 in adults. Weighted mean difference (WMD) were calculated and random effects meta-analysis was used to estimate the overall effects. Twenty-seven trials were included in the meta-analysis. Supplementation with native vitamin D (23 studies, n = 2247 participants; weighted mean difference [WMD] = 0.5 pg/mL, 95 % CI: -0.52 to 1.51, P = 0.33; I2 = 29.9 %), and active vitamin D (5 studies, n = 342 participants, WMD = 29.45 pg/mL, 95 % CI: -3.9 to 62.81, P = 0.08; I2 = 99.3%) had no significant effects on serum FGF-23 concentration. In subgroup analyses, supplementation with ergocalciferol (3 studies, n = 205 participants; WMD = 18.27 pg/mL, 95 % CI: 5.36-31.17, P = 0.006), and daily dosing regimens (9 studies, n = 1374 participants; WMD = 0.41 pg/mL, 95 % CI: 0.22 to 0.59, P < 0.001) increased serum FGF-23 levels compared to control. Overall, our findings revealed no significan effect of vitamin D supplementation on serum FGF-23 concentration. However, further high quality, large-scale studies are needed to better elucidate this relationship.

Cardiovascular remodeling as a result of fibroblast growth factor-23 (FGF-23)/Klotho imbalance in patients with CKD.

BACKGROUND: In chronic kidney disease (CKD) cardiovascular remodeling (CVR) is very frequent compared with general population and, as suppose, may be associated with «new¼ renal risk factors. The aim of study was to estimate association of new serum biomarkers (FGF-23, Klotho) and traditional biomarker of cardiac damage-serum Troponin I (sTr-I) with signs of CVR. METHODS: One hundred thirty CKD G1-5D patients without cardiovascular disease (CVD) clinical manifestation were included. We measured serum FGF-23, Klotho and sTr-I. The instrumental methods were: echocardiography, SphygmoCor test [Pulse Wave Velocity (PWV), Central (aortic) Blood Pressure (CBP), Subendocardial Blood Supply (SBS)]. RESULTS: FGF-23 level correlated with: sTr-I (r = 0.512; p < 0.01), eccentric left ventricular hypertrophy, LVH (r = 0.543; p < 0.01), SBS (r = - 0.499; p < 0.05). There were no differences of FGF-23 level in patients with normal and high CBP. Klotho correlated with concentric LVH (r = - 0.451; p < 0.01), PWV (r = - 0.667; p < 0.001), Cardiac Calcification Score, CCS (r = - 0.581; p < 0.01). Multivariate analysis revealed positive independent association of FGF-23 with eccentric LVH (OR = 1.036, 95% CI (1.004-1.068); p = 0.038). Klotho was a negative determinant for concentric LVH (OR = 0.990, 95% CI 0.987-0.994; p < 0.001), increased PWV (OR = 0.984, 95% CI (0.977-0.991); p < 0.001) and CCS (OR = 0.991, 95% CI (0.988-0.995); p < 0.001). In addition, multivariate analysis revealed a relationship between serum Klotho (OR = 0.980, 95% CI (0.964-0.996); p = 0.016), FGF-23 (OR = 3.145, 95% CI (1.020-9.695); p = 0.046) and troponin I level. CONCLUSION: In CKD patients without CVD clinical manifestation increased serum FGF-23 level and decreased Klotho are associated with CVR: FGF-23 with eccentric LVH (independently of CBP), Klotho determinate concentric LVH, PWV and CCS. Moderately elevated sTr-I levels may be a manifestation of FGF-23/Klotho imbalance in CKD.

The relationship between FGF23 and anemia in HD and renal transplant patients.

PURPOSE: Recent studies claim that FGF23 is also associated with anemia and inflammation. In this study, the relationship between FGF23 and anemia in hemodialysis (HD) and renal transplantation patients (RTx patients) patients was investigated. METHODS: This was a cross-sectional study involving 40 RTx patients (13 females, 27 males; mean age, 45.93 ± 12.49 years) who had transplantation at least 6 months before, 25 HD patients (12 females, 13 males; mean age, 54.72 ± 15.5 years), and 20 healthy control subjects (13 females, 7 males; mean age, 36.7 ± 9.38 years). FGF23 was studied using Elisa method. Parameters such as iron, ferritin, total iron binding capacity, and transferrin saturation were assessed. RESULTS: FGF23 level was significantly higher in HD patients when compared with the RTx patients and control groups. In the bivariate correlation analysis, hemoglobin was positively correlated with albumin (r = 0.681, p = 0.000), ferritin (r = 0.446, p = 0.043), and negatively correlated with CRP (r = - 0.476, p = 0.016) and FGF23 (r = 0.493, p = 0.043). FGF23 was found to be an independent predictor of decreased hemoglobin in HD patients. In addition, this association was observed to disappear after transplantation. CONCLUSION: While FGF23 is closely related to hemoglobin levels in HD patients, we have shown that this relationship disappears after transplantation.

The Successful Treatment of Deep Soft-tissue Calcifications with Topical Sodium Thiosulphate and Acetazolamide in a Boy with Hyperphosphatemic Familial Tumoral Calcinosis due to a Novel Mutation in FGF23

Hyperphosphatemic familial tumoral calcinosis (HFTC) is a rare autosomal recessive disorder. Topical sodium thiosulfate (STS) and acetazolamide can be a safe and effective treatment for patients who do not respond to conventional therapy for ectopic calcifications. We report the successful treatment of deep soft-tissue calcifications with topical STS and acetazolamide in a boy diagnosed with HFTC due to a novel homozygous mutation of FGF23.

Urinary Potassium Excretion, Fibroblast Growth Factor 23, and Incident Hypertension in the General Population-Based PREVEND Cohort.

High plasma fibroblast growth factor 23 (FGF23) and low potassium intake have each been associated with incident hypertension. We recently demonstrated that potassium supplementation reduces FGF23 levels in pre-hypertensive individuals. The aim of the current study was to address whether 24-h urinary potassium excretion, reflecting dietary potassium intake, is associated with FGF23, and whether FGF23 mediates the association between urinary potassium excretion and incident hypertension in the general population. At baseline, 4194 community-dwelling individuals without hypertension were included. Mean urinary potassium excretion was 76 (23) mmol/24 h in men, and 64 (20) mmol/24 h in women. Plasma C-terminal FGF23 was 64.5 (54.2-77.8) RU/mL in men, and 70.3 (56.5-89.5) RU/mL in women. Urinary potassium excretion was inversely associated with FGF23, independent of age, sex, urinary sodium excretion, bone and mineral parameters, inflammation, and iron status (St. ß -0.02, p < 0.05). The lowest sex-specific urinary potassium excretion tertile (HR 1.18 (95% CI 1.01-1.37)), and the highest sex-specific tertile of FGF23 (HR 1.17 (95% CI 1.01-1.37)) were each associated with incident hypertension, compared with the reference tertile. FGF23 did not mediate the association between urinary potassium excretion and incident hypertension. Increasing potassium intake, and reducing plasma FGF23 could be independent targets to reduce the risk of hypertension in the general population.

Bone marrow sinusoidal endothelium controls terminal erythroid differentiation and reticulocyte maturation.

Within the bone marrow microenvironment, endothelial cells (EC) exert important functions. Arterial EC support hematopoiesis while H-type capillaries induce bone formation. Here, we show that BM sinusoidal EC (BM-SEC) actively control erythropoiesis. Mice with stabilized ß-catenin in BM-SEC (Ctnnb1OE-SEC) generated by using a BM-SEC-restricted Cre mouse line (Stab2-iCreF3) develop fatal anemia. While activation of Wnt-signaling in BM-SEC causes an increase in erythroblast subsets (PII-PIV), mature erythroid cells (PV) are reduced indicating impairment of terminal erythroid differentiation/reticulocyte maturation. Transplantation of Ctnnb1OE-SEC hematopoietic stem cells into wildtype recipients confirms lethal anemia to be caused by cell-extrinsic, endothelial-mediated effects. Ctnnb1OE-SEC BM-SEC reveal aberrant sinusoidal differentiation with altered EC gene expression and perisinusoidal ECM deposition and angiocrine dysregulation with de novo endothelial expression of FGF23 and DKK2, elevated in anemia and involved in vascular stabilization, respectively. Our study demonstrates that BM-SEC play an important role in the bone marrow microenvironment in health and disease.

FGF23 and Hypophosphatemic Rickets/Osteomalacia.

PURPOSE OF REVIEW: X-linked hypophosphatemia and tumor-induced osteomalacia are diseases characterized by hypophosphatemia with impaired proximal tubular phosphate reabsorption. Complete resection of responsible tumors is the first-line therapy for patients with tumor-induced osteomalacia. In contrast, phosphate and active vitamin D have been used for patients with X-linked hypophosphatemia and inoperable ones with tumor-induced osteomalacia. The purpose of this review is to summarize the pathogenesis of these diseases and discuss about the new treatment. RECENT FINDINGS: Excessive FGF23 production has been shown to underline several kinds of hypophosphatemic rickets/osteomalacia including X-linked hypophosphatemia and tumor-induced osteomalacia. Burosumab, an anti-FGF23 monoclonal antibody, was approved for clinical use, while the indications of burosumab are different depending on countries. The inhibition of excessive FGF23 activity has been approved as a new therapy for several kinds of hypophosphatemic diseases. Further studies are necessary to clarify the long-term effects and safety of burosumab.

Inhibitory effect of Astragalus Membranaceus on osteoporosis in SAMP6 mice by regulating vitaminD/FGF23/Klotho signaling pathway.

Spontaneous senile osteoporosis severely threatens the health of the senior population which has emerged as a severe issue for society. A SAMP6 mouse model was utilized to estimate the impact of intragastrically administered Astragalus Membranaceus (AR) on spontaneous senile osteoporosis. Bone mineral density (BMD) and bone microstructure were measured using Micro-CT; contents of calcium and phosphorus were determined with the colorimetric method; and gene and protein expressions of fibroblast growth factor 23 (FGF23), Klotho, Vitamin D receptor (VDR), CYP27B1 and CYP24A1 were detected using qPCR, Western blot and ELISA assays, respectively. The findings indicated that AR could improve the femoral BMD and bone microstructure, elevate the contents of calcium and phosphorus, and increase the expression of Klotho, VDR, and CYP27B1 whereas decreasing the expression of FGF23 and CYP24A1 in SAMP6 mice in a dose independent manner. The present study has demonstrated that AR can promote osteogenesis and alleviate osteoporosis. It is also expected to provide a new insight for the treatment of spontaneous senile osteoporosis and to serve as a research basis for AR application.

In Vivo Analysis of the Contribution of Proprotein Convertases to the Processing of FGF23.

Fibroblast growth factor 23 (FGF23) is a hormone secreted from fully differentiated osteoblasts and osteocytes that inhibits phosphate reabsorption by kidney proximal tubules. The full-length (i.e., intact) protein mediates FGF23 endocrine functions, while endoproteolytic cleavage at a consensus cleavage sequence for the proprotein convertases (PCs) inactivates FGF23. Two PCs, furin and PC5, were shown to cleave FGF23 in vitro at RHTR179↓, but whether they are fulfilling this function in vivo is currently unknown. To address this question, we used here mice lacking either or both furin and PC5 in cell-specific manners and mice lacking the paired basic amino acid-cleaving enzyme 4 (PACE4) in all cells. Our analysis shows that furin inactivation in osteoblasts and osteocytes results in a 25% increase in circulating intact FGF23, without any significant impact on serum phosphate levels, whether mice are maintained on a normal or a low phosphate diet. Under conditions of iron deficiency, FGF23 is normally processed in control mice, but its processing is impaired in mice lacking furin in osteoblasts and osteocytes. In contrast, FGF23 is normally cleaved following erythropoietin or IL-1ß injections in mice lacking furin or both furin and PC5, and in PACE4-deficient mice. Altogether, these studies suggest that furin is only partially responsible for FGF23 cleavage under certain conditions in vivo. The processing of FGF23 may therefore involve the redundant action of multiple PCs or of other peptidases in osteoblasts, osteocytes and hematopoietic cells.

Myostatin regulates the production of fibroblast growth factor 23 (FGF23) in UMR106 osteoblast-like cells.

Myostatin is a signaling molecule produced by skeletal muscle cells (myokine) that inhibits muscle hypertrophy and has further paracrine and endocrine effects in other organs including bone. Myostatin binds to activin receptor type 2B which forms a complex with transforming growth factor-ß type I receptor (TGF-ßRI) and induces intracellular p38MAPK and NFκB signaling. Fibroblast growth factor 23 (FGF23) is a paracrine and endocrine mediator produced by bone cells and regulates phosphate and vitamin D metabolism in the kidney. P38MAPK and NFκB-dependent store-operated Ca2+ entry (SOCE) are positive regulators of FGF23 production. Here, we explored whether myostatin influences the synthesis of FGF23. Fgf23 gene expression was determined by qRT-PCR and FGF23 protein by ELISA in UMR106 osteoblast-like cells. UMR106 cells expressed activin receptor type 2A and B. Myostatin upregulated Fgf23 gene expression and protein production. The myostatin effect on Fgf23 was significantly attenuated by TGF-ßRI inhibitor SB431542, p38MAPK inhibitor SB202190, and NFκB inhibitor withaferin A. Moreover, SOCE inhibitor 2-APB blunted the myostatin effect on Fgf23. Taken together, myostatin is a stimulator of Fgf23 expression in UMR106 cells, an effect at least partially mediated by downstream TGF-ßRI/p38MAPK signaling as well as NFκB-dependent SOCE.

Orphan nuclear receptor ERR-γ regulates hepatic FGF23 production in acute kidney injury.

Fibroblast growth factor 23 (FGF23), a hormone generally derived from bone, is important in phosphate and vitamin D homeostasis. In acute kidney injury (AKI) patients, high-circulating FGF23 levels are associated with disease progression and mortality. However, the organ and cell type of FGF23 production in AKI and the molecular mechanism of its excessive production are still unidentified. For insight, we investigated folic acid (FA)-induced AKI in mice. Interestingly, simultaneous with FGF23, orphan nuclear receptor ERR-γ expression is increased in the liver of FA-treated mice, and ectopic overexpression of ERR-γ was sufficient to induce hepatic FGF23 production. In patients and in mice, AKI is accompanied by up-regulated systemic IL-6, which was previously identified as an upstream regulator of ERR-γ expression in the liver. Administration of IL-6 neutralizing antibody to FA-treated mice or of recombinant IL-6 to healthy mice confirms IL-6 as an upstream regulator of hepatic ERR-γ-mediated FGF23 production. A significant (P < 0.001) interconnection between high IL-6 and FGF23 levels as a predictor of AKI in patients that underwent cardiac surgery was also found, suggesting the clinical relevance of the finding. Finally, liver-specific depletion of ERR-γ or treatment with an inverse ERR-γ agonist decreased hepatic FGF23 expression and plasma FGF23 levels in mice with FA-induced AKI. Thus, inverse agonist of ERR-γ may represent a therapeutic strategy to reduce adverse plasma FGF23 levels in AKI.

Upstream Regulators of Fibroblast Growth Factor 23.

Fibroblast growth factor 23 (FGF23) has been described as an important regulator of mineral homeostasis, but has lately also been linked to iron deficiency, inflammation, and erythropoiesis. FGF23 is essential for the maintenance of phosphate homeostasis in the body and activating mutations in the gene itself or inactivating mutations in its upstream regulators can result in severe chronic hypophosphatemia, where an unbalanced mineral homeostasis often leads to rickets in children and osteomalacia in adults. FGF23 can be regulated by changes in transcriptional activity or by changes at the post-translational level. The balance between O-glycosylation and phosphorylation is an important determinant of how much active intact or inactive cleaved FGF23 will be released in the circulation. In the past years, it has become evident that iron deficiency and inflammation regulate FGF23 in a way that is not associated with its classical role in mineral metabolism. These conditions will not only result in an upregulation of FGF23 transcription, but also in increased cleavage, leaving the levels of active intact FGF23 unchanged. The exact mechanisms behind and function of this process are still unclear. However, a deeper understanding of FGF23 regulation in both the classical and non-classical way is important to develop better treatment options for diseases associated with disturbed FGF23 biology. In this review, we describe how the currently known upstream regulators of FGF23 change FGF23 transcription and affect its post-translational modifications at the molecular level.

Ap2s1 mutation causes hypercalcaemia in mice and impairs interaction between calcium-sensing receptor and adaptor protein-2.

Adaptor protein 2 (AP2), a heterotetrameric complex comprising AP2α, AP2ß2, AP2µ2 and AP2σ2 subunits, is ubiquitously expressed and involved in endocytosis and trafficking of membrane proteins, such as the calcium-sensing receptor (CaSR), a G-protein coupled receptor that signals via Gα11. Mutations of CaSR, Gα11 and AP2σ2, encoded by AP2S1, cause familial hypocalciuric hypercalcaemia types 1-3 (FHH1-3), respectively. FHH3 patients have heterozygous AP2S1 missense Arg15 mutations (p.Arg15Cys, p.Arg15His or p.Arg15Leu) with hypercalcaemia, which may be marked and symptomatic, and occasional hypophosphataemia and osteomalacia. To further characterize the phenotypic spectrum and calcitropic pathophysiology of FHH3, we used CRISPR/Cas9 genome editing to generate mice harboring the AP2S1 p.Arg15Leu mutation, which causes the most severe FHH3 phenotype. Heterozygous (Ap2s1+/L15) mice were viable, and had marked hypercalcaemia, hypermagnesaemia, hypophosphataemia, and increases in alkaline phosphatase activity and fibroblast growth factor-23. Plasma 1,25-dihydroxyvitamin D was normal, and no alterations in bone mineral density or bone turnover were noted. Homozygous (Ap2s1L15/L15) mice invariably died perinatally. Co-immunoprecipitation studies showed that the AP2S1 p.Arg15Leu mutation impaired protein-protein interactions between AP2σ2 and the other AP2 subunits, and also with the CaSR. Cinacalcet, a CaSR positive allosteric modulator, decreased plasma calcium and parathyroid hormone concentrations in Ap2s1+/L15 mice, but had no effect on the diminished AP2σ2-CaSR interaction in vitro. Thus, our studies have established a mouse model that is representative for FHH3 in humans, and demonstrated that the AP2S1 p.Arg15Leu mutation causes a predominantly calcitropic phenotype, which can be ameliorated by treatment with cinacalcet.

The relationship of indoxyl sulfate and p-cresyl sulfate with target cardiovascular proteins in hemodialysis patients.

Protein-bound uremic toxins (Indoxyl sulfate [IS] and p-cresyl sulfate [PCS]) are both associated with cardiovascular (CV) and all-cause mortality in subjects with chronic kidney disease (CKD). Possible mechanisms have not been elucidated. In hemodialysis patients, we investigated the relationship between the free form of IS and PCS and 181 CV-related proteins. First, IS or PCS concentrations were checked, and high levels were associated with an increased risk of acute coronary syndrome (ACS) in 333 stable HD patients. CV proteins were further quantified by a proximity extension assay. We examined associations between the free form protein-bound uremic toxins and the quantified proteins with correction for multiple testing in the discovery process. In the second step, the independent association was evaluated by multivariable-adjusted models. We rank the CV proteins related to protein-bound uremic toxins by bootstrapped confidence intervals and ascending p-value. Six proteins (signaling lymphocytic activation molecule family member 5, complement component C1q receptor, C-C motif chemokine 15 [CCL15], bleomycin hydrolase, perlecan, and cluster of differentiation 166 antigen) were negatively associated with IS. Fibroblast growth factor 23 [FGF23] was the only CV protein positively associated with IS. Three proteins (complement component C1q receptor, CCL15, and interleukin-1 receptor-like 2) were negatively associated with PCS. Similar findings were obtained after adjusting for classical CV risk factors. However, only higher levels of FGF23 was related to increased risk of ACS. In conclusion, IS and PCS were associated with several CV-related proteins involved in endothelial barrier function, complement system, cell adhesion, phosphate homeostasis, and inflammation. Multiplex proteomics seems to be a promising way to discover novel pathophysiology of the uremic toxin.

Up-Regulation of Fibroblast Growth Factor 23 Gene Expression in UMR106 Osteoblast-like Cells with Reduced Viability.

Fibroblast growth factor 23 (FGF23) controls vitamin D and phosphate homeostasis in the kidney and has additional paracrine effects elsewhere. As a biomarker, its plasma concentration is associated with progression of inflammatory, renal, and cardiovascular diseases. Major stimuli of FGF23 synthesis include active vitamin D and inflammation. Antineoplastic chemotherapy treats cancer by inducing cellular damage ultimately favoring cell death (apoptosis and necrosis) and causing inflammation. Our study explored whether chemotherapeutics and other apoptosis inducers impact on Fgf23 expression. Experiments were performed in osteoblast-like UMR106 cells, Fgf23 gene expression and protein synthesis were determined by qRT-PCR and ELISA, respectively. Viability was assessed by MTT assay and NFκB activity by Western Blotting. Antineoplastic drugs cisplatin and doxorubicin as well as apoptosis inducers procaspase-activating compound 1 (PAC-1), a caspase 3 activator, and serum depletion up-regulated Fgf23 transcripts while reducing cell proliferation and viability. The effect of cisplatin on Fgf23 transcription was paralleled by Il-6 up-regulation and NFκB activation and attenuated by Il-6 and NFκB signaling inhibitors. To conclude, cell viability-decreasing chemotherapeutics as well as apoptosis stimulants PAC-1 and serum depletion up-regulate Fgf23 gene expression. At least in part, Il-6 and NFκB may contribute to this effect.