RESUMEN
Fibroblast growth factor (FGF) 23 is a phosphate-regulating hormone that is elevated in patients with chronic kidney disease and associated with cardiovascular mortality. Experimental studies showed that elevated FGF23 levels induce cardiac hypertrophy by targeting cardiac myocytes via FGF receptor isoform 4 (FGFR4). A recent structural analysis revealed that the complex of FGF23 and FGFR1, the physiologic FGF23 receptor in the kidney, includes soluble α-klotho (klotho) and heparin, which both act as co-factors for FGF23/FGFR1 signaling. Here, we investigated whether soluble klotho, a circulating protein with cardio-protective properties, and heparin, a factor that is routinely infused into patients with kidney failure during the hemodialysis procedure, regulate FGF23/FGFR4 signaling and effects in cardiac myocytes. We developed a plate-based binding assay to quantify affinities of specific FGF23/FGFR interactions and found that soluble klotho and heparin mediate FGF23 binding to distinct FGFR isoforms. Heparin specifically mediated FGF23 binding to FGFR4 and increased FGF23 stimulatory effects on hypertrophic growth and contractility in isolated cardiac myocytes. When repetitively injected into two different mouse models with elevated serum FGF23 levels, heparin aggravated cardiac hypertrophy. We also developed a novel procedure for the synthesis and purification of recombinant soluble klotho, which showed anti-hypertrophic effects in FGF23-treated cardiac myocytes. Thus, soluble klotho and heparin act as independent FGF23 co-receptors with opposite effects on the pathologic actions of FGF23, with soluble klotho reducing and heparin increasing FGF23-induced cardiac hypertrophy. Hence, whether heparin injections during hemodialysis in patients with extremely high serum FGF23 levels contribute to their high rates of cardiovascular events and mortality remains to be studied.
Asunto(s)
Factor-23 de Crecimiento de Fibroblastos , Heparina , Proteínas Klotho , Insuficiencia Renal Crónica , Animales , Cardiomegalia , Glucuronidasa/metabolismo , Heparina/metabolismo , Humanos , Proteínas Klotho/metabolismo , Ratones , Insuficiencia Renal Crónica/complicaciones , Insuficiencia Renal Crónica/terapiaRESUMEN
Fibroblast growth factor (FGF) 21, a hormone that increases insulin sensitivity, has shown promise as a therapeutic agent to improve metabolic dysregulation. Here we report that FGF21 directly targets cardiac myocytes by binding ß-klotho and FGF receptor (FGFR) 4. In combination with high glucose, FGF21 induces cardiac myocyte growth in width mediated by extracellular signal-regulated kinase 1/2 (ERK1/2) signaling. While short-term FGF21 elevation can be cardio-protective, we find that in type 2 diabetes (T2D) in mice, where serum FGF21 levels are elevated, FGFR4 activation induces concentric cardiac hypertrophy. As T2D patients are at risk for heart failure with preserved ejection fraction (HFpEF), we propose that induction of concentric hypertrophy by elevated FGF21-FGFR4 signaling may constitute a novel mechanism promoting T2D-associated HFpEF such that FGFR4 blockade might serve as a cardio-protective therapy in T2D. In addition, potential adverse cardiac effects of FGF21 mimetics currently in clinical trials should be investigated.
Asunto(s)
Diabetes Mellitus Tipo 2 , Insuficiencia Cardíaca , Animales , Cardiomegalia/metabolismo , Diabetes Mellitus Tipo 2/complicaciones , Diabetes Mellitus Tipo 2/metabolismo , Modelos Animales de Enfermedad , Factores de Crecimiento de Fibroblastos/metabolismo , Insuficiencia Cardíaca/metabolismo , Humanos , Ratones , Miocitos Cardíacos/metabolismo , Receptor Tipo 4 de Factor de Crecimiento de Fibroblastos/metabolismo , Volumen SistólicoAsunto(s)
Enfermedades Renales , Fibrosis , Glucuronidasa , Humanos , Riñón/patología , Enfermedades Renales/patología , PéptidosRESUMEN
Elevations in plasma phosphate concentrations (hyperphosphatemia) occur in chronic kidney disease (CKD), in certain genetic disorders, and following the intake of a phosphate-rich diet. Whether hyperphosphatemia and/or associated changes in metabolic regulators, including elevations of fibroblast growth factor 23 (FGF23) directly contribute to specific complications of CKD is uncertain. Here, we report that similar to patients with CKD, mice with adenine-induced CKD develop inflammation, anemia, and skeletal muscle wasting. These complications are also observed in mice fed high phosphate diet even without CKD. Ablation of pathologic FGF23-FGFR4 signaling did not protect mice on an increased phosphate diet or mice with adenine-induced CKD from these sequelae. However, low phosphate diet ameliorated anemia and skeletal muscle wasting in a genetic mouse model of CKD. Our mechanistic in vitro studies indicate that phosphate elevations induce inflammatory signaling and increase hepcidin expression in hepatocytes, a potential causative link between hyperphosphatemia, anemia, and skeletal muscle dysfunction. Our study suggests that high phosphate intake, as caused by the consumption of processed food, may have harmful effects irrespective of pre-existing kidney injury, supporting not only the clinical utility of treating hyperphosphatemia in CKD patients but also arguing for limiting phosphate intake in healthy individuals.
Asunto(s)
Anemia , Hiperfosfatemia , Anemia/complicaciones , Animales , Factor-23 de Crecimiento de Fibroblastos/metabolismo , Factores de Crecimiento de Fibroblastos/metabolismo , Humanos , Hiperfosfatemia/complicaciones , Inflamación , Ratones , Músculo Esquelético/metabolismo , Receptor Tipo 4 de Factor de Crecimiento de FibroblastosRESUMEN
In chronic kidney disease (CKD), elevated serum levels of the phosphate regulating hormone fibroblast growth factor (FGF) 23 have emerged as powerful risk factors for cardiovascular disease and death. Mechanistically, FGF23 can bind and activate fibroblast growth factor receptor (FGFR) 4 independently of α-klotho, the canonical co-receptor for FGF23 in the kidney, which stimulates left ventricular hypertrophy and hepatic production of inflammatory cytokines. FGF23 has also been shown to independently predict progression of renal disease, however, whether FGF23 and FGFR4 also contribute to CKD remains unknown. Here, we generated a mouse model with dual deletions of FGFR4 and α-klotho, and we induced CKD in mice with either global deletion or constitutive activation of FGFR4. We demonstrate that FGF23 is not capable of inducing phosphaturia via FGFR4 and that FGFR4 does not promote or mitigate renal injury in animal models of CKD. Taken together our results suggest FGFR4 inhibition as a safe alternative strategy to target cardiovascular disease and chronic inflammation in patients with CKD without interrupting the necessary phosphaturic effects of FGF23.
Asunto(s)
Receptor Tipo 4 de Factor de Crecimiento de Fibroblastos/metabolismo , Insuficiencia Renal Crónica/patología , Animales , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Factor-23 de Crecimiento de Fibroblastos , Técnicas de Sustitución del Gen , Glucuronidasa/metabolismo , Humanos , Proteínas Klotho , Ratones , Ratones Noqueados , Ratones Transgénicos , Receptor Tipo 4 de Factor de Crecimiento de Fibroblastos/fisiología , Factores de RiesgoRESUMEN
BACKGROUND: In uremic animals, vitamin D receptor (VDR) agonists like paricalcitol (Pc) attenuate cardiac hypertrophy, but this effect has not been replicated consistently in humans with chronic kidney disease. Elevated fibroblast growth factor 23 (FGF23) levels cause cardiac hypertrophy with activation of the myocardial calcineurin/nuclear factor of activated T cell (NFAT) axis and may antagonize the cardioprotective effects of VDR agonist therapy. We hypothesized that the effectiveness of Pc may depend on the prevailing circulating levels of FGF23 and could be potentiated by the combined administration of a pan-FGF23 receptor (FGFR) blocker agent (PD173074). METHODS: In rats with 5/6 nephrectomy treated with Pc or PD173074 or both agents concurrently, myocardial mRNA expression of renin-angiotensin system, VDR, FGFR4, and calcineurin/NFAT target genes was determined. In adolescents on hemodialysis, we analyzed sequential echocardiograms, blood pressures and serial FGF23 measurements, and their relations to the cumulative administered dose of parenteral Pc. RESULTS: The ratio of Pc dose/plasma levels of FGF23 correlated inversely (P < 0.005) with the cardiac mass in uremic rats and in hemodialysis patients, independently of hypertension. Despite persistently elevated FGF23 levels and myocardial FGFR4 activation, Pc suppressed upregulated myocardial calcineurin/NFAT target genes, and the effects were amplified by coadministration of PD173074. CONCLUSIONS: The beneficial effects of Pc on uremic cardiac hypertrophy are counterbalanced by the increased FGF23 levels. Blockade of FGF23-mediated signaling increased the Pc-induced suppression of the myocardial calcineurin/NFAT system. Higher doses of Pc should be considered in the treatment of patients with uremic cardiomyopathy.
Asunto(s)
Síndrome Cardiorrenal/prevención & control , Cardiomiopatías/prevención & control , Ergocalciferoles/farmacología , Factores de Crecimiento de Fibroblastos/sangre , Ventrículos Cardíacos/efectos de los fármacos , Fallo Renal Crónico/tratamiento farmacológico , Pirimidinas/farmacología , Receptor Tipo 4 de Factor de Crecimiento de Fibroblastos/antagonistas & inhibidores , Receptores de Calcitriol/agonistas , Adolescente , Animales , Síndrome Cardiorrenal/metabolismo , Síndrome Cardiorrenal/fisiopatología , Cardiomiopatías/metabolismo , Cardiomiopatías/fisiopatología , Niño , Modelos Animales de Enfermedad , Quimioterapia Combinada , Femenino , Factor-23 de Crecimiento de Fibroblastos , Ventrículos Cardíacos/metabolismo , Ventrículos Cardíacos/fisiopatología , Humanos , Fallo Renal Crónico/metabolismo , Fallo Renal Crónico/fisiopatología , Masculino , Ratas Sprague-Dawley , Receptor Tipo 4 de Factor de Crecimiento de Fibroblastos/metabolismo , Receptores de Calcitriol/metabolismo , Estudios Retrospectivos , Transducción de Señal , Uremia/tratamiento farmacológico , Uremia/metabolismo , Uremia/patología , Uremia/fisiopatología , Función Ventricular Izquierda/efectos de los fármacos , Remodelación Ventricular/efectos de los fármacosRESUMEN
Circulating levels of fibroblast growth factor (FGF)23 are associated with systemic inflammation and increased mortality in chronic kidney disease. α-Klotho, a co-receptor for FGF23, is downregulated in chronic obstructive pulmonary disease (COPD). However, whether FGF23 and Klotho-mediated FGF receptor (FGFR) activation delineates a pathophysiological mechanism in COPD remains unclear. We hypothesised that FGF23 can potentiate airway inflammation via Klotho-independent FGFR4 activation.FGF23 and its effect were studied using plasma and transbronchial biopsies from COPD and control patients, and primary human bronchial epithelial cells isolated from COPD patients as well as a murine COPD model.Plasma FGF23 levels were significantly elevated in COPD patients. Exposure of airway epithelial cells to cigarette smoke and FGF23 led to a significant increase in interleukin-1ß release via Klotho-independent FGFR4-mediated activation of phospholipase Cγ/nuclear factor of activated T-cells signalling. In addition, Klotho knockout mice developed COPD and showed airway inflammation and elevated FGFR4 expression in their lungs, whereas overexpression of Klotho led to an attenuation of airway inflammation.Cigarette smoke induces airway inflammation by downregulation of Klotho and activation of FGFR4 in the airway epithelium in COPD. Inhibition of FGF23 or FGFR4 might serve as a novel anti-inflammatory strategy in COPD.
Asunto(s)
Factores de Crecimiento de Fibroblastos/sangre , Glucuronidasa/metabolismo , Pulmón/patología , Enfermedad Pulmonar Obstructiva Crónica/sangre , Receptor Tipo 4 de Factor de Crecimiento de Fibroblastos/metabolismo , Adulto , Anciano , Animales , Células Epiteliales/metabolismo , Femenino , Factor-23 de Crecimiento de Fibroblastos , Glucuronidasa/genética , Humanos , Inflamación/patología , Proteínas Klotho , Masculino , Ratones , Ratones Noqueados , Persona de Mediana Edad , Enfermedad Pulmonar Obstructiva Crónica/metabolismo , Humo/efectos adversosRESUMEN
Immune evasion and the suppression of antitumor responses during cancer progression are considered hallmarks of cancer and are typically attributed to tumor-derived factors. Although the molecular basis for the crosstalk between tumor and immune cells is an area of active investigation, whether host-specific germline variants can dictate immunosuppressive mechanisms has remained a challenge to address. A commonly occurring germline mutation (c.1162G>A/rs351855 G/A) in the FGFR4 (CD334) gene enhances signal transducer and activator of transcription 3 (STAT3) signaling and is associated with poor prognosis and accelerated progression of multiple cancer types. Here, using rs351855 SNP-knockin transgenic mice and Fgfr4-knockout mice, we reveal the genotype-specific gain of immunological function of suppressing the CD8/CD4+FOXP3+CD25+ regulatory T cell ratio in vivo. Furthermore, using knockin transgenic mouse models for lung and breast cancers, we establish the host-specific, tumor-extrinsic functions of STAT3-enhancing germline variants in impeding the tumor infiltration of CD8 T cells. Thus, STAT3-enhancing germline receptor variants contribute to immune evasion through their pleiotropic functions in immune cells.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Mutación de Línea Germinal/inmunología , Proteínas de Neoplasias/inmunología , Neoplasias Experimentales/inmunología , Factor de Transcripción STAT3/inmunología , Linfocitos T Reguladores/inmunología , Escape del Tumor , Animales , Linfocitos T CD8-positivos/patología , Mutación de Línea Germinal/genética , Ratones , Ratones Noqueados , Proteínas de Neoplasias/genética , Neoplasias Experimentales/genética , Neoplasias Experimentales/patología , Receptor Tipo 4 de Factor de Crecimiento de Fibroblastos/genética , Receptor Tipo 4 de Factor de Crecimiento de Fibroblastos/inmunología , Factor de Transcripción STAT3/genética , Linfocitos T Reguladores/patologíaRESUMEN
Fibroblast growth factor (FGF) 23 is a phosphaturic hormone that directly targets cardiac myocytes via FGF receptor (FGFR) 4 thereby inducing hypertrophic myocyte growth and the development of left ventricular hypertrophy (LVH) in rodents. Serum FGF23 levels are highly elevated in patients with chronic kidney disease (CKD), and it is likely that FGF23 directly contributes to the high rates of LVH and cardiac death in CKD. It is currently unknown if the cardiac effects of FGF23 are solely pathological, or if they potentially can be reversed. Here, we report that FGF23-induced cardiac hypertrophy is reversible in vitro and in vivo upon removal of the hypertrophic stimulus. Specific blockade of FGFR4 attenuates established LVH in the 5/6 nephrectomy rat model of CKD. Since CKD mimics a form of accelerated cardiovascular aging, we also studied age-related cardiac remodeling. We show that aging mice lacking FGFR4 are protected from LVH. Finally, FGF23 increases cardiac contractility via FGFR4, while known effects of FGF23 on aortic relaxation do not require FGFR4. Taken together, our data highlight a role of FGF23/FGFR4 signaling in the regulation of cardiac remodeling and function, and indicate that pharmacological interference with cardiac FGF23/FGFR4 signaling might protect from CKD- and age-related LVH.
Asunto(s)
Factores de Crecimiento de Fibroblastos/metabolismo , Hipertrofia Ventricular Izquierda/etiología , Hipertrofia Ventricular Izquierda/metabolismo , Receptor Tipo 4 de Factor de Crecimiento de Fibroblastos/metabolismo , Transducción de Señal , Animales , Biopsia , Dieta , Modelos Animales de Enfermedad , Factor-23 de Crecimiento de Fibroblastos , Factores de Crecimiento de Fibroblastos/sangre , Hipertrofia Ventricular Izquierda/patología , Ratones , Ratones Noqueados , Contracción Miocárdica/genética , Miocitos Cardíacos/metabolismo , Ratas , Receptor Tipo 4 de Factor de Crecimiento de Fibroblastos/antagonistas & inhibidoresRESUMEN
The liver plays a decisive role in the regulation of systemic inflammation. In chronic kidney disease in particular, the liver reacts in response to the uremic milieu, oxidative stress, endotoxemia and the decreased clearance of circulating proinflammatory cytokines by producing a large number of acute-phase reactants. Experimental tools to study inflammation and the underlying role of hepatocytes are crucial to understand the regulation and contribution of hepatic cytokines to a systemic acute phase response and a prolonged pro-inflammatory scenario, especially in an intricate setting such as chronic kidney disease. Since studying complex mechanisms of inflammation in vivo remains challenging, resource-intensive and usually requires the usage of transgenic animals, primary isolated hepatocytes provide a robust tool to gain mechanistic insights into the hepatic acute-phase response. Since this in vitro technique features moderate costs, high reproducibility and common technical knowledge, primary isolated hepatocytes can also be easily used as a screening approach. Here, we describe an enzymatic-based method to isolate primary murine hepatocytes, and we describe the assessment of an inflammatory response in these cells using ELISA and quantitative real-time PCR.
Asunto(s)
Hepatocitos/citología , Inflamación/patología , Hígado/citología , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Animales , Técnicas de Cultivo de Célula/métodos , Células Cultivadas , Citocinas/metabolismo , Ensayo de Inmunoadsorción Enzimática/métodos , Inflamación/inducido químicamente , Inflamación/metabolismo , Mediadores de Inflamación/metabolismo , Interleucina-6/efectos adversos , Lipopolisacáridos/toxicidad , Ratones , Perfusión , Reproducibilidad de los ResultadosRESUMEN
BACKGROUND: Vitamin D deficiency and excess of circulating fibroblast growth factor 23 (FGF23) contribute to cardiovascular mortality in patients with chronic kidney disease (CKD). FGF23 activates FGF receptor 4 and (FGFR4) calcineurin/nuclear factor of activated T cells (NFAT) signaling in cardiac myocytes, thereby causing left ventricular hypertrophy (LVH). Here, we determined if 1,25-dihydroxyvitamin D (calcitriol) inhibits FGF23-induced cardiac signaling and LVH. METHODS: 5/6 nephrectomized (5/6 Nx) rats were treated with different doses of calcitriol for 4 or 10 weeks and cardiac expression of FGF23/FGFR4 and activation of calcineurin/NFAT as well as LVH were analyzed. FGFR4 activation and hypertrophic cell growth were studied in cultured cardiac myocytes that were co-treated with FGF23 and calcitriol. RESULTS: In 5/6Nx rats with LVH, we detected elevated FGF23 expression in bone and myocardium, increased cardiac expression of FGFR4 and elevated cardiac activation of calcineurin/NFAT signaling. Cardiac expression levels of FGF23 and FGFR4 significantly correlated with the presence of LVH in uremic rats. Treatment with calcitriol reduced LVH as well as cardiac FGFR4 expression and calcineurin/NFAT activation. Bone and cardiac FGF23 expression were further stimulated by calcitriol in a dose-dependent manner, but levels of intact cardiac FGF23 protein were suppressed by high-dose calcitriol. In cultured cardiac myocytes, co-treatment with calcitriol blocked FGF23-induced activation of FGFR4 and hypertrophic cell growth. CONCLUSIONS: Our data suggest that in CKD, cardioprotective effects of calcitriol stem from its inhibitory actions on the cardiac FGF23/FGFR4 system, and based on their counterbalancing effects on cardiac myocytes, high FGF23 and low calcitriol synergistically contribute to cardiac hypertrophy.
Asunto(s)
Factores de Crecimiento de Fibroblastos/metabolismo , Hipertrofia Ventricular Izquierda/prevención & control , Receptor Tipo 4 de Factor de Crecimiento de Fibroblastos/metabolismo , Insuficiencia Renal Crónica/complicaciones , Vitamina D/administración & dosificación , Animales , Factor-23 de Crecimiento de Fibroblastos , Humanos , Hipertrofia Ventricular Izquierda/etiología , Hipertrofia Ventricular Izquierda/metabolismo , Masculino , Ratas , Ratas Sprague-Dawley , Transducción de Señal , Vitaminas/administración & dosificaciónRESUMEN
Patients with chronic kidney disease (CKD) develop increased levels of the phosphate-regulating hormone, fibroblast growth factor (FGF) 23, that are associated with a higher risk of mortality. Increases in inflammatory markers are another common feature that predicts poor clinical outcomes. Elevated FGF23 is associated with higher circulating levels of inflammatory cytokines in CKD, which can stimulate osteocyte production of FGF23. Here, we studied whether FGF23 can directly stimulate hepatic production of inflammatory cytokines in the absence of α-klotho, an FGF23 coreceptor in the kidney that is not expressed by hepatocytes. By activating FGF receptor isoform 4 (FGFR4), FGF23 stimulated calcineurin signaling in cultured hepatocytes, which increased the expression and secretion of inflammatory cytokines, including C-reactive protein. Elevating serum FGF23 levels increased hepatic and circulating levels of C-reactive protein in wild-type mice, but not in FGFR4 knockout mice. Administration of an isoform-specific FGFR4 blocking antibody reduced hepatic and circulating levels of C-reactive protein in the 5/6 nephrectomy rat model of CKD. Thus, FGF23 can directly stimulate hepatic secretion of inflammatory cytokines. Our findings indicate a novel mechanism of chronic inflammation in patients with CKD and suggest that FGFR4 blockade might have therapeutic anti-inflammatory effects in CKD.
Asunto(s)
Citocinas/metabolismo , Factores de Crecimiento de Fibroblastos/metabolismo , Hepatocitos/metabolismo , Inflamación/metabolismo , Insuficiencia Renal Crónica/metabolismo , Animales , Calcineurina/metabolismo , Factor-23 de Crecimiento de Fibroblastos , Glucuronidasa/metabolismo , Humanos , Proteínas Klotho , Ratones , Factores de Transcripción NFATC/metabolismo , Fosfolipasa C gamma/metabolismo , Cultivo Primario de Células , Ratas , Receptor Tipo 4 de Factor de Crecimiento de Fibroblastos/metabolismo , Transducción de SeñalRESUMEN
Chronic kidney disease (CKD) is a worldwide public health threat that increases risk of death due to cardiovascular complications, including left ventricular hypertrophy (LVH). Novel therapeutic targets are needed to design treatments to alleviate the cardiovascular burden of CKD. Previously, we demonstrated that circulating concentrations of fibroblast growth factor (FGF) 23 rise progressively in CKD and induce LVH through an unknown FGF receptor (FGFR)-dependent mechanism. Here, we report that FGF23 exclusively activates FGFR4 on cardiac myocytes to stimulate phospholipase Cγ/calcineurin/nuclear factor of activated T cell signaling. A specific FGFR4-blocking antibody inhibits FGF23-induced hypertrophy of isolated cardiac myocytes and attenuates LVH in rats with CKD. Mice lacking FGFR4 do not develop LVH in response to elevated FGF23, whereas knockin mice carrying an FGFR4 gain-of-function mutation spontaneously develop LVH. Thus, FGF23 promotes LVH by activating FGFR4, thereby establishing FGFR4 as a pharmacological target for reducing cardiovascular risk in CKD.