RESUMEN
The activation of mixed lineage kinase-like (MLKL) by receptor-interacting protein kinase-3 (RIPK3) results in plasma membrane (PM) disruption and a form of regulated necrosis, called necroptosis. Here, we show that, during necroptosis, MLKL-dependent calcium (Ca2+) influx and phosphatidylserine (PS) exposure on the outer leaflet of the plasma membrane preceded loss of PM integrity. Activation of MLKL results in the generation of broken, PM "bubbles" with exposed PS that are released from the surface of the otherwise intact cell. The ESCRT-III machinery is required for formation of these bubbles and acts to sustain survival of the cell when MLKL activation is limited or reversed. Under conditions of necroptotic cell death, ESCRT-III controls the duration of plasma membrane integrity. As a consequence of the action of ESCRT-III, cells undergoing necroptosis can express chemokines and other regulatory molecules and promote antigenic cross-priming of CD8+ T cells.
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Membrana Celular/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Necrosis/metabolismo , Animales , Calcio/metabolismo , Supervivencia Celular , Células HT29 , Humanos , Células Jurkat , Ratones , Células 3T3 NIH , Fosfatidilserinas , Proteínas Quinasas/metabolismo , Transducción de SeñalRESUMEN
The pathogenesis of parathyroid gland hyperplasia is poorly understood, and a better understanding is essential if there is to be improvement over the current strategies for prevention and treatment of secondary hyperparathyroidism. Here we investigate the specific role of Klotho expressed in the parathyroid glands (PTGs) in mediating parathyroid hormone (PTH) and serum calcium homeostasis, as well as the potential interaction between calcium-sensing receptor (CaSR) and Klotho. We generated mouse strains with PTG-specific deletion of Klotho and CaSR and dual deletion of both genes. We show that ablating CaSR in the PTGs increases PTH synthesis, that Klotho has a pivotal role in suppressing PTH in the absence of CaSR, and that CaSR together with Klotho regulates PTH biosynthesis and PTG growth. We utilized the tdTomato gene in our mice to visualize and collect PTGs to reveal an inhibitory function of Klotho on PTG cell proliferation. Chronic hypocalcemia and ex vivo PTG culture demonstrated an independent role for Klotho in mediating PTH secretion. Moreover, we identify an interaction between PTG-expressed CaSR and Klotho. These findings reveal essential and interrelated functions for CaSR and Klotho during parathyroid hyperplasia.
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Glucuronidasa/fisiología , Glándulas Paratiroides/metabolismo , Hormona Paratiroidea/biosíntesis , Receptores Acoplados a Proteínas G/fisiología , Animales , Huesos/patología , Calcio/metabolismo , Calcio de la Dieta/administración & dosificación , Femenino , Factor-23 de Crecimiento de Fibroblastos , Glucuronidasa/deficiencia , Glucuronidasa/genética , Homeostasis , Hipercalcemia/genética , Hipercalcemia/patología , Hiperparatiroidismo/genética , Hiperparatiroidismo/patología , Hiperplasia , Hipocalcemia/metabolismo , Hipofosfatemia/genética , Hipofosfatemia/patología , Inmunoprecipitación , Riñón/patología , Proteínas Klotho , Masculino , Ratones , Glándulas Paratiroides/patología , Hormona Paratiroidea/genética , Mapeo de Interacción de Proteínas , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Receptores Sensibles al Calcio , Receptores Acoplados a Proteínas G/deficiencia , Receptores Acoplados a Proteínas G/genéticaRESUMEN
Hyalinosis is a vascular lesion affecting the renal vasculature and contributing to aging-related renal function decline. We assessed whether arteriolar hyalinosis is caused by Klotho deficiency, a state known to induce both renal and vascular phenotypes associated with aging. Histochemistry was used to assess hyalinosis in Klotho-/- kidneys, compared with Klotho+/- and wild-type littermates. Immunohistochemistry was used to investigate vascular lesion composition and the different layers of the vascular wall. Finally, spironolactone was used to inhibit calcification in kl/kl mice, and vascular lesions were characterized in the kidney. Arteriolar hyalinosis was detected in Klotho-/- mice, which was present up to the afferent arterioles. Hyalinosis was accompanied by loss of α-smooth muscle actin expression, whereas the endothelial lining was mostly intact. Hyalinous lesions were positive for IgM and iC3b/c/d, indicating subendothelial leakage of plasma proteins. The presence of extracellular matrix proteins suggested increased production by smooth muscle cells (SMCs). Finally, in Klotho-/- mice with marked vascular calcification, treatment with spironolactone allowed for replacement of calcification by hyalinosis. Klotho deficiency potentiates both endothelial hyperpermeability and SMC dedifferentiation. In the absence of a calcification-inducing stimulus, SMCs assume a synthetic phenotype in response to subendothelial leakage of plasma proteins. In the kidney, this results in arteriolar hyalinosis, which contributes to the decline in renal function. Klotho may play a role in preventing aging-related arteriolar hyalinosis.
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Arterioloesclerosis/patología , Glucuronidasa/fisiología , Riñón/patología , Músculo Liso Vascular/patología , Calcificación Vascular/patología , Animales , Arterioloesclerosis/metabolismo , Células Cultivadas , Riñón/metabolismo , Proteínas Klotho , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Músculo Liso Vascular/metabolismo , Calcificación Vascular/metabolismoRESUMEN
BACKGROUND: Recent studies suggest that the phosphaturic hormone fibroblast growth factor 23 (FGF23) is involved in regulation of renal sodium excretion and blood pressure. There is evidence of both direct effects via regulation of the sodium-chloride symporter (NCC) in the distal tubule, and indirect effects through interactions with the renin-angiotensin-aldosterone system. However, clinical data on the association between FGF23 and renal sodium regulation is lacking. Herein, we investigated the associations of FGF23 with renal sodium handling and blood pressure in non-dialysis CKD patients. METHODS: This was a cross-sectional study encompassing 180 CKD patients Stage 1-5, undergoing renal biopsy. Plasma intact FGF23, 24-h urinary sodium excretion, fractional excretion of sodium (FENa) and blood pressure were measured at baseline. The association between FGF23 and renal sodium handling was explored by multivariate regression analysis. RESULTS: The median age was 52.8 years, 60.6% were men and the median estimated glomerular filtration rate (eGFR) was 50.6 mL/min/1.73 m2. In univariate analysis, FGF23 was positively associated with FENa (Spearman's rho = 0.47; P < 0.001) and systolic blood pressure (rho = 0.17, P < 0.05), but not with plasma sodium, 24-h urinary sodium excretion or mean arterial blood pressure. The association between FGF23 and FENa remained significant after adjustment for potential confounders (multivariable adjusted ß coefficient 0.60, P < 0.001). This association was stronger among the 107 individuals with eGFR <60 mL/min/1.73 m2 (ß = 0.47, P = 0.04) and in the 73 individuals on any diuretics (ß = 0.88, P < 0.001). Adjustment for measured GFR instead of eGFR did not alter the relationship. CONCLUSIONS: FGF23 is independently associated with increased FENa in non-dialysis CKD patients. These data do not support the notion that FGF23 causes clinically significant sodium retention. Further studies are warranted to explore the mechanism underlying this association.
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Factores de Crecimiento de Fibroblastos/metabolismo , Insuficiencia Renal Crónica/orina , Sodio/orina , Adulto , Anciano , Presión Sanguínea , Estudios Transversales , Femenino , Factor-23 de Crecimiento de Fibroblastos , Tasa de Filtración Glomerular , Humanos , Masculino , Persona de Mediana Edad , Pronóstico , Insuficiencia Renal Crónica/sangre , Insuficiencia Renal Crónica/patología , Sodio/sangreRESUMEN
Phosphate homeostasis is primarily maintained in the renal proximal tubules, where the expression of sodium/phosphate cotransporters (Npt2a and Npt2c) is modified by the endocrine actions of both fibroblast growth factor 23 (FGF23) and parathyroid hormone (PTH). However, the specific contribution of each regulatory pathway in the proximal tubules has not been fully elucidated in vivo. We have previously demonstrated that proximal tubule-specific deletion of the FGF23 coreceptor Klotho results in mild hyperphosphatemia with little to no change in serum levels of FGF23, 1,25(OH)2D3, and PTH. In the present study, we characterized mice in which the PTH receptor PTH1R was specifically deleted from the proximal tubules, either alone or in combination with Klotho ( PT-PTH1R-/- and PT-PTH1R/KL-/-, respectively). PT-PTH1R-/- mice showed significant increases in serum FGF23 and PTH levels, whereas serum phosphate levels were maintained in the normal range, and Npt2a and Npt2c expression in brush border membrane (BBM) did not change compared with control mice. In contrast, PT-PTH1R/KL-/- mice displayed hyperphosphatemia and an increased abundance of Npt2a and Npt2c in the renal BBM, along with increased circulating FGF23 levels. While serum calcium was normal, 1,25(OH)2D3 levels were significantly decreased, leading to extremely high levels of PTH. Collectively, mice with a deletion of PTH1R alone in proximal tubules results in only minor changes in phosphate regulation, whereas deletion of both PTH1R and Klotho leads to a severe disturbance, including hyperphosphatemia with increased sodium/phosphate cotransporter expression in BBM. These results suggest an important interplay between the PTH/PTH1R and FGF23/Klotho pathways to affect renal phosphate handling in the proximal tubules.
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Factores de Crecimiento de Fibroblastos/sangre , Glucuronidasa/metabolismo , Hiperfosfatemia/sangre , Túbulos Renales Proximales/metabolismo , Hormona Paratiroidea/sangre , Fosfatos/sangre , Reabsorción Renal , Animales , Calcitriol/sangre , Calcio/sangre , Células Cultivadas , Factor-23 de Crecimiento de Fibroblastos , Predisposición Genética a la Enfermedad , Glucuronidasa/deficiencia , Glucuronidasa/genética , Hiperfosfatemia/genética , Hiperfosfatemia/fisiopatología , Túbulos Renales Proximales/fisiopatología , Proteínas Klotho , Ratones Endogámicos C57BL , Ratones Noqueados , Fenotipo , Receptor de Hormona Paratiroídea Tipo 1/deficiencia , Receptor de Hormona Paratiroídea Tipo 1/genética , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo IIa/metabolismo , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo IIc/metabolismo , Regulación hacia ArribaRESUMEN
Circulating levels of bone-derived fibroblast growth factor 23 (FGF23) increase early during acute and chronic kidney disease and are associated with adverse outcomes. Membrane-bound Klotho acts as a permissive coreceptor for FGF23, and its expression was recently found in osteoblasts/osteocytes. We hypothesized that Klotho in bone cells is part of an autocrine feedback loop that regulates FGF23 expression during renal failure. Thus, we induced renal failure in mice with targeted deletion of Klotho in long bones. Uremic wild-type (KLfl/fl ) and knockout (Prx1-Cre;KLfl/fl ) mice both responded with reduced body weight, kidney atrophy, hyperphosphatemia, and increased bone turnover. Importantly, long bones of Prx1-Cre;KLfl/fl mice but not their axial skeleton failed to increase FGF23 expression as observed in uremic KLfl/fl mice. Consequently, Prx1-Cre;KLfl/fl mice had significantly lower serum FGF23 and parathyroid hormone levels, and higher renal 1-α-hydroxylase expression, serum 1,25-dihydroxyvitamin D, and calcium levels than KLfl/fl mice. These results were confirmed in two independent models of renal failure, adenine diet induced and 5/6 nephrectomy. Moreover, FGF23-treated bone cells required Klotho to increase FGF23 mRNA and ERK phosphorylation. In summary, our novel findings show that Klotho in bone is crucial for inducing FGF23 production upon renal failure. We propose the presence of an autocrine feedback loop in which Klotho senses the need for FGF23.-Kaludjerovic, J., Komaba, H., Sato, T., Erben, R. G., Baron, R., Olauson, H., Larsson, T. E., Lanske, B. Klotho expression in long bones regulates FGF23 production during renal failure.
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Factores de Crecimiento de Fibroblastos/metabolismo , Glucuronidasa/metabolismo , Riñón/metabolismo , Insuficiencia Renal/metabolismo , Animales , Huesos/metabolismo , Calcitriol/metabolismo , Calcio/metabolismo , Factor-23 de Crecimiento de Fibroblastos , Glucuronidasa/deficiencia , Proteínas Klotho , Ratones Noqueados , Osteoblastos/metabolismo , Hormona Paratiroidea/genética , Fosfatos/metabolismoRESUMEN
BACKGROUND: Chronic kidney disease-associated mineral bone disorder (CKD-MBD) is common in pediatric kidney disease patients and a risk factor for future cardiovascular disease (CVD). Fibroblast growth factor-23 (FGF23) and Klotho are novel key players in CKD-MBD, and has been suggested to be involved in the development of CVD. METHODS: This prospective cohort study included 74 pediatric patients; 31 with CKD (age range 0.8-18.8 years, glomerular filtration rate (GFR) range 9-68 mL/min/1.73 m2) and 43 transplanted patients (CKD-T; age range 3.3-17.7 years, GFR range 10-99 mL/min/1.73 m2) examined annually for 3 years. We assessed longitudinal patterns and predictors of FGF23 and soluble Klotho, as well as associations to cardiac remodeling and function using echocardiographic pulse wave Doppler (PWD) and color-coded tissue Doppler imaging (cc-TDI). RESULTS: The prevalence of high FGF23 levels (≥95th percentile) was 60% in CKD and 42% in CKD-T patients, despite a low prevalence of hyperphosphatemia and normal Klotho levels. Low GFR at baseline was a predictor for high mean log FGF23 during follow-up in CKD and CKD-T patients (ß = -0.2, p < 0.001). A high log FGF23 z-score longitudinally was borderline significantly associated with elevated left ventricular mass index (LVMI) in CKD patients (ß = 1.8, p = 0.06). In addition, high log FGF23 (ß = -0.43, p = 0.01) and low log Klotho (ß = 0.44, p = 0.006) over time were associated with a worse left ventricular diastolic function (cc-TDI e'/a') in CKD-T patients. CONCLUSIONS: In pediatric CKD and CKD-T patients, the FGF23 level increase and Klotho level decrease with progressing renal failure, despite well-controlled phosphate levels. Following adjustments, both high FGF23 and low Klotho levels were strongly associated with a worse left ventricular diastolic function longitudinally. The potential role of FGF23 and Klotho in cardiac morbidity in pediatric CKD requires further investigation.
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Enfermedades Cardiovasculares/diagnóstico por imagen , Factores de Crecimiento de Fibroblastos/sangre , Glucuronidasa/sangre , Insuficiencia Renal Crónica/complicaciones , Adolescente , Biomarcadores/sangre , Enfermedades Cardiovasculares/sangre , Enfermedades Cardiovasculares/etiología , Niño , Preescolar , Estudios de Cohortes , Ecocardiografía Doppler , Femenino , Factor-23 de Crecimiento de Fibroblastos , Tasa de Filtración Glomerular , Corazón/diagnóstico por imagen , Humanos , Lactante , Proteínas Klotho , Masculino , Estudios Prospectivos , Insuficiencia Renal Crónica/sangreRESUMEN
Osteocytes within the mineralized bone matrix control bone remodeling by regulating osteoblast and osteoclast activity. Osteocytes express the aging suppressor Klotho, but the functional role of this protein in skeletal homeostasis is unknown. Here we identify Klotho expression in osteocytes as a potent regulator of bone formation and bone mass. Targeted deletion of Klotho from osteocytes led to a striking increase in bone formation and bone volume coupled with enhanced osteoblast activity, in sharp contrast to what is observed in Klotho hypomorphic (kl/kl) mice. Conversely, overexpression of Klotho in cultured osteoblastic cells inhibited mineralization and osteogenic activity during osteocyte differentiation. Further, the induction of chronic kidney disease with high-turnover renal osteodystrophy led to downregulation of Klotho in bone cells. This appeared to offset the skeletal impact of osteocyte-targeted Klotho deletion. Thus, our findings establish a key role of osteocyte-expressed Klotho in regulating bone metabolism and indicate a new mechanism by which osteocytes control bone formation.
Asunto(s)
Envejecimiento/metabolismo , Trastorno Mineral y Óseo Asociado a la Enfermedad Renal Crónica/patología , Glucuronidasa/metabolismo , Osteocitos/metabolismo , Osteogénesis/fisiología , Animales , Densidad Ósea , Huesos/metabolismo , Huesos/patología , Diferenciación Celular , Modelos Animales de Enfermedad , Regulación hacia Abajo , Femenino , Factor-23 de Crecimiento de Fibroblastos , Factores de Crecimiento de Fibroblastos/metabolismo , Glucuronidasa/genética , Humanos , Inmunohistoquímica , Proteínas Klotho , Ratones , Ratones Noqueados , Osteoblastos/fisiología , Osteoclastos/fisiología , Cultivo Primario de Células , Transducción de SeñalRESUMEN
Klotho is a transmembrane protein expressed in the renal tubules where it acts as a permissive coreceptor for fibroblast growth factor 23 (FGF23). FGF23 signaling reduces the abundance of CYP27b1 and phosphate cotransporters NPT2a and NPT2c, leading to a decrease in 1,25(OH)2D3 synthesis and a rise in urinary phosphate excretion, respectively. Systemic or whole-nephron deletion of Klotho in mice results in renal FGF23 resistance characterized by high 1,25(OH)2D3 and phosphate levels and premature aging. Expression of Klotho is highest in the distal tubules, whereas 25OH vitamin D 1α hydroxylation and phosphate reabsorption predominantly occur in the proximal tubules. Currently, the segment-specific roles of Klotho in renal tubules are not fully understood. Here we have generated mice with Klotho specifically ablated from the proximal tubules using 3 different Cre mouse strains. All 3 models displayed impaired urinary phosphate excretion and increased abundance of NPT2a in the brush border membrane. Notably, hyperphosphatemia in knockout mice was mild or nonexistent under basal conditions but occurred upon high phosphate loading, indicating the presence of compensatory mechanisms. Effects on 1,25(OH)2D3 varied between mouse strains but were modest overall. Thus, Klotho expressed in the proximal tubules has a defined but limited role in renal phosphate handling in vivo.
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25-Hidroxivitamina D3 1-alfa-Hidroxilasa/metabolismo , Factores de Crecimiento de Fibroblastos/metabolismo , Glucuronidasa/metabolismo , Túbulos Renales/fisiología , Fosfatos/metabolismo , Eliminación Renal , Envejecimiento Prematuro/metabolismo , Animales , Calcitriol/metabolismo , Femenino , Factor-23 de Crecimiento de Fibroblastos , Glucuronidasa/genética , Humanos , Hiperfosfatemia/sangre , Hiperfosfatemia/genética , Inmunohistoquímica , Túbulos Renales/citología , Proteínas Klotho , Ratones , Ratones Endogámicos C57BL , Fosfatos/orina , Cultivo Primario de Células , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo IIa/metabolismo , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo IIc/metabolismoRESUMEN
Klotho acts as a co-receptor for and dictates tissue specificity of circulating FGF23. FGF23 inhibits PTH secretion, and reduced Klotho abundance is considered a pathogenic factor in renal secondary hyperparathyroidism. To dissect the role of parathyroid gland resident Klotho in health and disease, we generated mice with a parathyroid-specific Klotho deletion (PTH-KL(-/-)). PTH-KL(-/-) mice had a normal gross phenotype and survival; normal serum PTH and calcium; unaltered expression of the PTH gene in parathyroid tissue; and preserved PTH response and sensitivity to acute changes in serum calcium. Their PTH response to intravenous FGF23 delivery or renal failure did not differ compared to their wild-type littermates despite disrupted FGF23-induced activation of the MAPK/ERK pathway. Importantly, calcineurin-NFAT signaling, defined by increased MCIP1 level and nuclear localization of NFATC2, was constitutively activated in PTH-KL(-/-) mice. Treatment with the calcineurin-inhibitor cyclosporine A abolished FGF23-mediated PTH suppression in PTH-KL(-/-) mice whereas wild-type mice remained responsive. Similar results were observed in thyro-parathyroid explants ex vivo. Collectively, we present genetic and functional evidence for a novel, Klotho-independent, calcineurin-mediated FGF23 signaling pathway in parathyroid glands that mediates suppression of PTH. The presence of Klotho-independent FGF23 effects in a Klotho-expressing target organ represents a paradigm shift in the conceptualization of FGF23 endocrine action.
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Factores de Crecimiento de Fibroblastos/genética , Proteínas de la Membrana/genética , Hormona Paratiroidea/genética , Transducción de Señal/genética , Animales , Calcineurina/metabolismo , Inhibidores de la Calcineurina , Calcio/sangre , Ciclosporina/farmacología , Factor-23 de Crecimiento de Fibroblastos , Factores de Crecimiento de Fibroblastos/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Riñón/metabolismo , Proteínas Klotho , Proteínas de la Membrana/metabolismo , Ratones , Glándulas Paratiroides/metabolismo , Hormona Paratiroidea/sangre , Eliminación de Secuencia , Vitamina D/metabolismoRESUMEN
Sclerostin, an osteocyte-derived inhibitor of bone formation, is linked to mineral bone disorder. In order to validate its potential as a predictor of vascular calcification, we explored associations of circulating sclerostin with measures of calcification in 89 epigastric artery biopsies from patients with end-stage renal disease. Significantly higher sclerostin levels were found in the serum of patients with epigastric and coronary artery calcification (calcification score 100 or more). In Spearman's rank correlations, sclerostin levels significantly associated with age, intact parathyroid hormone, bone-specific alkaline phosphatase, and percent calcification. Multivariable regression showed that age, male gender, and sclerostin each significantly associated with the presence of medial vascular calcification. Receiver operating characteristic curve analysis showed that sclerostin (AUC 0.68) predicted vascular calcification. Vascular sclerostin mRNA and protein expressions were low or absent, and did not differ between calcified and non-calcified vessels, suggesting that the vasculature is not a major contributor to circulating levels. Thus, high serum sclerostin levels associate with the extent of vascular calcification as evaluated both by coronary artery CT and scoring of epigastric artery calcification. Among circulating biomarkers of mineral bone disorder, only sclerostin predicted vascular calcification.
RESUMEN
Klotho was discovered as an antiaging gene, and α-Klotho (Klotho) is expressed in multiple tissues with a broad set of biologic functions. Membrane-bound Klotho binds fibroblast growth factor 23 (FGF23), but a soluble form of Klotho is also produced by alternative splicing or cleavage of the extracellular domain of the membrane-bound protein. The relative organ-specific contributions to the levels and effects of circulating Klotho remain unknown. We explored these issues by generating a novel mouse strain with Klotho deleted throughout the nephron (Six2-KL(-/-)). Klotho shedding from Six2-KL(-/-) kidney explants was undetectable and the serum Klotho level was reduced by approximately 80% in Six2-KL(-/-) mice compared with wild-type littermates. Six2-KL(-/-) mice exhibited severe growth retardation, kyphosis, and premature death, closely resembling the phenotype of systemic Klotho knockout mice. Notable biochemical changes included hyperphosphatemia, hypercalcemia, hyperaldosteronism, and elevated levels of 1,25-dihydroxyvitamin D and Fgf23, consistent with disrupted renal Fgf23 signaling. Kidney histology demonstrated interstitial fibrosis and nephrocalcinosis in addition to absent dimorphic tubules. A direct comparative analysis between Six2-KL(-/-) and systemic Klotho knockout mice supports extensive, yet indistinguishable, extrarenal organ manifestations. Thus, our data reveal the kidney as the principal contributor of circulating Klotho and Klotho-induced antiaging traits.
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Glucuronidasa/fisiología , Riñón/metabolismo , Envejecimiento/metabolismo , Animales , Femenino , Factor-23 de Crecimiento de Fibroblastos , Factores de Crecimiento de Fibroblastos/metabolismo , Riñón/patología , Proteínas Klotho , Masculino , Ratones NoqueadosRESUMEN
Klotho regulates many pathways in the aging process, but it remains unclear how it is physiologically regulated. Because Klotho is synthesized, cleaved, and released from the kidney; activates the chief urinary K+ secretion channel (ROMK) and stimulates urinary K+ secretion, we explored if Klotho protein is regulated by dietary K+ and the potassium-regulatory hormone, Aldosterone. Klotho protein along the nephron was evaluated in humans and in wild-type (WT) mice; and in mice lacking components of Aldosterone signaling, including the Aldosterone-Synthase KO (AS-KO) and the Mineralocorticoid-Receptor KO (MR-KO) mice. We found the specific cells of the distal nephron in humans and mice that are chief sites of regulated K+ secretion have the highest Klotho protein expression along the nephron. WT mice fed K+-rich diets increased Klotho expression in these cells. AS-KO mice exhibit normal Klotho under basal conditions but could not upregulate Klotho in response to high-K+ intake in the K+-secreting cells. Similarly, MR-KO mice exhibit decreased Klotho protein expression. Together, i) Klotho is highly expressed in the key sites of regulated K+ secretion in humans and mice, ii) In mice, K+-rich diets increase Klotho expression specifically in the potassium secretory cells of the distal nephron, iii) Aldosterone signaling is required for Klotho response to high K+ intake.
Asunto(s)
Aldosterona , Glucuronidasa , Proteínas Klotho , Ratones Noqueados , Potasio , Proteínas Klotho/metabolismo , Animales , Humanos , Ratones , Potasio/metabolismo , Aldosterona/metabolismo , Glucuronidasa/metabolismo , Glucuronidasa/genética , Masculino , Nefronas/metabolismo , Potasio en la Dieta/metabolismo , Potasio en la Dieta/administración & dosificación , Femenino , Receptores de Mineralocorticoides/metabolismo , Receptores de Mineralocorticoides/genética , Ratones Endogámicos C57BLRESUMEN
PURPOSE OF REVIEW: The wealth of data regarding fibroblast growth factor-23 (FGF23) and Klotho in chronic kidney disease (CKD) has risen exponentially over the past decade. This review is an attempt to summarize pivotal aspects of previous research, provide an update of recent findings and define important areas for future investigation. RECENT FINDINGS: The phosphaturic hormone FGF23 increases dramatically as renal function declines. Identification of contributing stimuli to the rise in FGF23 is fundamental and recent evidence suggest a multifactorial cause which entails perturbed osteocyte function and renal mechanisms such as Klotho deficiency and, somewhat paradoxically, systemic Klotho excess. Circulating FGF23 predicts adverse outcomes, particularly cardiovascular disease, in CKD as well as in the general population. The concept of FGF23 merely as a biomarker and regulator of mineral metabolism is currently challenged by data linking FGF23 to pathological processes such as cardiac hypertrophy. Conversely, tissue level of the FGF23 coreceptor Klotho declines in early CKD and this deficiency is linked to accelerated ageing, cellular senescence, vascular calcification, oxidative stress and renal fibrosis. At present, methodological difficulties limit the utility of soluble Klotho measurements. Animal proof-of-concept studies have demonstrated beneficial effects of Klotho delivery in CKD, whereas anti-FGF23 therapy using neutralizing antibodies improved biochemical and bone parameters at the expense of increased vascular calcification and mortality. SUMMARY: Pathological alterations of FGF23-Klotho in CKD are implicated as clinical biomarkers and may provide novel therapeutic strategies to alleviate the cardiovascular risk and slow CKD progression.
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Factores de Crecimiento de Fibroblastos/sangre , Glucuronidasa/sangre , Riñón/metabolismo , Insuficiencia Renal Crónica/sangre , Animales , Biomarcadores/metabolismo , Progresión de la Enfermedad , Factor-23 de Crecimiento de Fibroblastos , Humanos , Riñón/patología , Riñón/fisiopatología , Proteínas Klotho , Pronóstico , Insuficiencia Renal Crónica/mortalidad , Insuficiencia Renal Crónica/patología , Insuficiencia Renal Crónica/fisiopatología , Insuficiencia Renal Crónica/terapia , Transducción de SeñalRESUMEN
FGF23 is a bone-derived hormone that regulates mineral metabolism by inhibiting renal tubular phosphate reabsorption and suppressing circulating 1,25(OH)2D and PTH levels. These effects are mediated by FGF-receptor binding and activation in the presence of its coreceptor Klotho, which is expressed in the distal tubules of the kidney. Recently, expression of Klotho in skeletal tissues has been reported, indicating a direct, yet unclear, extrarenal effect of FGF23 on cells involved with bone development and remodeling. In the present study, we found that bone marrow stromal cells harvested from Klotho null mice developed fewer osteoblastic but more adipocytic colonies than cells from wild-type mice. The underlying mechanism was explored by experiments on mouse C3H10T1/2 cells. We found that Klotho was weakly expressed and that FGF23 dose-dependently affected the lineage fate determination. The effects of FGF23 on cell differentiation can be diminished by SU 5402, a specific tyrosine kinase inhibitor for FGF receptors. Our results indicate that FGF23 directly affects the differentiation of bone marrow stromal cells.
Asunto(s)
Linaje de la Célula , Factores de Crecimiento de Fibroblastos/fisiología , Regulación de la Expresión Génica , Células Madre Mesenquimatosas/citología , Adipocitos/citología , Envejecimiento , Animales , Huesos/metabolismo , Diferenciación Celular , Proliferación Celular , Relación Dosis-Respuesta a Droga , Factor-23 de Crecimiento de Fibroblastos , Glucuronidasa/metabolismo , Túbulos Renales/metabolismo , Proteínas Klotho , Ratones , Ratones Endogámicos C3H , Osteoblastos/metabolismo , Osteoporosis/metabolismo , Fosfatos/metabolismo , Unión Proteica , Proteínas Recombinantes/metabolismoRESUMEN
BACKGROUND: In vivo models of uremia are important tools to study numerous aspects of acute and chronic kidney disease. Mouse models are pivotal because most genetically engineered animal models are mice, which allow dissecting the impact of selected target genes in renal failure. Adenine-based protocols to induce renal failure are available in rats, but have not been adapted in mice due to their reluctance to consume adenine. In the current paper we developed a novel method for induction of renal failure through dietary delivery of adenine mixed in a casein-based diet. RESULTS: After an induction phase, a stable model of renal impairment was obtained (target urea range 80-100 mg/dL), mimicking several aspects of chronic kidney disease - mineral and bone disorder including secondary hyperparathyroidism, bone abnormalities and pathological elevation of FGF23. No deaths occurred and the level of uremia was adaptable through adjustments of the adenine content, providing significant advantages compared to existing models. In an 8-week proof-of-concept study, renal histology showed mainly a tubulointerstitial damage with infiltrating leukocytes, interstitial edema and widening of the Bownman's space. Fibrosis was present in most animals as defined by histology and gene expression changes of fibrosis markers. Parathyroid cell proliferation was markedly increased but without signs of glandular hypertrophy. Skeletal histology showed increased trabecular bone and bone marrow adiposity whereas bone biomarkers (CTX and PINP) suggested higher bone formation, but surprisingly, lower bone resorption and perturbations in mineral metabolism. CONCLUSIONS: We present a novel, non-surgical method for induction of renal failure in mice. This is an important complement to existing uremic models for pathophysiological studies in acute and chronic kidney disease, especially in terms of tubulointerstitial lesions.
Asunto(s)
Adenina/administración & dosificación , Adenina/toxicidad , Modelos Animales de Enfermedad , Nefritis Intersticial/inducido químicamente , Nefritis Intersticial/patología , Animales , Factor-23 de Crecimiento de Fibroblastos , Ratones , Ratones Endogámicos C57BL , Nefritis Intersticial/metabolismoRESUMEN
Renal Klotho controls mineral metabolism by directly modulating tubular reabsorption of phosphate and calcium and by acting as a co-receptor for the phosphaturic and vitamin D-regulating hormone fibroblast growth factor-23 (FGF23). Klotho null mice have a markedly abnormal phenotype. We sought to determine effects of renal-specific and partial deletion of Klotho to facilitate investigation of its roles in health and disease. We generated a mouse model with partial deletion of Klotho in distal tubular segments (Ksp-KL(-/-)). In contrast to Klotho null mice, Ksp-KL(-/-) mice were fertile, had a normal gross phenotype, and did not have vascular or tubular calcification on renal histology. However, Ksp-KL(-/-) mice were hyperphosphatemic with elevated FGF23 levels and abundant expression of the sodium-phosphate cotransporter Npt2a at the brush border membrane. Serum calcium and 1,25-dihydroxyvitamin D(3) levels were normal but parathyroid hormone levels were decreased. TRPV5 protein was reduced with a parallel mild increase in urinary calcium excretion. Renal expression of vitamin D regulatory enzymes and vitamin D receptor was higher in Ksp-KL(-/-) mice than controls, suggesting increased turnover of vitamin D metabolites and a functional increase in vitamin D signaling. There was a threshold effect of residual renal Klotho expression on FGF23: deletion of >70% of Klotho resulted in FGF23 levels 30-250 times higher than in wild-type mice. A subgroup of Ksp-KL(-/-) mice with normal phosphate levels had elevated FGF23, suggesting a Klotho-derived renal-bone feedback loop. Taken together, renal FGF23-Klotho signaling, which is disrupted in CKD, is essential for homeostatic control of mineral metabolism.
Asunto(s)
Glucuronidasa/deficiencia , Túbulos Renales Distales/metabolismo , Minerales/metabolismo , Animales , Calcio/metabolismo , Femenino , Factor-23 de Crecimiento de Fibroblastos , Factores de Crecimiento de Fibroblastos/metabolismo , Expresión Génica , Marcación de Gen , Glucuronidasa/genética , Glucuronidasa/metabolismo , Homeostasis , Hiperfosfatemia/genética , Hiperfosfatemia/metabolismo , Proteínas Klotho , Masculino , Ratones , Ratones Noqueados , Hormona Paratiroidea/sangre , Transducción de Señal , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo IIa/metabolismo , Vitamina D/metabolismoRESUMEN
αKlotho (Klotho) has well established renoprotective effects; however, the molecular pathways mediating its glomerular protection remain incompletely understood. Recent studies have reported that Klotho is expressed in podocytes and protects glomeruli through auto- and paracrine effects. Here, we examined renal expression of Klotho in detail and explored its protective effects in podocyte-specific Klotho knockout mice, and by overexpressing human Klotho in podocytes and hepatocytes. We demonstrate that Klotho is not significantly expressed in podocytes, and transgenic mice with either a targeted deletion or overexpression of Klotho in podocytes lack a glomerular phenotype and have no altered susceptibility to glomerular injury. In contrast, mice with hepatocyte-specific overexpression of Klotho have high circulating levels of soluble Klotho, and when challenged with nephrotoxic serum have less albuminuria and less severe kidney injury compared to wildtype mice. RNA-seq analysis suggests an adaptive response to increased endoplasmic reticulum stress as a putative mechanism of action. To evaluate the clinical relevance of our findings, the results were validated in patients with diabetic nephropathy, and in precision cut kidney slices from human nephrectomies. Together, our data reveal that the glomeruloprotective effects of Klotho is mediated via endocrine actions, which increases its therapeutic potential for patients with glomerular diseases.
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Nefropatías Diabéticas , Podocitos , Humanos , Ratones , Animales , Glomérulos Renales , Nefropatías Diabéticas/metabolismo , Riñón/metabolismo , Albuminuria/metabolismo , Ratones Transgénicos , Ratones NoqueadosRESUMEN
Background: Disturbances in magnesium homeostasis are common in patients with chronic kidney disease (CKD) and are associated with increased mortality. The kidney is a key organ in maintaining normal serum magnesium concentrations. To this end, fractional excretion of magnesium (FEMg) increases as renal function declines. Despite recent progress, the hormonal regulation of renal magnesium handling is incompletely understood. Fibroblast Growth Factor 23 (FGF23) is a phosphaturic hormone that has been linked to renal magnesium handling. However, it has not yet been reported whether FGF23 is associated with renal magnesium handling in CKD patients. Methods: The associations between plasma FGF23 levels, plasma and urine magnesium concentrations and FEMg was investigated in a cross-sectional cohort of 198 non-dialysis CKD patients undergoing renal biopsy. Results: FGF23 was significantly correlated with FEMg (Pearson's correlation coefficient = 0.37, p<0.001) and urinary magnesium (-0.14, p=0.04), but not with plasma magnesium. The association between FGF23 and FEMg remained significant after adjusting for potential confounders, including estimated glomerular filtration rate (eGFR), parathyroid hormone and 25-hydroxyvitamin D. Conclusions: We report that plasma FGF23 is independently associated with measures of renal magnesium handling in a cohort of non-dialysis CKD patients. A potential causal relationship should be investigated in future studies.
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Magnesio , Insuficiencia Renal Crónica , Humanos , Factor-23 de Crecimiento de Fibroblastos , Estudios Transversales , Factores de Crecimiento de Fibroblastos/metabolismo , Riñón/metabolismo , Insuficiencia Renal Crónica/complicacionesRESUMEN
OBJECTIVE: Hyperphosphatemia is a cardiovascular risk factor in patients with chronic kidney disease. Relations of circulating calcium (Ca) and phosphorus (Pi) to long-term mortality risk in the community require further investigation. METHODS AND RESULTS: Associations of serum Ca and Pi to mortality were evaluated in a community-based cohort of 2176 men (mean age, 50.1 years). During follow-up (median, 29.8 years), 1009 men died, and 466 of these deaths resulted from cardiovascular causes. In Cox proportional hazards models, serum Pi and [CaxPi] were independent predictors of total mortality (hazard ratio per SD, 1.06; 95% CI, 1.01-1.12; P=0.03; 1.07; 95% CI, 1.01-1.12; P=0.01) and cardiovascular mortality (1.10; 95% CI, 1.02-1.18; P=0.01; 1.10; 95% CI, 1.03-1.19; P=0.008). Serum Ca was associated with risk of total mortality (1.08; 95% CI, 1.01-1.16; P=0.02) and noncardiovascular mortality (1.10; 95% CI, 1.01-1.21; P=0.04). Results were consistent after multivariate adjustments in subsamples of individuals with estimated glomerular filtration rate >90 mL/min and low-to-normal serum Ca and Pi. CONCLUSIONS: Circulating Ca and Pi levels are associated with risks of total, cardiovascular, and noncardiovascular mortality in the community, and their conjoint effects are additive. Additional studies are warranted to evaluate whether Ca and Pi are modifiable risk factors in the general population.