RESUMEN
Acute renal depletion of sorting nexin 1 (SNX1) in mice results in blunted natriuretic response and hypertension due to impaired dopamine D5 receptor (D5 R) activity. We elucidated the molecular mechanisms for these phenotypes in Snx1-/- mice. These mice had increased renal expressions of angiotensin II type 1 receptor (AT1 R), NADPH oxidase (NOX) subunits, D5 R, and NaCl cotransporter. Basal reactive oxygen species (ROS), NOX activity, and blood pressure (BP) were also higher in Snx1-/- mice, which were normalized by apocynin, a drug that prevents NOX assembly. Renal proximal tubule (RPT) cells from hypertensive (HT) Euro-American males had deficient SNX1 activity, impaired D5 R endocytosis, and increased ROS compared with cells from normotensive (NT) Euro-American males. siRNA-mediated depletion of SNX1 in RPT cells from NT subjects led to a blunting of D5 R agonist-induced increase in cAMP production and decrease in Na+ transport, effects that were normalized by over-expression of SNX1. Among HT African-Americans, three of the 12 single nucleotide polymorphisms interrogated for the SNX1 gene were associated with a decrease in systolic BP in response to hydrochlorothiazide (HCTZ). The results illustrate a new paradigm for the development of hypertension and imply that the trafficking protein SNX1 may be a crucial determinant for hypertension and response to antihypertensive therapy.
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Hipertensión/metabolismo , Estrés Oxidativo/fisiología , Nexinas de Clasificación/metabolismo , Animales , Presión Sanguínea/fisiología , Línea Celular , Femenino , Humanos , Riñón/metabolismo , Túbulos Renales Proximales/metabolismo , Masculino , Ratones , NADPH Oxidasas/metabolismo , Oxidación-Reducción , Transporte de Proteínas/fisiología , ARN Interferente Pequeño/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Receptor de Angiotensina Tipo 1/metabolismoRESUMEN
Na+/H+ exchanger regulatory factor 1 (NHERF1; also known as ezrin-radixin-moesin-binding phosphoprotein 50) is a PSD-95, disc large, zona occludens-1 adapter that acts as a scaffold for signaling complexes and cytoskeletal-plasma membrane interactions. NHERF1 is crucial to ß-2-adrenoceptor (ß2AR)-mediated activation of cystic fibrosis transmembrane conductance regulator (CFTR) in epithelial cells, and NHERF1 has been proposed to mediate the recycling of internalized ß2AR back to the cell membrane. In the current study, we assessed the role of NHERF1 in regulating cAMP-mediated signaling and immunomodulatory functions in airway smooth muscle (ASM). NHERF1 knockdown attenuated the induction of (protein kinase A) phospho-vasodilator-stimulated phosphoprotein (p-VASP) by isoproterenol (ISO), prostaglandin E2 (PGE2), or forskolin (FSK) as well as the induction of p-heat shock protein 20 after 4 h of stimulation with ISO and FSK. NHERF1 knockdown fully abrogated the ISO-, PGE2-, and FSK-induced IL-6 gene expression and cytokine production without affecting cAMP-mediated phosphodiesterase 4D (PDE4D) gene expression, phospho-cAMP response element-binding protein (p-CREB), and cAMP response element (CRE)-Luc, or PDGF-induced cyclin D1 expression. Interestingly, NHERF1 knockdown prevented ISO-induced chromatin-binding of the transcription factor CCAAT-enhancer-binding protein-ß (c/EBPß). c/EBPß knockdown almost completely abrogated the cAMP-mediated IL-6 but not PDE4D gene expression. The differential regulation of cAMP-induced signaling and gene expression in our study indicates a role for NHERF1 in the compartmentalization of cAMP signaling in ASM.-Pera, T., Tompkins, E., Katz, M., Wang, B., Deshpande, D. A., Weinman, E. J., Penn, R. B. Specificity of NHERF1 regulation of GPCR signaling and function in human airway smooth muscle.
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Músculo Liso/metabolismo , Fosfoproteínas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Sistema Respiratorio/metabolismo , Intercambiadores de Sodio-Hidrógeno/metabolismo , Proteína beta Potenciadora de Unión a CCAAT/metabolismo , AMP Cíclico/metabolismo , Ciclina D1/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Músculo Liso/inmunología , Músculo Liso/fisiología , Fosfoproteínas/antagonistas & inhibidores , Fosfoproteínas/genética , Fenómenos Fisiológicos Respiratorios , Sistema Respiratorio/inmunología , Transducción de Señal , Intercambiadores de Sodio-Hidrógeno/antagonistas & inhibidores , Intercambiadores de Sodio-Hidrógeno/genéticaRESUMEN
BACKGROUND: Cystinuria is an inherited disorder of renal amino acid transport that causes recurrent nephrolithiasis and significant morbidity in humans. It has an incidence of 1 in 7000 worldwide making it one of the most common genetic disorders in man. We phenotypically characterized a mouse model of cystinuria type A resultant from knockout of Slc3a1. METHODS: Knockout of Slc3a1 at RNA and protein levels was evaluated using real-time quantitative PCR and immunofluorescence. Slc3a1 knockout mice were placed on normal or breeder chow diets and evaluated for cystine stone formation over time suing x-ray analysis, and the development of kidney injury by measuring injury biomarkers. Kidney injury was also evaluated via histologic analysis. Amino acid levels were measured in the blood of mice using high performance liquid chromatography. Liver glutathione levels were measured using a luminescent-based assay. RESULTS: We confirmed knockout of Slc3a1 at the RNA level, while Slc7a9 RNA representing the co-transporter was preserved. As expected, we observed bladder stone formation in Slc3a1-/- mice. Male Slc3a1-/- mice exhibited lower weights compared to Slc3a1+/+. Slc3a1-/- mice on a regular diet demonstrated elevated blood urea nitrogen (BUN) without elevation of serum creatinine. However, placing the knockout animals on a breeder chow diet, containing a higher cystine concentration, resulted in the development of elevation of both BUN and creatinine indicative of more severe chronic kidney disease. Histological examination revealed that these dietary effects resulted in worsened kidney tubular obstruction and interstitial inflammation as well as worsened bladder inflammation. Cystine is a precursor for the antioxidant molecule glutathione, so we evaluated glutathione levels in the livers of Slc3a1-/- mice. We found significantly lowered levels of both reduced and total glutathione in the knockout animals. CONCLUSIONS: Our results suggest that that diet can affect the development and progression of chronic kidney disease in an animal model of cystinuria, which may have important implications for patients with this disease. Additionally, reduced glutathione may predispose those with cystinuria to injury caused by oxidative stress. Word count: 327.
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Nitrógeno de la Urea Sanguínea , Cistinuria/diagnóstico por imagen , Cistinuria/metabolismo , Sistemas de Transporte de Aminoácidos Básicos/deficiencia , Sistemas de Transporte de Aminoácidos Básicos/genética , Sistemas de Transporte de Aminoácidos Neutros/deficiencia , Sistemas de Transporte de Aminoácidos Neutros/genética , Animales , Cistinuria/genética , Femenino , Masculino , Ratones , Ratones NoqueadosRESUMEN
Dopamine decreases Na-K-ATPase (NKA) activity by PKC-dependent phosphorylation and endocytosis of the NKA α1. Dopamine-mediated regulation of NKA is impaired in aging and some forms of hypertension. Using opossum (OK) proximal tubule cells (PTCs), we demonstrated that sodium-hydrogen exchanger regulatory factor-1 (NHERF-1) associates with NKA α1 and dopamine-1 receptor (D1R). This association is required for the dopamine-mediated regulation of NKA. In OK cells, dopamine decreases NHERF-1 association with NKA α1 but increases its association with D1R. However, it is not known whether NHERF-1 plays a role in dopamine-mediated NKA regulation in animal models of hypertension. We hypothesized that defective dopamine-mediated regulation of NKA results from the decrease in NHERF-1 expression in rat renal PTCs isolated from animal models of hypertension [spontaneously hypertensive rats (SHRs) and aged F344 rats]. To test this hypothesis, we isolated and cultured renal PTCs from 22-mo-old F344 rats and their controls, normotensive 4-mo-old F344 rats, and SHRs and their controls, normotensive Wistar-Kyoto (WKY) rats. The results demonstrate that in both hypertensive models (SHR and aged F344), NHERF-1 expression, dopamine-mediated phosphorylation of NKA, and ouabain-inhibitable K+ transport are reduced. Transfection of NHERF-1 into PTCs from aged F344 and SHRs restored dopamine-mediated inhibition of NKA. These results suggest that decreased renal NHERF-1 expression contributes to the impaired dopamine-mediated inhibition of NKA in PTCs from animal models of hypertension.
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Hipertensión/genética , Túbulos Renales Proximales/metabolismo , Fosfoproteínas/biosíntesis , Intercambiadores de Sodio-Hidrógeno/biosíntesis , ATPasa Intercambiadora de Sodio-Potasio/biosíntesis , Animales , Presión Sanguínea/genética , Línea Celular , Modelos Animales de Enfermedad , Dopamina/metabolismo , Regulación de la Expresión Génica/genética , Humanos , Hipertensión/metabolismo , Hipertensión/patología , Riñón/metabolismo , Riñón/patología , Túbulos Renales Proximales/patología , Masculino , Fosfoproteínas/genética , Ratas , Ratas Endogámicas SHR , Transducción de Señal/genética , Intercambiadores de Sodio-Hidrógeno/genética , ATPasa Intercambiadora de Sodio-Potasio/genéticaRESUMEN
UNLABELLED: The intercellular adhesion molecule 1 (ICAM-1) is induced in mouse liver after bile duct ligation (BDL) and plays a key role in neutrophil-mediated liver injury in BDL mice. ICAM-1 has been shown to interact with cytoskeletal ezrin-radixin-moesin (ERM) proteins that also interact with the PDZ protein, Na(+) /H(+) exchanger regulatory factor 1 (NHERF-1/EBP50). In NHERF-1(-/-) mice, ERM proteins are significantly reduced in brush-border membranes from kidney and small intestine. ERM knockdown reduces ICAM-1 expression in response to tumor necrosis factor alpha. Here we show that NHERF-1 assembles ERM proteins, ICAM-1 and F-actin into a macromolecule complex that is increased in mouse liver after BDL. Compared to wild-type (WT) mice, both sham-operated and BDL NHERF-1(-/-) mice have lower levels of activated ERM and ICAM-1 protein in the liver accompanied by significantly reduced hepatic neutrophil accumulation, serum alanine aminotransferase, and attenuated liver injury after BDL. However, total bile acid concentrations in serum and liver of sham and BDL NHERF-1(-/-) mice were not significantly different from WT controls, although hepatic tetrahydroxylated bile acids and Cyp3a11 messenger RNA levels were higher in NHERF-1(-/-) BDL mice. CONCLUSION: NHERF-1 participates in the inflammatory response that is associated with BDL-induced liver injury. Deletion of NHERF-1 in mice leads to disruption of the formation of ICAM-1/ERM/NHERF-1 complex and reduction of hepatic ERM proteins and ICAM-1, molecules that are up-regulated and are essential for neutrophil-mediated liver injury in cholestasis. Further study of the role of NHERF-1 in the inflammatory response in cholestasis and other forms of liver injury should lead to discovery of new therapeutic targets in hepatic inflammatory diseases.
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Colestasis Intrahepática/etiología , Molécula 1 de Adhesión Intercelular/fisiología , Hepatopatías/etiología , Fosfoproteínas/fisiología , Intercambiadores de Sodio-Hidrógeno/fisiología , Animales , Hepatitis/etiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neurofibromina 2/fisiología , Fosfoproteínas/genética , Intercambiadores de Sodio-Hidrógeno/genéticaRESUMEN
Na+/H+ exchanger regulatory factor (NHERF1) plays a critical role in the renal transport of phosphate by binding to Na+-Pi cotransporter (NpT2a) in the proximal tubule. While the association between NpT2a and NHERF1 in the apical membrane is known, the role of NHERF1 to regulate the trafficking of NpT2a has not been studied. To address this question, we performed cell fractionation by sucrose gradient centrifugation in opossum kidney (OK) cells placed in low-Pi medium to stimulate forward trafficking of NpT2a. Immunoblot analysis demonstrated expression of NpT2a and NHERF1 in the endoplasmic reticulum (ER)/Golgi. Coimmunoprecipitation demonstrated a NpT2a-NHERF1 interaction in the ER/Golgi. Low-Pi medium for 4 and 8 h triggered a decrease in NHERF1 in the plasma membrane with a corresponding increase in the ER/Golgi. Time-lapse total internal reflection fluorescence imaging of OK cells placed in low-Pi medium, paired with particle tracking and mean square displacement analysis, indicated active directed movement of NHERF1 at early and late time points, whereas NpT2a showed active movement only at later times. Silence of NHERF1 in OK cells expressing green fluorescent protein (GFP)-NpT2a resulted in an intracellular accumulation of GFP-NpT2a. Transfection with GFP-labeled COOH-terminal (TRL) PDZ-binding motif deleted or wild-type NpT2a in OK cells followed by cell fractionation and immunoprecipitation confirmed that the interaction between NpT2a and NHERF1 was dependent on the TRL motif of NpT2a. We conclude that appropriate trafficking of NpT2a to the plasma membrane is dependent on the initial association between NpT2a and NHERF1 through the COOH-terminal TRL motif of NpT2a in the ER/Golgi and requires redistribution of NHERF1 to the ER/Golgi.
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Retículo Endoplásmico/metabolismo , Aparato de Golgi/metabolismo , Riñón/metabolismo , Fosfoproteínas/metabolismo , Intercambiadores de Sodio-Hidrógeno/metabolismo , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo IIa/metabolismo , Animales , Línea Celular , DidelphisRESUMEN
Sodium-coupled SLC12 cation chloride cotransporters play important roles in cell volume and chloride homeostasis, epithelial fluid secretion, and renal tubular salt reabsorption. These cotransporters are phosphorylated and activated indirectly by With-No-Lysine (WNK) kinases through their downstream effector kinases, Ste20- and SPS1-related proline alanine-rich kinase (SPAK) and oxidative stress-responsive kinase 1 (OSR1). Multiple WNK kinases can coexist within a single cell type, although their relative contributions to SPAK/OSR1 activation and salt transport remain incompletely understood. Deletion of specific WNKs from cells that natively express a functional WNK-SPAK/OSR1 network will help resolve these knowledge gaps. Here, we outline a simple method to selectively knock out full-length WNK1 expression from mammalian cells using RNA-guided clustered regularly interspaced short palindromic repeats/Cas9 endonucleases. Two clonal cell lines were generated by using a single-guide RNA (sgRNA) targeting exon 1 of the WNK1 gene, which produced indels that abolished WNK1 protein expression. Both cell lines exhibited reduced endogenous WNK4 protein abundance, indicating that WNK1 is required for WNK4 stability. Consistent with an on-target effect, the reduced WNK4 abundance was associated with increased expression of the KLHL3/cullin-3 E3 ubiquitin ligase complex and was rescued by exogenous WNK1 overexpression. Although the morphology of the knockout cells was indistinguishable from control, they exhibited low baseline SPAK/OSR1 activity and failed to trigger regulatory volume increase after hypertonic stress, confirming an essential role for WNK1 in cell volume regulation. Collectively, our data show how this new, powerful, and accessible gene-editing technology can be used to dissect and analyze WNK signaling networks.
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Sistemas CRISPR-Cas , Técnicas de Silenciamiento del Gen/métodos , Genoma Humano , Péptidos y Proteínas de Señalización Intracelular/deficiencia , Proteínas Serina-Treonina Quinasas/deficiencia , Proteínas Adaptadoras Transductoras de Señales , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Forma de la Célula , Tamaño de la Célula , Proteínas Cullin/genética , Proteínas Cullin/metabolismo , Regulación hacia Abajo , Exones , Genotipo , Células HEK293 , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas de Microfilamentos , Antígenos de Histocompatibilidad Menor , Presión Osmótica , Fenotipo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Estabilidad Proteica , Transducción de Señal , Factores de Tiempo , Transfección , Proteína Quinasa Deficiente en Lisina WNK 1RESUMEN
P(i) uptake in the small intestine occurs predominantly through the NaPi-2b (SLC34a2) co-transporter. NaPi-2b is regulated by changes in dietary P(i) but the mechanisms underlying this regulation are largely undetermined. Sequence analyses show NaPi-2b has a PDZ binding motif at its C terminus. Immunofluorescence imaging shows NaPi-2b and two PDZ domain containing proteins, NHERF1 and PDZK1, are expressed in the apical microvillar domain of rat small intestine enterocytes. Co-immunoprecipitation studies in rat enterocytes show that NHERF1 associates with NaPi-2b but not PDZK1. In HEK co-expression studies, GFP-NaPi-2b co-precipitates with FLAG-NHERF1. This interaction is markedly diminished when the C-terminal four amino acids are truncated from NaPi-2b. FLIM-FRET analyses using tagged proteins in CACO-2(BBE) cells show a distinct phasor shift between NaPi-2b and NHERF1 but not between NaPi-2b and the PDZK1 pair. This shift demonstrates that NaPi-2b and NHERF1 reside within 10 nm of each other. NHERF1(-/-) mice, but not PDZK1(-/-) mice, had a diminished adaptation of NaPi-2b expression in response to a low P(i) diet. Together these studies demonstrate that NHERF1 associates with NaPi-2b in enterocytes and regulates NaPi-2b adaptation.
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Enterocitos/metabolismo , Regulación de la Expresión Génica/fisiología , Fosfoproteínas/metabolismo , Intercambiadores de Sodio-Hidrógeno/metabolismo , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo IIb/biosíntesis , Animales , Células CACO-2 , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Proteínas del Citoesqueleto , Enterocitos/citología , Células HEK293 , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Masculino , Proteínas de la Membrana , Ratones , Ratones Noqueados , Microvellosidades/genética , Microvellosidades/metabolismo , Fosfoproteínas/genética , Estructura Terciaria de Proteína , Ratas , Ratas Sprague-Dawley , Intercambiadores de Sodio-Hidrógeno/genética , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo IIb/genéticaRESUMEN
Increases in serum concentrations of parathyroid hormone (PTH) and fibroblast growth factor 23 (FGF-23) and ultimately phosphate and decreases in 1,25-dihydroxyvitamin D level are thought to play a central role in the progressive nature of kidney disease and the development of cardiovascular disease in patients with chronic kidney disease. The initial changes in PTH and FGF-23 levels are adaptive to maintain serum phosphate concentration and phosphate load within defined levels by increasing urinary excretion of phosphate. Less well appreciated is the unanticipated finding that absorption of phosphate from the gastrointestinal tract is not downregulated in chronic kidney disease. This maladaptive response maintains higher levels of phosphate absorption, thereby contributing to the phosphate burden. Moreover, in response to a low-phosphate diet, as often is prescribed to such patients, gut phosphate absorption may be enhanced, undermining the potential beneficial effects of this intervention. Given the poor response to limiting phosphate intake and the use of phosphate binders, we suggest that research efforts be oriented toward better understanding of the factors that affect phosphate absorption in the gastrointestinal tract and the development of agents that directly inhibit phosphate transporters in the small intestine and/or their associated binding proteins.
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Enfermedades Cardiovasculares/epidemiología , Tracto Gastrointestinal/metabolismo , Hiperfosfatemia/metabolismo , Fosfatos/metabolismo , Insuficiencia Renal Crónica/metabolismo , Adulto , Enfermedades Cardiovasculares/metabolismo , Dieta , Progresión de la Enfermedad , Factor-23 de Crecimiento de Fibroblastos , Humanos , Hiperfosfatemia/complicaciones , Hiperfosfatemia/prevención & control , Masculino , Cooperación del Paciente , Proteínas de Unión a Fosfato/uso terapéutico , Insuficiencia Renal Crónica/complicaciones , Factores de RiesgoRESUMEN
While the classic electrocardiographic (EKG) findings of hyperkalemia are well known to clinicians, the association between hyperkalemia and bradycardia is not widely appreciated. Three cases of profound bradycardia due to hyperkalemia in patients with End Stage Renal Disease (ESRD) on hemodialysis are described to provide a base for discussion of specific issues in the management of such patients. The patients presented with hyperkalemia and severe bradycardia that did not improve after administration of atropine. Urgent hemodialysis in two cases led to resolution of the bradycardia. In the third case, the failure to recognize that bradycardia was the consequence of the hyperkalemia led to unnecessary interventions and delays in initiating dialysis. These cases highlight the causal relation between hyperkalemia and bradycardia in ESRD patients and emphasize the need for increased awareness of this association.
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Bradicardia/etiología , Electrocardiografía , Hiperpotasemia/complicaciones , Diálisis Renal/efectos adversos , Humanos , Fallo Renal Crónico/complicaciones , Masculino , Persona de Mediana EdadRESUMEN
Fibroblast growth factor-23 (FGF-23) inhibits sodium-dependent phosphate transport in brush border membrane vesicles derived from hormone-treated kidney slices of the mouse and in mouse proximal tubule cells by processes involving mitogen-activated protein kinase (MAPK) but not protein kinase A (PKA) or protein kinase C (PKC). By contrast, phosphate transport in brush border membrane vesicles and proximal tubule cells from sodium-hydrogen exchanger regulatory factor-1 (NHERF-1)-null mice were resistant to the inhibitory effect of FGF-23 (10(-9) m). Infection of NHERF-1-null proximal tubule cells with wild-type adenovirus-GFP-NHERF-1 increased basal phosphate transport and restored the inhibitory effect of FGF-23. Infection with adenovirus-GFP-NHERF-1 containing a S77A or T95D mutation also increased basal phosphate transport, but the cells remained resistant to FGF-23 (10(-9) m). Low concentrations of FGF-23 (10(-13) m) and PTH (10(-11) m) individually did not inhibit phosphate transport or activate PKA, PKC, or MAPK. When combined, however, these hormones markedly inhibited phosphate transport associated with activation of PKC and PKA but not MAPK. These studies indicate that FGF-23 inhibits phosphate transport in the mouse kidney by processes that involve the scaffold protein NHERF-1. In addition, FGF-23 synergizes with PTH to inhibit phosphate transport by facilitating the activation of the PTH signal transduction pathway.
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Factores de Crecimiento de Fibroblastos/metabolismo , Túbulos Renales Proximales/metabolismo , Hormona Paratiroidea/metabolismo , Fosfatos/metabolismo , Fosfoproteínas/metabolismo , Intercambiadores de Sodio-Hidrógeno/metabolismo , Adenoviridae , Sustitución de Aminoácidos , Animales , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/genética , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Factor-23 de Crecimiento de Fibroblastos , Factores de Crecimiento de Fibroblastos/agonistas , Factores de Crecimiento de Fibroblastos/genética , Factores de Crecimiento de Fibroblastos/farmacología , Transporte Iónico/efectos de los fármacos , Transporte Iónico/fisiología , Ratones , Ratones Noqueados , Mutación Missense , Hormona Paratiroidea/agonistas , Hormona Paratiroidea/genética , Hormona Paratiroidea/farmacología , Fosfoproteínas/genética , Proteína Quinasa C/genética , Proteína Quinasa C/metabolismo , Intercambiadores de Sodio-Hidrógeno/genética , Transducción GenéticaRESUMEN
The renal excretion of inorganic phosphate is regulated in large measure by three hormones, namely, parathyroid hormone, dopamine, and fibroblast growth factor-23. Recent experiments have indicated that the major sodium-dependent phosphate transporter in the renal proximal tubule, Npt2a, binds to the adaptor protein sodium-hydrogen exchanger regulatory factor-1 (NHERF-1) and in the absence of NHERF-1, the inhibitory effect of these three hormones is absent. From these observations, a new model for the hormonal regulation of renal phosphate transport was developed. The downstream signaling pathways of these hormones results in the phosphorylation of the PDZ 1 domain of NHERF-1 and the dissociation of Npt2a/NHERF-1 complexes. In turn, this dissociation facilitates the endocytosis of Npt2a with a subsequent decrease in the apical membrane abundance of the transporter and a decrease in phosphate reabsorption. The current review outlines the experimental observations supporting the operation of this unique regulatory system.
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Dopamina/fisiología , Factores de Crecimiento de Fibroblastos/fisiología , Hormonas/metabolismo , Riñón/metabolismo , Hormona Paratiroidea/fisiología , Fosfatos/metabolismo , Fosfoproteínas/fisiología , Intercambiadores de Sodio-Hidrógeno/fisiología , Animales , Transporte Biológico Activo/fisiología , Factor-23 de Crecimiento de Fibroblastos , Humanos , Modelos Moleculares , Dominios PDZ/fisiología , Fosfoproteínas/química , Fosfoproteínas/genética , Fosforilación , Conformación Proteica , Transducción de Señal/efectos de los fármacos , Intercambiadores de Sodio-Hidrógeno/química , Intercambiadores de Sodio-Hidrógeno/genética , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo IIa/metabolismoRESUMEN
Sorting of intracellular G-protein-coupled receptors (GPCRs) either to lysosomes for degradation or to plasma membrane for surface insertion and functional expression is a key process regulating signaling strength of GPCRs across the plasma membrane in adult mammalian cells. However, little is known about the molecular mechanisms governing the dynamic process of receptor sorting to the plasma membrane for functional expression under normal and pathological conditions. In this study, we demonstrate that delta-opioid receptor (DOPr), a GPCR constitutively targeted to intracellular compartments, is driven to the surface membrane of central synaptic terminals and becomes functional by the neurotrophin nerve growth factor (NGF) in native brainstem neurons. The NGF-triggered DOPr translocation is predominantly mediated by the signaling pathway involving the tyrosine receptor kinase A, Ca(2+)-mobilizing phospholipase C, and Ca(2+)/calmodulin-dependent protein kinase II. Importantly, it requires interactions with the cytoplasmic sorting protein NHERF-1 (Na(+)/H(+) exchange regulatory factor-1) and N-ethyl-maleimide-sensitive factor-regulated exocytosis. In addition, this NGF-mediated mechanism is likely responsible for the emergence of functional DOPr induced by chronic opioids. Thus, NGF may function as a key molecular switch that redirects the sorting of intracellularly targeted DOPr to plasma membrane, resulting in new functional DOPr on central synapses under chronic opioid conditions.
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Factor de Crecimiento Nervioso/farmacología , Factor de Crecimiento Nervioso/fisiología , Receptores Opioides delta/fisiología , Animales , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Potenciales Postsinápticos Excitadores/fisiología , Masculino , Ratones , Ratones Noqueados , Morfina/farmacología , Ratas , Ratas Wistar , Receptores Opioides delta/agonistasRESUMEN
Na(+)-K(+)-ATPase activity in renal proximal tubule is regulated by several hormones including parathyroid hormone (PTH) and dopamine. The current experiments explore the role of Na(+)/H(+) exchanger regulatory factor 1 (NHERF-1) in dopamine-mediated regulation of Na(+)-K(+)-ATPase. We measured dopamine regulation of ouabain-sensitive (86)Rb uptake and Na(+)-K(+)-ATPase α1 subunit phosphorylation in wild-type opossum kidney (OK) (OK-WT) cells, OKH cells (NHERF-1-deficient), and OKH cells stably transfected with full-length human NHERF-1 (NF) or NHERF-1 constructs with mutated PDZ-1 (Z1) or PDZ-2 (Z2) domains. Treatment with 1 µM dopamine decreased ouabain-sensitive (86)Rb uptake, increased phosphorylation of Na(+)-K(+)-ATPase α1-subunit, and enhanced association of NHERF-1 with D1 receptor in OK-WT cells but not in OKH cells. Transfection with wild-type, full-length, or PDZ-1 domain-mutated NHERF-1 into OKH cells restored dopamine-mediated regulation of Na(+)-K(+)-ATPase and D1-like receptor association with NHERF-1. Dopamine did not regulate Na(+)-K(+)-ATPase or increase D1-like receptor association with NHERF-1 in OKH cells transfected with mutated PDZ-2 domain. Dopamine stimulated association of PKC-ζ with NHERF-1 in OK-WT and OKH cells transfected with full-length or PDZ-1 domain-mutated NHERF-1 but not in PDZ-2 domain-mutated NHERF-1-transfected OKH cells. These results suggest that NHERF-1 mediates Na(+)-K(+)-ATPase regulation by dopamine through its PDZ-2 domain.
Asunto(s)
Dopamina/fisiología , Células Epiteliales/metabolismo , Túbulos Renales Proximales/metabolismo , Dominios PDZ/fisiología , Fosfoproteínas/fisiología , Intercambiadores de Sodio-Hidrógeno/fisiología , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Animales , Línea Celular Transformada , Didelphis , Dopamina/farmacología , Células Epiteliales/citología , Humanos , Túbulos Renales Proximales/citología , Mutación , Dominios PDZ/efectos de los fármacos , Dominios PDZ/genética , Fosfoproteínas/química , Fosfoproteínas/genética , Proteína Quinasa C/metabolismo , Subunidades de Proteína/metabolismo , Receptores de Dopamina D1/agonistas , Receptores de Dopamina D1/metabolismo , Proteína de Retinoblastoma/metabolismo , Intercambiadores de Sodio-Hidrógeno/química , Intercambiadores de Sodio-Hidrógeno/genéticaRESUMEN
The phosphorylation of the sodium-hydrogen exchanger regulatory factor-1 (NHERF-1) plays a key role in the regulation of renal phosphate transport by parathyroid hormone (PTH) and dopamine. Ser(77) in the first PDZ domain of NHERF-1 is a downstream target of both hormones. The current experiments explore the role of Thr(95), another phosphate acceptor site in the PDZ I domain, on hormone-mediated regulation of phosphate transport in the proximal tubule of the kidney. The substitution of alanine for threonine at position 95 (T95A) significantly decreased the rate and extent of in vitro phosphorylation of Ser(77) by PKC. In NHERF-1-null proximal tubule cells, neither PTH nor dopamine inhibited sodium-dependent phosphate transport. Infection of the cells with adenovirus expressing full-length WT GFP-NHERF-1 increased basal phosphate transport and restored the inhibitory effect of both PTH and dopamine. Infection with full-length NHERF-1 containing a T95A mutation, however, increased basal phosphate transport but not the responsiveness to either hormone. As determined by surface plasmon resonance, the substitution of serine for aspartic acid (S77D) in the PDZ I domain decreased the binding affinity to the sodium-dependent phosphate transporter 2a (Npt2a) as compared with WT PDZ I, but a T95D mutation had no effect on binding. Finally, cellular studies indicated that both PTH and dopamine treatment increased the phosphorylation of Thr(95). These studies indicate a remarkable cooperativity between the phosphorylation of Thr(95) and Ser(77) of NHERF-1 in the hormonal regulation of renal phosphate transport. The phosphorylation of Thr(95) facilitates the phosphorylation of Ser(77). This, in turn, results in the dissociation of NHERF-1 from Npt2a and a decrease in phosphate transport in renal proximal tubule cells.
Asunto(s)
Transporte Biológico/efectos de los fármacos , Dopamina/farmacología , Hormona Paratiroidea/farmacología , Fosfatos/metabolismo , Fosfoproteínas/metabolismo , Serina/metabolismo , Intercambiadores de Sodio-Hidrógeno/metabolismo , Treonina/metabolismo , Animales , Western Blotting , Línea Celular , Células Cultivadas , Electroforesis en Gel de Poliacrilamida , Humanos , Ratones , Fosfoproteínas/genética , Fosforilación/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Intercambiadores de Sodio-Hidrógeno/genética , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo IIa/metabolismoRESUMEN
Dopamine inhibited phosphate transport in isolated renal brush border membrane vesicles and in cultured renal proximal tubule cells from wild-type but not from NHERF-1 null mice. Co-immunoprecipitation experiments established that NHERF-1 associated with D1-like receptors. In wild-type mice, dopamine stimulated cAMP accumulation and protein kinase C (PKC) activity in renal proximal tubule cells, an effect that was abolished by SCH-23390, a D1-like receptor antagonist. In NHERF-1 null kidney tissue; however, dopamine failed to stimulate either cAMP accumulation or PKC activity. Infection of proximal tubule cells from NHERF-1 null mice with adenovirus-green fluorescent protein-NHERF-1 restored the ability of dopamine to stimulate cAMP and PKC. Finally, in (32)P-labeled wild-type proximal tubule cells and in opossum kidney cells, dopamine increased NHERF-1 phosphorylation at serine 77 of the PDZ I domain of NHERF-1, a site previously shown to attenuate binding of cellular targets including the Npt2a (sodium-dependent phosphate transporter 2a). Together, these studies establish that NHERF-1 plays a key role in dopamine signaling and is also a downstream target of D1-like receptors in the mouse kidney. These studies suggest a novel role for the PDZ adapter protein NHERF-1 in coordinating dopamine signals that inhibit renal phosphate transport.
Asunto(s)
Dopaminérgicos/farmacología , Dopamina/farmacología , Túbulos Renales Proximales/metabolismo , Fosfatos/metabolismo , Fosfoproteínas/metabolismo , Transducción de Señal/efectos de los fármacos , Intercambiadores de Sodio-Hidrógeno/metabolismo , Sodio/metabolismo , Adenoviridae , Animales , Transporte Iónico/efectos de los fármacos , Transporte Iónico/genética , Ratones , Ratones Noqueados , Zarigüeyas , Fosfoproteínas/genética , Fosforilación , Proteína Quinasa C/genética , Proteína Quinasa C/metabolismo , Receptores de Dopamina D1/genética , Receptores de Dopamina D1/metabolismo , Transducción de Señal/genética , Intercambiadores de Sodio-Hidrógeno/genética , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo IIa/genética , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo IIa/metabolismoRESUMEN
Multidrug resistance-associated protein 2 (Mrp2, Abcc2) is an ATP-binding cassette transporter localized at the canalicular membrane of hepatocytes that plays an important role in bile formation and detoxification. Prior in vitro studies suggest that Mrp2 can bind to Na(+)/H(+) exchanger regulatory factor 1 (NHERF-1), a PDZ protein that cross-links membrane proteins to actin filaments. However the role of NHERF-1 in the expression and functional regulation of Mrp2 remains largely unknown. Here we examine the interaction of Mrp2 and NHERF-1 and its physiological significance in HEK293 cells and NHERF-1 knock-out mice. Mrp2 co-precipitated with NHERF-1 in co-transfected HEK293 cells, an interaction that required the PDZ-binding motif of Mrp2. In NHERF-1(-/-) mouse liver, Mrp2 mRNA was unchanged but Mrp2 protein was reduced in whole cell lysates and membrane-enriched fractions to approximately 50% (p < 1 x 10(-6)) and approximately 70% (p < 0.05), respectively, compared with wild-type mice, suggesting that the down-regulation of Mrp2 expression was caused by post-transcriptional events. Mrp2 remained localized at the apical/canalicular membrane of NHERF-1(-/-) mouse hepatocytes, although its immunofluorescent labeling was noticeably weaker. Bile flow in NHERF-1(-/-) mice was reduced to approximately 70% (p < 0.001) in association with a 50% reduction in glutathione excretion (p < 0.05) and a 60% reduction in glutathione-methylfluorescein (GS-MF) excretion in isolated mouse hepatocyte (p < 0.01). Bile acid and bilirubin excretion remained unchanged compared with wild-type mice. These findings strongly suggest that NHERF-1 binds to Mrp2, and plays a critical role in the canalicular expression of Mrp2 and its function as a determinant of glutathione-dependent, bile acid-independent bile flow.
Asunto(s)
Proteínas Portadoras/metabolismo , Regulación de la Expresión Génica , Hígado/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Fosfoproteínas/metabolismo , Intercambiadores de Sodio-Hidrógeno/metabolismo , Animales , Ácidos y Sales Biliares/química , Glutatión/metabolismo , Hepatocitos/citología , Hepatocitos/metabolismo , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína 2 Asociada a Resistencia a Múltiples Medicamentos , RatasRESUMEN
Parathyroid hormone (PTH) inhibits the reabsorption of phosphate in the renal proximal tubule by disrupting the binding of the sodium-dependent phosphate transporter 2A (Npt2a) to the adapter protein sodium-hydrogen exchanger regulatory factor-1 (NHERF-1), a process initiated by activation of protein kinase C (PKC). To gain additional insights into the dynamic sequence of events, the time course of these responses was studied in living opossum kidney (OK) cells. Using a FRET-based biosensor, we found that PTH activated intracellular PKC within seconds to minutes. In cells expressing GFP-Npt2a and mCherry-NHERF, PTH did not affect the relative abundance of NHERF-1 but there was a significant and time-dependent decrease in the Npt2a/NHERF-1 ratio. The half-time to maximal dissociation was 15 to 20 min. By contrast, PTH had no effect on the fluorescence ratio for GFP-ezrin compared with mCherry-NHERF-1 at the apical surface. These experiments establish that PTH treatment of proximal tubule OK cells leads to rapid activation of PKC with the subsequent dissociation of Npt2a/NHERF-1 complexes. The association of NHERF-1 with Ezrin and their localization at the apical membrane, however, was unperturbed by PTH, thereby enabling the rapid recruitment and membrane reinsertion of Npt2a and other NHERF-1 targets on termination of the hormone response.
Asunto(s)
Riñón/metabolismo , Complejos Multiproteicos/metabolismo , Hormona Paratiroidea/farmacología , Fosfoproteínas/metabolismo , Intercambiadores de Sodio-Hidrógeno/metabolismo , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo IIa/metabolismo , Animales , Línea Celular , Proteínas del Citoesqueleto/metabolismo , Femenino , Complejos Multiproteicos/efectos de los fármacos , ZarigüeyasRESUMEN
The current experiments explore the role of dopamine in facilitating the acute increase in renal phosphate excretion in response to a high-phosphate diet. Compared with a low-phosphate (0.1%) diet for 24 h, mice fed a high-phosphate (1.2%) diet had significantly higher rates of phosphate excretion in the urine associated with a two- to threefold increase in the dopamine content of the kidney and in the urinary excretion of dopamine. Animals fed a high-phosphate diet had a significant increase in the abundance and activity of renal DOPA (l-dihydroxyphenylalanine) decarboxylase and significant reductions in renalase, monoamine oxidase A, and monoamine oxidase B. The activity of protein kinase A and protein kinase C, markers of activation of renal dopamine receptors, were significantly higher in animals fed a high-phosphate vs. a low-phosphate diet. Treatment of rats with carbidopa, an inhibitor of DOPA decarboxylase, impaired adaptation to a high-phosphate diet. These experiments indicate that the rapid adaptation to a high-phosphate diet involves alterations in key enzymes involved in dopamine synthesis and degradation, resulting in increased renal dopamine content and activation of the signaling cascade used by dopamine to inhibit the renal tubular reabsorption of phosphate.
Asunto(s)
Dopamina/metabolismo , Riñón/metabolismo , Fósforo Dietético/metabolismo , Adaptación Fisiológica , Análisis de Varianza , Animales , Inhibidores de Descarboxilasas de Aminoácidos Aromáticos , Carbidopa/farmacología , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Dopa-Decarboxilasa/metabolismo , Dopamina/orina , Inhibidores Enzimáticos/farmacología , Riñón/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Monoaminooxidasa/metabolismo , Fósforo Dietético/administración & dosificación , Fósforo Dietético/orina , Proteína Quinasa C/metabolismo , Ratas , Ratas Sprague-Dawley , Transducción de Señal , Factores de Tiempo , Regulación hacia ArribaRESUMEN
Parathyroid hormone (PTH), via activation of PKC and/or protein kinase A, inhibits renal proximal tubular phosphate reabsorption by facilitating the internalization of the major sodium-dependent phosphate transporter, Npt2a. Herein, we explore the hypothesis that the effect of PTH is mediated by phosphorylation of serine 77 (S77) of the first PDZ domain of the Npt2a-binding protein sodium-hydrogen exchanger regulatory factor-1 (NHERF-1). Using recombinant polypeptides representing PDZ I, S77 of NHERF-1 is phosphorylated by PKC but not PKA. When expressed in primate kidney epithelial cells (BSC-1 cells), however, activation of either protein kinase phosphorylates S77, suggesting that the phosphorylation of PDZ I by PKC and PKA proceeds by different biochemical pathways. PTH and other activators of PKC and PKA dissociate NHERF-1/Npt2a complexes, as assayed using quantitative coimmunoprecipitation, confocal microscopy, and sucrose density gradient ultracentrifugation in mice. Murine NHERF-1-/- renal proximal tubule cells infected with adenovirus-GFP-NHERF-1 containing an S77A mutation showed significantly increased phosphate transport compared with a phosphomimetic S77D mutation and were resistant to the inhibitory effect of PTH compared with cells infected with wild-type NHERF-1. These results indicate that PTH-mediated inhibition of renal phosphate transport involves phosphorylation of S77 of the NHERF-1 PDZ I domain and the dissociation of NHERF-1/Npt2a complexes.