Total Fibroblast Growth Factor 23 Research Articles

Gα11 deficiency increases fibroblast growth factor 23 levels in a mouse model of familial hypocalciuric hypercalcemia.

Fibroblast growth factor 23 (FGF23) production has recently been shown to increase downstream of Gαq/11-PKC signaling in osteocytes. Inactivating mutations in the gene encoding Gα11 (GNA11) cause familial hypocalciuric hypercalcemia (FHH) due to impaired calcium-sensing receptor signaling. We explored the effect of Gα11 deficiency on FGF23 production in mice with heterozygous (Gna11+/-) or homozygous (Gna11-/-) ablation of Gna11. Both Gna11+/- and Gna11-/- mice demonstrated hypercalcemia and mildly raised parathyroid hormone levels, consistent with FHH. Strikingly, these mice also displayed increased serum levels of total and intact FGF23 and hypophosphatemia. Gna11-/- mice showed augmented Fgf23 mRNA levels in the liver and heart, but not in bone or bone marrow, and also showed evidence of systemic inflammation with elevated serum IL-1β levels. Furin gene expression was significantly increased in the Gna11-/- liver, suggesting enhanced FGF23 cleavage despite the observed rise in circulating intact FGF23 levels. Gna11-/- mice had normal renal function and reduced serum levels of glycerol-3-phosphate, excluding kidney injury as the primary cause of elevated intact FGF23 levels. Thus, Gα11 ablation caused systemic inflammation and excess serum FGF23 in mice, suggesting that patients with FHH - at least those with GNA11 mutations - may be at risk for these complications.

Bone Marrow Sinusoidal Endothelial Cells Are a Site of Fgf23 Upregulation in Murine ß-Thalassemia

Fibroblast growth factor-23 (FGF23) is a bone-derived hormonal regulator of mineral metabolism with emerging functions in the bone marrow. FGF23 has been reported to negatively regulate erythropoiesis [PMID:24509850] and promote hematopoietic progenitor mobilization from the bone marrow [PMID:33512467]. Elevated FGF23 levels in the circulation have been described in iron-deficient subjects and also in subjects with the iron-loading anemia β-thalassemia. Recently, we reported bone marrow sinusoidal endothelial cells (BM-SEC) as a novel site of Fgf23 upregulation in mice with chronic iron-deficiency anemia and acute, phlebotomy-induced anemia [PMID:37417950]. We and others have detected Fgf23 mRNA elevation in total bone marrow of Hbb th3/+ mice, a model of non-transfusion dependent β-thalassemia that exhibits systemic iron loading. In the present study, we sought to clarify sites of Fgf23 promoter activity in Hbb th3/+ bone marrow using a reporter allele in which the enhanced green fluorescent protein (eGFP) coding sequence is knocked into the endogenous Fgf23 locus ( Fgf23 eGFP). We generated Hbb +/+ and Hbb th3/+ female littermates that carried either one ( Fgf23 +/eGFP) or zero ( Fgf23 +/+) copies of the Fgf23 reporter allele, which were raised on a standard rodent diet (200 mg/kg iron, 1.0% calcium, 0.7% phosphorus). Fgf23 eGFP/eGFP homozygotes were not generated due to known early growth retardation and mortality in Fgf23 null mice. After blood and tissue collection at 8 weeks of age, phenotypes of the 4 Hbb-Fgf23 genotype combinations were compared by two-way ANOVA. Compared to mice with intact Hbb and Fgf23 alleles ( Hbb +/+ Fgf23 +/+), Hbb th3/+ Fgf23 +/+ mice showed markedly elevated plasma levels of “total” FGF23 (sum of intact, active hormone and C-terminal cleaved fragments). Plasma total FGF23 levels in Hbb th3/+ Fgf23 +/+ and Hbb th3/+ Fgf23 +/eGFP mice were similar, indicating utility of the latter genotype for interrogating sites of increased Fgf23 expression in murine b-thalassemia. We therefore used anti-GFP immunohistochemistry in formalin-fixed bone marrow sections to assess Fgf23 eGFP reporter allele expression in the context of tissue architecture. Hbb th3/+ Fgf23 +/eGFP mice showed GFP expression in BM-SEC throughout the bone marrow, which was more intense than in non-anemic Hbb +/+ Fgf23 +/eGFP mice; controls lacking the Fgf23 eGFP allele confirmed anti-GFP antibody specificity. Hbb th3/+ mice also showed Fgf23 eGFP reporter expression in the thymic vasculature but not vasculature of the liver, spleen, or kidney. In contrast to their marked elevation in plasma total FGF23, Hbb th3/+ Fgf23 +/+ mice showed plasma levels of the intact, biologically-active FGF23 hormone that overlapped with levels in Hbb +/+ Fgf23 +/+ controls. Interestingly, compared with Hbb th3/+ Fgf23 +/+ mice, Hbb th3/+ Fgf23 +/eGFP mice showed 45% lower plasma intact FGF23, suggesting an effect of Fgf23 gene dosage. Given prior reported effects of FGF23 on hematopoiesis, we assessed if Fgf23 gene dosage modulated the severity of ineffective erythropoiesis in Hbb th3/+ mice. Compared to Hbb +/+ Fgf23 +/+ controls, Hbb th3/+ Fgf23 +/+ mice showed reductions in RBC, hemoglobin, and mean corpuscular hemoglobin, as well as increases in RDW and in spleen weight (reflecting extramedullary hematopoiesis). However, the severity of these parameters was not modified by heterozygous Fgf23 disruption ( Hbb th3/+ Fgf23 +/eGFP).While FGF23 has been reported to induce left ventricular hypertrophy [PMID:21985788], Hbb th3/+ Fgf23 +/+ and Hbb th3/+ Fgf23 +/eGFP mice showed similarly increased heart-to-body weight ratios compared to Hbb +/+ Fgf23 +/+ controls. In non-thalassemic mice, heterozygous Fgf23 disruption ( Hbb +/+ Fgf23 +/eGFP) caused a trend ( P=0.10) towards lower plasma intact FGF23 while not impacting hematological parameters or spleen weight. In summary, by showing that Fgf23 gene dosage modulates plasma intact FGF23 levels in Hbb th3/+ mice without altering severity of anemia or splenomegaly, our data suggest that moderate reductions in circulating levels of the intact FGF23 hormone do not alter the severity of ineffective erythropoiesis in Hbb th3/+mice at 8 weeks of age. Our finding that β-thalassemic mice exhibit Fgf23 upregulation in BM-SEC lays the groundwork for future investigation of the functional effects of locally-produced FGF23 in the thalassemic hematopoietic stem cell niche.

Kidney phosphate wasting predicts poor outcome in polycystic kidney disease.

Patients with ADPKD have disproportionately high levels of fibroblast growth factor-23 (FGF-23) for their CKD-stage with only a subgroup that develops kidney phosphate wasting. We assessed factors associated with phosphate wasting and hypothesize that it identifies patients with more severe disease and predicts disease progression. We included 604 patients with ADPKD from a multi-center prospective observational (DIPAK) cohort in 4 university medical centers in the Netherlands. We measured parathyroid hormone (PTH), total plasma FGF-23 levels and calculated the ratio of tubular maximum reabsorption rate of phosphate to glomerular filtration rate (TmP/GFR) with < 0.8 mmol/L defined as kidney phosphate wasting. We analysed the association of TmP/GFR with eGFR decline over time and the risk for a composite kidney outcome (≥ 30% eGFR decline, kidney failure or kidney replacement therapy). In our cohort (age 48 ± 12 years, 39% male, eGFR 63 ± 28 mL/min/1.73m2), 59% of patients had phosphate wasting. Male sex (coefficient -0.2, 95% confidence interval [CI] -0.2; -0.1), eGFR (0.002, 0.001-0.004), FGF-23 (0.1, 0.03-0.2), PTH(-0.2, -0.3; -0.06) and Copeptin(-0.08, -0.1; -0.08) were associated with TmP/GFR. Corrected for PTH, FGF-23 and eGFR, every 0.1 mmol/L decrease in TmP/GFR was associated with a greater eGFR decline of 0.2 ml/min/1.73m2/year (95% CI 0.01-0.3) and an increased hazard ratio of 1.09 (95% CI 1.01-1.18) of the composite kidney outcome. Our study shows that in patients with ADPKD phosphate wasting is prevalent and associated with more rapid disease progression. Phosphate wasting may be a consequence of early proximal tubular dysfunction and insufficient suppression of PTH.

Circulating Levels of Fibroblast Growth Factor 23 Selective for C-Terminal (FGF23-CT) in Hemodialysis Patients

Background: In hemodialysis patients, fibroblast growth factor 23 (FGF23) has reportedly been associated with the development of cardiovascular complications and a high risk of mortality. Our objective here was to study the cleavage characteristics of FGF23 in hemodialysis patients. Methods: This study design is a cross-sectional observational investigation of three facilities without intervention. To assess FGF23 concentrations, we obtained plasma samples from 97 hemodialysis patients before the hemodialysis session and from 16 healthy volunteers. We measured the FGF23 C-terminal fragment and intact FGF23 concentrations by using a commercial enzyme-linked immunosorbent assay. Results: Serum levels of the FGF23 C-terminal fragment were 189 ± 121 ng/mL in healthy volunteers and 306 ± 206 ng/mL in hemodialysis patients. The ratios of intact FGF23 to total FGF23 were 0.03 ± 0.03 in healthy volunteers and 0.44 ± 0.28 in hemodialysis patients. The ratios were positively correlated with levels of inorganic phosphate in hemodialysis patients (p &lt; 0.001, r = 0.52). Conclusion: We measured actual levels of the serum FGF23 C-terminal fragment in hemodialysis patients by using a new commercial kit for the first time. The ratio of intact FGF23 to total FGF23 was lower in healthy controls than the ratio in hemodialysis patients. The cleavage percentage of FGF23 was considerably higher in both groups of subjects than previously thought. We suggest that hyperphosphatemia in hemodialysis patients was associated with impaired cleavage of FGF23.

Associations between anemia and FGF23 in the CKiD study

BackgroundFibroblast growth factor 23 (FGF23) is a bone-derived hormone that plays a central role in chronic kidney disease-mineral bone disorder and is associated with CKD progression and cardiovascular morbidity. Factors related to CKD-associated anemia, including iron deficiency, can increase FGF23 production. This study aimed to assess whether anemia and/or iron deficiency are associated with increased circulating concentrations of FGF23 in the large, well-characterized Chronic Kidney Disease in Children (CKiD) study cohort.MethodsHemoglobin concentrations, iron parameters, C-terminal (total) FGF23, intact FGF23, and relevant covariables were measured in cross-sectional analysis of CKiD study subjects.ResultsIn 493 pediatric patients with CKD (median [interquartile range] age 13 [9, 16] years), the median estimated glomerular filtration rate was 48 [35, 61] ml/min/1.73 m2, and 103 patients (21%) were anemic. Anemic subjects had higher total FGF23 concentrations than non-anemic subjects (204 [124, 390] vs. 109 [77, 168] RU/ml, p < 0.001). In multivariable linear regression modeling, anemia was independently associated with higher total FGF23, after adjustment for demographic, kidney-related, mineral metabolism, and inflammatory covariables (standardized β (95% confidence interval) 0.10 (0.04, 0.17), p = 0.002). In the subset of subjects with available iron parameters (n = 191), iron deficiency was not associated with significantly higher total FGF23 concentrations. In the subgroup that had measurements of both total and intact FGF23 (n = 185), in fully adjusted models, anemia was significantly associated with higher total FGF23 (standardized β (95% CI) 0.16 (0.04, 0.27), p = 0.008) but not intact FGF23 (standardized β (95% CI) 0.02 (−0.12, 0.15), p = 0.81).ConclusionsIn this cohort of pediatric patients with CKD, anemia was associated with increased total FGF23 levels but was not independently associated with elevated intact FGF23, suggesting possible effects on both FGF23 production and cleavage. Further studies are warranted to investigate non-mineral factors affecting FGF23 production and metabolism in CKD.Graphical abstractA higher resolution version of the Graphical abstract is available as Supplementary information

PD03-01 FGF-23 PRODUCTION COUPLING WITH ALDOSTERONE AND PREDICTING CLINICAL OUTCOMES IN PRIMARY ALDOSTERONISM

You have accessJournal of UrologyCME1 Apr 2023PD03-01 FGF-23 PRODUCTION COUPLING WITH ALDOSTERONE AND PREDICTING CLINICAL OUTCOMES IN PRIMARY ALDOSTERONISM Jeff Chueh, Shuo-Meng Wang, VinCent Wu, and Kang-Yung Peng Jeff ChuehJeff Chueh More articles by this author , Shuo-Meng WangShuo-Meng Wang More articles by this author , VinCent WuVinCent Wu More articles by this author , and Kang-Yung PengKang-Yung Peng More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003220.01AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Higher fibroblast growth factor-23 (FGF-23) levels are associated with activating renin-angiotensin-aldosterone system (RAAS), more volume overload, and hypertension. The roles of FGF-23 in primary aldosteronism (PA) haven't been studied. METHODS: Unilateral PA (uPA) patients who underwent adrenalectomy were enrolled and followed for their clinical outcomes from Aug 15 to Jan 19. The FGF-23 expression in a rat osteoblast-like cell culture was evaluated after the addition of aldosterone. RESULTS: A total of 121 uPA patients & 69 matched essential hypertension (EH) controls were enrolled. The log [carboxyl-terminal FGF-23 (cFGF-23)] level was higher in uPA patients (p=0.001).] In Cox proportional hazard model, the preoperative Log [plasma cFGF-23]>1.98 was a risk factor for either all-cause mortality and cardiovascular or kidney events (HR, 4.01, p=0.038). Plasma cFGF-23 levels were increased in a nonlinear manner re: plasma aldosterone concentration (PAC). Log [plasma cFGF-23]>1.75 independently predicted post-op remission of hypertension (odds ratio (OR), 3.8, p=0.004). The plasma total FGF-23 level was significantly higher in PA patients (p<0.001), and it was significantly reduced post-op (p<0.001). When osteoblast-like UMR-106 cells were cultured with aldosterone, the cFGF-23 concentration in the culture medium was elevated (p=0.028), but it was abrogated after family with sequence similarity 20, member C (FAM20c) short hairpin RNA was added (p= 0.659). Docking experiments showed that aldosterone may bind to the potential drug binding pocket of FAM20c (Pdb: 5YH3; Figure 1A, B). CONCLUSIONS: Compared with EH controls, uPA patients had higher cFGF-23 levels--associated with a higher risk of cardiovascular composite outcomes, though such higher cFGF-23 levels predicted hypertension remission after adrenalectomy. Their total FGF-23 levels were significantly attenuated after adrenalectomy. An increase in cFGF-23 production among the uPA patients was secondary to increased cleavage of intact FGF-23 evidenced by higher FAM20c activity modulated by aldosterone. Source of Funding: Taiwan (NSC 101-2314-B-002-132-MY3, NSC100-2314-B-002-119, NSC 101-2314-B-002-085-MY3, MOST 104-2314-B-002 -125 -MY3; 111-2314-B-002-232-MY3) and NTUH 100-N1776, 101-M1953, 102-S2097 © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e74 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Jeff Chueh More articles by this author Shuo-Meng Wang More articles by this author VinCent Wu More articles by this author Kang-Yung Peng More articles by this author Expand All Advertisement PDF downloadLoading ...

Bone Marrow Sinusoidal Endothelial Cells Are a Site of Fgf23 Upregulation in Iron Deficiency Anemia

Iron deficiency anemia (IDA) has been identified as a potent stimulator of FGF23 (fibroblast growth factor 23), a phosphaturic hormone classically thought to be produced by bone-embedded osteocytes. Recently, both phlebotomy and erythropoietin administration have been shown to upregulate FGF23 production in bone marrow. However, the cell type(s) mediating FGF23 upregulation in states of perturbed erythropoiesis require further clarification.Tmprss6-/- mice exhibit hepcidin elevation leading to systemic iron deficiency and iron-restricted anemia. We previously reported that Tmprss6-/- mice exhibit altered phosphate balance, elevated circulating FGF23, and Fgf23 mRNA upregulation in bone marrow but not cortical bone. Here, we clarify the sites of Fgf23 promoter activity in Tmprss6 -/- bone marrow using a reporter allele in which the enhanced green fluorescent protein (eGFP) coding sequence has been knocked into the endogenous Fgf23 locus. We generated Tmprss6 +/+,Tmprss6 +/-, and Tmprss6 -/- littermates of both sexes that carried either one (Fgf23 +/eGFP) or zero (Fgf23 +/+) copies of the reporter allele. Tmprss6-/- mice showed hyperhepcidinemia, hypoferremia, microcytic anemia, and tissue iron deficiency, which were not altered by heterozygous Fgf23 disruption (Figure 1A-C). By ELISA, Tmprss6-/- Fgf23 +/eGFP mice showed plasma levels of “total” FGF23 (intact, active hormone and C-terminal cleaved fragments) that remained markedly elevated compared to Tmprss6+/+ littermates (Figure 1D). Total FGF23 elevation in Tmprss6-/- Fgf23 +/eGFP mice was slightly less pronounced than Tmprss6-/- Fgf23 +/+ mice, suggesting an effect of Fgf23 gene dosage. In mice with 2 intact Fgf23 alleles, serum erythropoietin showed a strong linear correlation with plasma total FGF23.By confocal imaging, femurs of mice carrying the Fgf23 eGFP allele showed green fluorescence in vascular regions of the bone marrow but not in the bone cortex. Green fluorescence was more intense in Tmprss6-/- Fgf23+/eGFP mice than non-anemic controls. By flow cytometry of enzymatically digested bone marrow, we observed bright green fluorescence in a subset of endothelial cells (CD45 - Ter119 - CD31 +) exclusively in mice carrying the Fgf23 eGFP reporter allele (Figure 1E). The percentage of endothelial cells that were GFP bright was higher in Tmprss6-/- Fgf23 +/eGFP versus non-anemic mice. To clarify the endothelial cell subtype that expresses Fgf23, we mined published transcriptomic datasets from mice of normal iron balance and discovered higher Fgf23 mRNA in bone marrow sinusoidal endothelial cells compared to other bone marrow endothelial cell populations. Accordingly, we used anti-GFP immunohistochemistry in formalin-fixed bone marrow sections to assess Fgf23 eGFP reporter allele expression in the context of tissue architecture. Tmprss6-/- Fgf23 +/eGFP mice showed GFP expression in bone marrow sinusoidal endothelial cells, which was more intense than in non-anemic controls (Figure 1F). GFP reporter expression was also detected in rare cells of the thymus but not in liver, spleen, heart, muscle, or kidney. Collectively, our data reveal that bone marrow sinusoidal endothelial cells are a site of Fgf23 upregulation in chronic IDA.Because IDA in Tmprss6-/- mice results from pathologic hepcidin elevation, we also sought to determine if bone marrow sinusoidal endothelial cells are a site of Fgf23 upregulation in anemic mice with intact hepcidin regulation. We therefore subjected Fgf23 +/eGFP mice (with 2 intact Tmprss6 alleles) to a 500µl phlebotomy regimen (with saline volume replacement) known to induce marked anemia and hepcidin suppression. Compared to non-phlebotomized Fgf23 +/eGFP controls, phlebotomized Fgf23 +/eGFP mice showed severe anemia, elevated serum erythropoietin, and elevated plasma FGF23 18 hours after blood loss. Additionally, immunohistochemistry revealed more intense GFP expression in bone marrow sinusoidal endothelial cells of phlebotomized Fgf23 +/eGFP mice than non-phlebotomized controls. Taken together, our results show for the first time that bone marrow sinusoidal endothelial cells are a site of Fgf23 upregulation in both acute and chronic anemia. Given the serum erythropoietin elevation in both models, our findings suggest that erythropoietin may act directly or indirectly on sinusoidal endothelial cells to promote FGF23 production during anemia. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Clinical progression of cats with early-stage chronic kidney disease fed diets with varying protein and phosphorus contents and calcium to phosphorus ratios.

BackgroundDietary protein and phosphorus (P) restriction is the mainstay for nutritional management of chronic kidney disease (CKD). However, adequate restriction levels for cats with early CKD remain unclear.ObjectivesTo investigate responses in cats with early CKD to varying dietary protein, P, and calcium (Ca) : P ratio.AnimalsNineteen research colony cats with International Renal Interest Society stages 1‐2 CKD.MethodsIn an opportunistic longitudinal case study, cats were fed a low protein (59 g/Mcal), low P (0.84 g/Mcal) dry diet (LP‐LP; Ca : P = 1.9) for 18 months and later transitioned onto a moderate protein (76‐98 g/Mcal), moderate P (1.4‐1.6 g/Mcal) dry‐wet diet regimen (MP‐MP; Ca : P = 1.4‐1.6) for 22 months. Fold‐changes in serum creatinine, total Ca (tCa) and P (primary outcomes) and fibroblast growth factor 23 (FGF23) were assessed by linear‐mixed models.ResultsWhile feeding LP‐LP, mean serum creatinine decreased (0.87‐fold, 95% confidence interval [CI] 0.81, 0.93, P < .001) to within reference range after 6 months, while increases in total Ca (tCa; 1.16‐fold, 95% CI 1.11, 1.22, P < .001) and FGF23 (2.72‐fold, 95% CI 1.72, 4.31, P < .001), but not in P (1.03‐fold, 95% CI 0.945, 1.124, P = .94), were observed after 17 months. On MP‐MP, mean creatinine, tCa and P remained within reference ranges and did not significantly change (P = .11, P = .98, and P = 1, respectively), while FGF23 significantly decreased (0.58‐fold, 95% CI 0.36, 0.95, P = .02) after 22 months.Conclusions and Clinical ImportanceCats with early CKD developed hypercalcemia after long‐term feeding of a highly P‐restricted diet. Increasing dietary P and reducing Ca : P ratio maintained renal markers, while improving Ca‐P balance. Cats with early CKD could benefit from moderately protein‐ and P‐restricted diets.

Excretion of urine extracellular vesicles bearing markers of activated immune cells and calcium/phosphorus physiology differ between calcium kidney stone formers and non-stone formers

Backgrounds:Previous studies have demonstrated that excretion of urinary extracellular vesicles (EVs) from different nephron segments differs between kidney stone formers and non-stone formers (NSFs), and could reflect pathogenic mechanisms of urinary stone disease. In this study we quantified selected populations of specific urinary EVs carrying protein markers of immune cells and calcium/phosphorus physiology in calcium oxalate stone formers (CSFs) compared to non-stone formers (NSFs).MethodsBiobanked urine samples from CSFs (n = 24) undergoing stone removal surgery and age- and sex- matched NSFs (n = 21) were studied. Urinary EVs carrying proteins related to renal calcium/phosphorus physiology (phosphorus transporters (PiT1 and PiT2), Klotho, and fibroblast growth factor 23 (FGF23); markers associated with EV generation (anoctamin-4 (ANO4) and Huntington interacting protein 1 (HIP1)), and markers shed from activated immune cells were quantified by standardized and published method of digital flow cytometry.ResultsUrine excretion of calcium, oxalate, phosphorus, and calcium oxalate supersaturation (SS) were significantly higher in CSFs compared to NSFs (P < 0.05). Urinary excretion of EVs with markers of total leukocytes (CD45), neutrophils (CD15), macrophages (CD68), Klotho, FGF23, PiT1, PiT2, and ANO4 were each markedly lower in CSFs than NSFs (P < 0.05) whereas excretion of those with markers of monocytes (CD14), T-Lymphocytes (CD3), B-Lymphocytes (CD19), plasma cells (CD138 plus CD319 positive) were not different between the groups. Urinary excretion of EVs expressing PiT1 and PiT2 negatively (P < 0.05) correlated with urinary phosphorus excretion, whereas excretion of EVs expressing FGF23 negatively (P < 0.05) correlated with both urinary calcium and phosphorus excretion. Urinary EVs with markers of HIP1 and ANO4 correlated negatively (P < 0.05) with clinical stone events and basement membrane calcifications on papillary tip biopsies.ConclusionsUrinary excretion of EVs derived from specific types of activated immune cells and EVs with proteins related to calcium/phosphorus regulation differed between CSFs and NSFs. Further validation of these and other populations of urinary EVs in larger cohort could identify biomarkers that elucidate novel pathogenic mechanisms of calcium stone formation in specific subsets of patients.

Associations among erythropoietic, iron-related, and FGF23 parameters in pediatric kidney transplant recipients.

In pediatric kidney transplant recipients, anemia is common and oftentimes multifactorial. Hemoglobin concentrations may be affected by traditional factors, such as kidney function and iron status, as well as novel parameters, such as fibroblast growth factor 23 (FGF23). Here, we evaluated associations among erythropoietic, iron-related, and FGF23 parameters in a cohort of pediatric kidney transplant recipients, hypothesizing that multiple factors are associated with hemoglobin concentrations. In a cross-sectional analysis of 59 pediatric kidney transplant recipients (median (interquartile range) age 16.3 (13.5, 18.6) years, median estimated glomerular filtration rate (eGFR) 67 (54, 87) ml/min/1.73 m2), the median age-related hemoglobin standard deviation score (SDS) was -2.1 (-3.3, -1.1). Hemoglobin SDS was positively associated with eGFR and calcium, and was inversely associated with erythropoietin (EPO), mycophenolate dose, and total, but not intact, FGF23. In multivariable analysis, total FGF23 remained inversely associated with hemoglobin SDS, independent of eGFR, iron parameters, EPO, and inflammatory markers, suggesting a novel FGF23-hemoglobin association in pediatric kidney transplant patients. In a subset of patients with repeat measurements, only delta hepcidin was inversely associated with delta hemoglobin SDS. Also, delta EPO positively correlated with delta erythroferrone (ERFE), and delta ERFE inversely correlated with delta hepcidin, suggesting a possible physiologic role for the EPO-ERFE-hepcidin axis in the setting of chronic kidney disease (CKD). Our study provides further insight into factors potentially associated with erythropoiesis in pediatric kidney transplant recipients. A higher resolution version of the Graphical abstract is available as Supplementary information.

Amelioration of chronic kidney disease-associated anemia by vadadustat in mice is not dependent on erythroferrone

Vadadustat is an investigational hypoxia-inducible factor prolyl hydroxylase inhibitor that increases endogenous erythropoietin production and has been shown to decrease hepcidin levels, ameliorate iron restriction, and increase hemoglobin concentrations in anemic patients with chronic kidney disease (CKD). In studies of physiological responses to other erythropoietic stimuli, erythropoietin induced erythroblast secretion of erythroferrone (ERFE), which acts on the liver to suppress hepcidin production and mobilize iron for erythropoiesis. We therefore investigated whether vadadustat effects on erythropoiesis and iron metabolism are dependent on ERFE. Wild type and ERFE knockout mice with and without CKD were treated with vadadustat or vehicle. In both wild type and ERFE knockout CKD models, vadadustat was similarly effective, as evidenced by normalized hemoglobin concentrations, increased expression of duodenal iron transporters, lower serum hepcidin levels, and decreased tissue iron concentrations. This is consistent with ERFE-independent increased iron mobilization. Vadadustat treatment also lowered serum urea nitrogen and creatinine concentrations and decreased expression of kidney fibrosis markers. Lastly, vadadustat affected fibroblast growth factor 23 (FGF23) profiles: in non-CKD mice, vadadustat increased plasma total FGF23 out of proportion to intact FGF23, consistent with the known effects of hypoxia-inducible factor-1α and erythropoietin on FGF23 production and metabolism. However, in the mice with CKD, vadadustat markedly decreased both total and intact FGF23, effects likely contributed to by the reduced loss of kidney function. Thus, in this CKD model, vadadustat ameliorated anemia independently of ERFE, improved kidney parameters, and decreased FGF23. How vadadustat affects CKD progression in humans warrants future studies.

Interplay of erythropoietin, fibroblast growth factor 23, and erythroferrone in patients with hereditary hemolytic anemia

AbstractRecently, erythropoietin (EPO) was identified as regulator of fibroblast growth factor 23 (FGF23). Proteolytic cleavage of biologically active intact FGF23 (iFGF23) results in the formation of C-terminal fragments (cFGF23). An increase in cFGF23 relative to iFGF23 suppresses FGF receptor signaling by competitive inhibition. EPO lowers the i:cFGF23 ratio, thereby overcoming iFGF23-mediated suppression of erythropoiesis. We investigated EPO-FGF23 signaling and levels of erythroferrone (ERFE) in 90 patients with hereditary hemolytic anemia (www.trialregister.nl [NL5189]). We show, for the first time, the importance of EPO-FGF23 signaling in hereditary hemolytic anemia: there was a clear correlation between total FGF23 and EPO levels (r = +0.64; 95% confidence interval [CI], 0.09-0.89), which persisted after adjustment for iron load, inflammation, and kidney function. There was no correlation between iFGF23 and EPO. Data are consistent with a low i:cFGF23 ratio. Therefore, as expected, we report a correlation between EPO and ERFE in a diverse set of hereditary hemolytic anemias (r = +0.47; 95% CI, 0.14-0.69). There was no association between ERFE and total FGF23 or iFGF23, which suggests that ERFE does not contribute to the connection between FGF23 and EPO. These findings open a new area of research and might provide potentially new druggable targets with the opportunity to ameliorate ineffective erythropoiesis and the development of disease complications in hereditary hemolytic anemias.

Erythropoietin Is Associated with a Decline in the iFGF23/cFGF23 Ratio in Patients with Various Hereditary Hemolytic Anemias

Background Recently, erythropoietin (EPO) was identified as an important regulator of production and cleavage of fibroblast growth factor 23 (FGF23). Since erythroid progenitor cells express high levels of FGF23 and carry the FGF receptor, they are involved in the FGF23 metabolic pathway. FGF23 is a bone-derived hormone, a key player in phosphate and vitamin D metabolism and regulator of bone mineralization. FGF23 is formed as intact, biologically active protein (iFGF23). Proteolytic cleavage results in formation of an allegedly assumed inactive C-terminal tail FGF23 (cFGF23). FGF23-knockouts or iFGF23 blocking peptides increase erythropoiesis, reduce erythroid cell apoptosis and increase EPO mRNA and circulating EPO. By competitive inhibition, increase in cFGF23 relative to iFGF23 leads to suppression of FGF receptor signaling. (Recombinant) EPO increases the amount of circulating FGF23 (iFGF23 plus cFGF23) and, more importantly, alters the iFGF23/cFGF23 ratio in favor of cFGF23, thereby overcoming suppression of erythropoiesis by iFGF23. As recently discussed, (van Vuren et al. Front. Physiol. 2019) we hypothesize that EPO-FGF23 signaling plays a role in hereditary hemolytic anemias and is herein related to iron metabolism, inflammation and bone health. We explored this concept further in the current study. Methods FGF23 was measured in 90 patients and 10 healthy controls of the ZEbRA trial, an observational study at the UMC Utrecht on clinical sequelae and pathophysiology of rare congenital hemolytic (Netherlands Trial Register [NL5189]). An overview of relevant patient characteristics is provided in the Table. Two FGF23 assays were used: one that detects the C-terminal and therefore quantifies cFGF23 and (full-length) iFGF23 (total FGF23; Immunotopics/Quidel) and one that only detects iFGF23 (Kainos Laboratories). Chemokines/cytokines were measured simultaneously with a Luminex assay. Results We observed a strong correlation between levels of total FGF23 (cFGF23 and iFGF23) and EPO in patients with various hemolytic anemias (r=0.64, 95% confidence interval [0.09; 0.89]) (Figure). As expected, there was neither a correlation between iFGF23 and EPO values nor between iFGF23 and total FGF23. These data are consistent with a decline of the iFGF23/cFGF23 ratio in response to EPO. iFGF23 and total FGF23 were not correlated to serum calcium, phosphate, creatinine, parathyroid hormone and 25-OH vitamin D (p&gt;0.05). We did not identify a correlation of iFGF23 and total FGF23 with bone health (hip and spine T-scores). EPO was associated with transferrin saturation (r=0.45, 95% CI [0.28; 0.63]) and ferritin (r=0.47, 95% CI [0.06; 0.79]), whereas total FGF23 did not correlate with iron status. The influence of inflammation on the EPO-FGF23 axis was clearly illustrated by higher mean total FGF23 in SCD patients (549 RU/L) compared to other patients (172 RU/L) (95% CI [87; 747]), without a difference in EPO or iFGF23. We identified a correlation of cFGF23 with CCL4 (r=0.49, 95% CI [0.15; 0.72]) and with sICAM (r=0.54, 95% CI [0.16; 0.75]), markers of endothelial dysfunction and immune players in pulmonary hypertension. (Kato et al. Br J Haematol. 2005; Sweatt et al. Circ Res. 2019) Conclusion We show for the first time the importance of EPO in FGF23 signaling in hemolytic anemias and investigated its relation with iron, inflammation and bone health. Absence of a relation between iron and FGF23 suggests that EPO is not simply an intermediary in FGF23 regulation by iron. FGF23 data of SCD patients suggest that inflammation is associated with an additional increase in FGF23 cleavage. Absence of a correlation of FGF23 with bone health might be attributable to treatment for osteoporosis in many patients. Alternatively, systemic FGF23 might underestimate paracrine signaling in the bone. Additionally, we hypothesize on a contribution of decline in the iFGF23/cFGF23 ratio to vasculopathic complications. Our hypothesis aligns with previous observations that too little or too much FGF signaling is related to endothelial dysregulation and endothelial-to-mesenchymal transition, which contributes to development of pulmonary hypertension. (Chen et al. Cell. Rep. 2012; Morrell et al. J. Am. Coll. 2009) Current findings open a new area for research and treatment in hemolytic anemias. Therapeutic targeting of the FGF23 pathway might provide an opportunity to intervene in the development of complications. Disclosures Glenthøj: Agios Pharmaceuticals, Inc.: Consultancy; Celgene: Consultancy; Alexion: Research Funding; Novartis: Consultancy; Novo Nordisk: Honoraria. van Wijk:Agios Pharmaceuticals: Consultancy, Research Funding; RR Mechatronics: Research Funding. van Beers:Novartis: Consultancy, Research Funding; Agios Pharmaceuticals, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Research Funding; RR Mechatronics: Research Funding.

Plasma total fibroblast growth factor 23 levels are associated with acute kidney injury and mortality in children with acute respiratory distress syndrome.

Acute respiratory distress syndrome (ARDS) has high rates of mortality and multisystem morbidity. Pre-clinical data suggest that fibroblast growth factor 23 (FGF23) may contribute to pulmonary pathology, and FGF23 is associated with mortality and morbidity, including acute kidney injury (AKI), in non-ARDS cohorts. Here, we assess whether FGF23 is associated with AKI and/or mortality in a cohort of 161 pediatric ARDS patients. Plasma total (intact + C-terminal) FGF23 and intact FGF23 concentrations were measured within 24 hours of ARDS diagnosis (Day 1), and associations with Day 3 AKI and 60-day mortality were evaluated. 35 patients (22%) developed AKI by 3 days post-ARDS diagnosis, and 25 (16%) died by 60 days post-ARDS diagnosis. In unadjusted models, higher Day 1 total FGF23 was associated with Day 3 AKI (odds ratio (OR) 2.22 [95% confidence interval (CI) 1.62, 3.03], p<0.001), but Day 1 intact FGF23 was not. In a model adjusted for demographics and disease severity, total FGF23 remained associated with AKI (OR 1.52 [95% CI 1.02, 2.26], p = 0.039). In unadjusted models, both higher Day 1 total and intact FGF23 were associated with 60-day mortality (OR 1.43 [95% CI 1.07, 1.91], p = 0.014; and OR 1.44 [95% CI 1.02, 2.05], p = 0.039, respectively). In the adjusted model, only total FGF23 remained associated with 60-day mortality (OR 1.62 [95% CI 1.07, 2.45], p = 0.023). In a subgroup analysis of patients with Day 1 plasma IL-6 concentrations available, inflammation partially mediated the association between total FGF23 and AKI. Our data suggest both inflammation-dependent and inflammation-independent associations between total FGF23 and clinical outcomes in pediatric ARDS patients.

Iron deficiency, elevated erythropoietin, fibroblast growth factor 23, and mortality in the general population of the Netherlands: A cohort study.

BackgroundEmerging data in chronic kidney disease (CKD) patients suggest that iron deficiency and higher circulating levels of erythropoietin (EPO) stimulate the expression and concomitant cleavage of the osteocyte-derived, phosphate-regulating hormone fibroblast growth factor 23 (FGF23), a risk factor for premature mortality. To date, clinical implications of iron deficiency and high EPO levels in the general population, and the potential downstream role of FGF23, are unclear. Therefore, we aimed to determine the associations between iron deficiency and higher EPO levels with mortality, and the potential mediating role of FGF23, in a cohort of community-dwelling subjects.Methods and findingsWe analyzed 6,544 community-dwelling subjects (age 53 ± 12 years; 50% males) who participated in the Prevention of Renal and Vascular End-Stage Disease (PREVEND) study—a prospective population-based cohort study, of which we used the second survey (2001–2003)—and follow-up was performed for a median of 8 years. We measured circulating parameters of iron status, EPO levels, and plasma total FGF23 levels. Our primary outcome was all-cause mortality. In multivariable linear regression analyses, ferritin (ß = –0.43), transferrin saturation (TSAT) (ß = −0.17), hepcidin (ß = −0.36), soluble transferrin receptor (sTfR; ß = 0.33), and EPO (ß = 0.28) were associated with FGF23 level, independent of potential confounders. During median (interquartile range [IQR]) follow-up of 8.2 (7.7–8.8) years, 379 (6%) subjects died. In multivariable Cox regression analyses, lower levels of TSAT (hazard ratio [HR] per 1 standard deviation [SD], 0.84; 95% confidence interval [CI], 0.75–0.95; P = 0.004) and higher levels of sTfR (HR, 1.15; 95% CI 1.03–1.28; P = 0.01), EPO (HR, 1.17; 95% CI 1.05–1.29; P = 0.004), and FGF23 (HR, 1.20; 95% CI 1.10–1.32; P < 0.001) were each significantly associated with an increased risk of death, independent of potential confounders. Adjustment for FGF23 levels markedly attenuated the associations of TSAT (HR, 0.89; 95% CI 0.78–1.01; P = 0.06), sTfR (HR, 1.08; 95% CI 0.96–1.20; P = 0.19), and EPO (HR, 1.10; 95% CI 0.99–1.22; P = 0.08) with mortality. FGF23 remained associated with mortality (HR, 1.15; 95% CI 1.04–1.27; P = 0.008) after adjustment for TSAT, sTfR, and EPO levels. Mediation analysis indicated that FGF23 explained 31% of the association between TSAT and mortality; similarly, FGF23 explained 32% of the association between sTfR and mortality and 48% of the association between EPO and mortality (indirect effect P < 0.05 for all analyses). The main limitations of this study were the observational study design and the absence of data on intact FGF23 (iFGF23), precluding us from discerning whether the current results are attributable to an increase in iFGF23 or in C-terminal FGF23 fragments.Conclusions and relevanceIn this study, we found that functional iron deficiency and higher EPO levels were each associated with an increased risk of death in the general population. Our findings suggest that FGF23 could be involved in the association between functional iron deficiency and increased EPO levels and death. Investigation of strategies aimed at correcting iron deficiency and reducing FGF23 levels is warranted.

Effects of erythropoietin on fibroblast growth factor 23 in mice and humans.

Erythropoietin (EPO) has been reported as a novel determinant of fibroblast growth factor 23 (FGF23) production; however, it is unknown whether FGF23 is stimulated by chronic exposure to EPO or by EPO administration in nonpolycystic chronic kidney disease (CKD) models. We analyzed the effects of chronic EPO on FGF23 in murine models with chronically high EPO levels and normal kidney function. We studied the effects of exogenous EPO on FGF23 in wild-type mice, with and without CKD, injected with EPO. Also, in four independent human CKD cohorts, we evaluated associations between FGF23 and serum EPO levels or exogenous EPO dose. Mice with high endogenous EPO have elevated circulating total FGF23, increased disproportionately to intact FGF23, suggesting coupling of increased FGF23 production with increased proteolytic cleavage. Similarly, in wild-type mice with and without CKD, a single exogenous EPO dose acutely increases circulating total FGF23 out of proportion to intact FGF23. In these murine models, the bone marrow is shown to be a novel source of EPO-stimulated FGF23 production. In humans, serum EPO levels and recombinant human EPO dose are positively and independently associated with total FGF23 levels across the spectrum of CKD and after kidney transplantation. In our largest cohort of 680 renal transplant recipients, serum EPO levels are associated with total FGF23, but not intact FGF23, consistent with the effects of EPO on FGF23 production and metabolism observed in our murine models. EPO affects FGF23 production and metabolism, which may have important implications for CKD patients.

Fibroblast growth factor 23 and lipid metabolism association in chronic kidney disease

Recent scientificstudies have demonstrated the effect of fibroblast growth factor 23 (FGF-23) on the volume and distribution of body fat. The aim of our study was to investigate of lipid metabolism in patients with chronic kidney disease (CKD) and its relationship with FGF-23.&#x0D; Methods. We conducted a single-center, cohort retrospective study involved 106 patients with CKD 1-5 stages. Among the patients were 47 women (44%) and 59 men (56%) aged (49.6±13.9) years. All patients were determined the blood lipid spectrum: total cholesterol level (LDL), high density lipoproteins (HDL) and triglycerides (TG). The lipid profile was examined using a biochemical analyzer Cobas Integra 400 Plus. The C-terminal FGF-23 fragment was determined using a set of reagents for the enzyme immunoassay “Biomedica” (Astria). The glomerular filtration rate (GFR) was calculated using the CKD EPI formula (KDIGO 2012). All the statistical analyses were performed using Statistica 10.0.&#x0D; Results. In patients with CKD, progressive decrease in the level of total cholesterol, LDL cholesterol, HDL cholesterol and the increase in TG concurrent with the fall in GFR was detected (p&lt;0.001). The concentration of the C-terminal FGF-23 fragment progressively increased in parallel with the fall in GFR, reaching the highest values at CKD stage 5 (p&lt;0.001). A significant relationship was found between FGF-23 and total cholesterol (r =-0.45, p&lt;0.05), LDL (r=-0.29, p&lt;0.05), HDL (r=-0.54, p&lt;0.05), FGF-23 and TG (r=0.28, p&lt;0.05). Conclusions. CKD is characterized by a significant growth in TG levels, which increases with progression of renal dysfunction. The level of FGF-23 in CKD steadily increases in parallel with the decrease in GFR. The parameters of lipid metabolism, namely, total cholesterol, LDL, TG and HDL, have a reliable relationship with FGF-23 in CKD.

Mechanism of FGF23 processing in fibrous dysplasia

Fibroblast growth factor-23 (FGF23) is a phosphate- and vitamin D-regulating hormone derived from osteoblasts/osteocytes that circulates in both active (intact, iFGF23) and inactive (C-terminal, cFGF23) forms. O-glycosylation by O-glycosyl transferase N-acetylgalactosaminyltransferase 3 (ppGalNAcT3) and differential cleavage by furin have been shown to be involved in regulating the ratio of active to inactive FGF23. Elevated iFGF23 levels are observed in a number of hypophosphatemic disorders, such as X-linked, autosomal recessive, and autosomal dominant hypophosphatemic rickets, whereas low iFGF23 levels are found in the hyperphosphatemic disorder familial tumoral calcinosis/hyperphosphatemic hyperostosis syndrome. Fibrous dysplasia of bone (FD) is associated with increased total FGF23 levels (cFGF23 + iFGF23); however, classic hypophosphatemic rickets is uncommon. Our results suggest that it can be explained by increased FGF23 cleavage leading to an increase in inactive cFGF23 relative to active iFGF23. Given the fact that FD is caused by activating mutations in the small G-protein G(s) α that results in increased cyclic adenosine monophosphate (cAMP) levels, we postulated that there may be altered FGF23 cleavage in FD and that the mechanism may involve alterations in cAMP levels and ppGalNacT3 and furin activities. Analysis of blood specimens from patients with FD confirmed that the elevated total FGF23 levels are the result of proportionally increased cFGF23 levels, consistent with less glycosylation and enhanced cleavage by furin. Analysis of primary cell lines of normal and mutation-harboring bone marrow stromal cells (BMSCs) from patients with FD demonstrated that BMSCs harboring the causative G(s) α mutation had higher cAMP levels, lower ppGalNAcT3, and higher furin activity. These data support the model wherein glycosylation by ppGalNAcT3 inhibits FGF23 cleavage by furin and suggest that FGF23 processing is a regulated process that controls overall FGF23 activity in FD patients.

Fibroblast Growth Factor 23 in Patients Undergoing Peritoneal Dialysis

Fibroblast growth factor 23 (FGF23) is an independent risk factor for mortality in patients with ESRD. Before FGF23 testing can be integrated into clinical practice of ESRD, further understanding of its determinants is needed. In a study of 67 adults undergoing peritoneal dialysis, we tested the hypothesis that longer dialysis vintage and lower residual renal function and renal phosphate clearance are associated with higher FGF23. We also compared the monthly variability of FGF23 versus parathyroid hormone (PTH) and serum phosphate. In unadjusted analyses, FGF23 correlated with serum phosphate (r = 0.66, P < 0.001), residual renal function (r = -0.37, P = 0.002), dialysis vintage (r = 0.31, P = 0.01), and renal phosphate clearance (r = -0.38, P = 0.008). In adjusted analyses, absence of residual renal function and greater dialysis vintage associated with higher FGF23, independent of demographics, laboratory values, peritoneal dialysis modality and adequacy, and treatment with vitamin D analogs and phosphate binders. Urinary and dialysate FGF23 clearances were minimal. In three serial monthly measurements, within-subject variability accounted for only 10% of total FGF23 variability compared with 50% for PTH and 60% for serum phosphate. Increased serum phosphate, loss of residual renal function, longer dialysis vintage, and lower renal phosphate clearance are associated with elevated FGF23 levels in ESRD patients undergoing peritoneal dialysis. FGF23 may be a more stable marker of phosphate metabolism in ESRD than PTH or serum phosphate.

The association of circulating ferritin with serum concentrations of fibroblast growth factor-23 measured by three commercial assays

The measurement of the serum concentration of fibroblast growth factor-23 (FGF-23) is beginning to be used as a diagnostic tool in renal phosphate wasting disorders. Having observed an increased serum FGF-23 in three subjects with low circulating ferritin concentrations we investigated the association between low ferritin and raised serum FGF-23. We measured FGF-23 in 150 random anonymized serum samples with ferritin concentrations between <5 and 50 microg/L using three commercially available enzyme-linked immunosorbent assay (ELISA) kits. One kit, Human FGF-23[C-term] (Immutopics Inc, USA) measures total FGF-23 whereas the other two kits, Immutopics intact and FGF-23 ELISA (Kainos, Japan) are reported to measure only the biologically active intact molecule. We have detected a significant inverse correlation of -0.565 (P<0.0001) between serum ferritin when <50 microg/L and FGF-23 using the C-terminal assay. This relationship is also shown with the Immutopics intact assay but is not demonstrated with the Kainos intact assay. The measurement of FGF-23 by both Immutopics assays is altered in the presence of low circulating concentrations of serum ferritin whereas with the Kainos intact assay this effect was not demonstrated. Serum ferritin should be measured when an elevated FGF-23 is obtained using the Immutopics C-terminal or intact FGF-23 assay to prevent misdiagnosis of the cause of this abnormality.