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1.
Int J Mol Sci ; 22(4)2021 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-33673381

RESUMO

Tumor necrosis factor (TNF) is known to activate the epithelial Na+ channel (ENaC) in A549 cells. A549 cells are widely used model for ENaC research. The role of δ-ENaC subunit in TNF-induced activation has not been studied. In this study we hypothesized that δ-ENaC plays a major role in TNF-induced activation of ENaC channel in A549 cells which are widely used model for ENaC research. We used CRISPR/Cas 9 approach to knock down (KD) the δ-ENaC in A549 cells. Western blot and immunofluorescence assays were performed to analyze efficacy of δ-ENaC protein KD. Whole-cell patch clamp technique was used to analyze the TNF-induced activation of ENaC. Overexpression of wild type δ-ENaC in the δ-ENaC KD of A549 cells restored the TNF-induced activation of whole-cell Na+ current. Neither N-linked glycosylation sites nor carboxyl terminus domain of δ-ENaC was necessary for the TNF-induced activation of whole-cell Na+ current in δ-ENaC KD of A549 cells. Our data demonstrated that in A549 cells the δ-ENaC plays a major role in TNF-induced activation of ENaC.


Assuntos
Sistemas CRISPR-Cas , Canais Epiteliais de Sódio , Fator de Necrose Tumoral alfa/metabolismo , Células A549 , Canais Epiteliais de Sódio/genética , Canais Epiteliais de Sódio/metabolismo , Humanos , Fator de Necrose Tumoral alfa/genética
2.
Int J Mol Sci ; 22(3)2021 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-33498219

RESUMO

K+ loading inhibits NKCC2 (Na-K-Cl cotransporter) and NCC (Na-Cl cotransporter) in the early distal tubules, resulting in Na+ delivery to the late distal convoluted tubules (DCTs). In the DCTs, Na+ entry through ENaC (epithelial Na channel) drives K+ secretion through ROMK (renal outer medullary potassium channel). WNK4 (with-no-lysine 4) regulates the NCC/NKCC2 through SAPK (Ste20-related proline-alanine-rich kinase)/OSR1 (oxidative stress responsive). K+ loading increases intracellular Cl-, which binds to the WNK4, thereby inhibiting autophosphorylation and downstream signals. Acute K+ loading-deactivated NCC was not observed in Cl--insensitive WNK4 mice, indicating that WNK4 was involved in K+ loading-inhibited NCC activity. However, chronic K+ loading deactivated NCC in Cl--insensitive WNK4 mice, indicating that other mechanisms may be involved. We previously reported that mammalian Ste20-like protein kinase 3 (MST3/STK24) was expressed mainly in the medullary TAL (thick ascending tubule) and at lower levels in the DCTs. MST3 -/- mice exhibited higher ENaC activity, causing hypernatremia and hypertension. To investigate MST3 function in maintaining Na+/K+ homeostasis in kidneys, mice were fed diets containing various concentrations of Na+ and K+. The 2% KCl diets induced less MST3 expression in MST3 -/- mice than that in wild-type (WT) mice. The MST3 -/- mice had higher WNK4, NKCC2-S130 phosphorylation, and ENaC expression, resulting in lower urinary Na+ and K+ excretion than those of WT mice. Lower urinary Na+ excretion was associated with elevated plasma [Na+] and hypertension. These results suggest that MST3 maintains Na+/K+ homeostasis in response to K+ loading by regulation of WNK4 expression and NKCC2 and ENaC activity.


Assuntos
Homeostase , Potássio na Dieta/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Sódio/metabolismo , Animais , Canais Epiteliais de Sódio/genética , Canais Epiteliais de Sódio/metabolismo , Rim/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Serina-Treonina Quinases/genética , Eliminação Renal , Membro 1 da Família 12 de Carreador de Soluto/genética , Membro 1 da Família 12 de Carreador de Soluto/metabolismo
3.
Biochim Biophys Acta Mol Basis Dis ; 1867(1): 165989, 2021 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-33065235

RESUMO

We previously showed that increased epithelial sodium channel (ENaC) activity in endothelial cells induced by oxidized low-density lipoprotein (ox-LDL) contributes to vasculature dysfunction. Here, we investigated whether ENaC participates in the pathological process of atherosclerosis using LDL receptor-deficient (LDLr-/-) mice. Male C57BL/6 and LDLr-/- mice were fed a normal diet (ND) or high fat diet (HFD) for 10 weeks. Our data show that treatment of LDLr-/- mice with a specific ENaC blocker, benzamil, significantly decreased atherosclerotic lesion formation and expression of matrix metalloproteinase 2 (MMP2) and metalloproteinase 9 (MMP9) in aortic arteries. Furthermore, benzamil ameliorated HFD-induced impairment of aortic endothelium-dependent dilation by reducing expression of proinflammatory cytokines, including TNF-α, IL-1ß, and IL-6 and production of adhesion molecules including VCAM-1 and ICAM-1 in both C57BL/6 and LDLr-/- mice fed with HFD. In addition, HFD significantly increased ENaC activity and the levels of serum lipids, including ox-LDL. Our in vitro data further demonstrated that exogenous ox-LDL significantly increased the production of TNF-α, IL-1ß, IL-6, VCAM-1 and ICAM-1. This ox-LDL-induced increase in inflammatory cytokines and adhesion molecules was reversed by γ-ENaC silencing or by treatment with the cyclooxygenase-2 (COX-2) antagonist celecoxib. Benzamil inhibited HFD-induced increase in COX-2 expression in aortic tissue in both C57BL/6 and LDLr-/- mice, and γ-ENaC gene silencing attenuated ox-LDL-induced COX-2 expression in HUVECs. These data together suggest that HFD-induced activation of ENaC stimulates inflammatory signaling, thereby contributes to HFD-induced endothelial dysfunction and atherosclerotic lesion formation. Thus, targeting endothelial ENaC may be a promising strategy to halt atherogenesis.


Assuntos
Aterosclerose , Dieta Hiperlipídica/efeitos adversos , Canais Epiteliais de Sódio/metabolismo , Receptores de LDL/deficiência , Animais , Aterosclerose/induzido quimicamente , Aterosclerose/genética , Aterosclerose/metabolismo , Aterosclerose/patologia , Citocinas/genética , Citocinas/metabolismo , Canais Epiteliais de Sódio/genética , Células Endoteliais da Veia Umbilical Humana , Humanos , Molécula 1 de Adesão Intercelular/genética , Molécula 1 de Adesão Intercelular/metabolismo , Lipoproteínas LDL/genética , Lipoproteínas LDL/metabolismo , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Camundongos Knockout , Receptores de LDL/metabolismo , Molécula 1 de Adesão de Célula Vascular/genética , Molécula 1 de Adesão de Célula Vascular/metabolismo
4.
Am J Physiol Regul Integr Comp Physiol ; 319(4): R387-R400, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-32783689

RESUMO

The conquest of freshwater and terrestrial habitats was a key event during vertebrate evolution. Occupation of low-salinity and dry environments required significant osmoregulatory adaptations enabling stable ion and water homeostasis. Sodium is one of the most important ions within the extracellular liquid of vertebrates, and molecular machinery for urinary reabsorption of this electrolyte is critical for the maintenance of body osmoregulation. Key ion channels involved in the fine-tuning of sodium homeostasis in tetrapod vertebrates are epithelial sodium channels (ENaCs), which allow the selective influx of sodium ions across the apical membrane of epithelial cells lining the distal nephron or the colon. Furthermore, ENaC-mediated sodium absorption across tetrapod lung epithelia is crucial for the control of liquid volumes lining the pulmonary surfaces. ENaCs are vertebrate-specific members of the degenerin/ENaC family of cation channels; however, there is limited knowledge on the evolution of ENaC within this ion channel family. This review outlines current concepts and hypotheses on ENaC phylogeny and discusses the emergence of regulation-defining sequence motifs in the context of osmoregulatory adaptations during tetrapod terrestrialization. In light of the distinct regulation and expression of ENaC isoforms in tetrapod vertebrates, we discuss the potential significance of ENaC orthologs in osmoregulation of fishes as well as the putative fates of atypical channel isoforms in mammals. We hypothesize that ancestral proton-sensitive ENaC orthologs might have aided the osmoregulatory adaptation to freshwater environments whereas channel regulation by proteases evolved as a molecular adaptation to lung liquid homeostasis in terrestrial tetrapods.


Assuntos
Evolução Biológica , Canais Epiteliais de Sódio/metabolismo , Evolução Molecular , Osmorregulação/fisiologia , Animais , Humanos
5.
Am J Physiol Cell Physiol ; 319(3): C589-C604, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32639874

RESUMO

The epithelial sodium channel (ENaC) regulates blood pressure by fine-tuning distal nephron sodium reabsorption. Our previous work has shown that ENaC gating is regulated by anionic phospholipid phosphates, including phosphatidylinositol 4,5-bisphosphate (PIP2). The PIP2-dependent regulation of ENaC is mediated by the myristoylated alanine-rich protein kinase C substrate-like protein-1 (MLP-1). MLP-1 binds to and is a reversible source of PIP2 at the plasma membrane. We examined MLP-1 regulation of ENaC in distal convoluted tubule clonal cell line DCT-15 cells. Wild-type MLP-1 runs at an apparent molecular mass of 52 kDa despite having a predicted molecular mass of 21 kDa. Native MLP-1 consists of several distinct structural elements: an effector domain that is highly positively charged, sequesters PIP2, contains serines that are the target of PKC, and controls MLP-1 association with the membrane; a myristoylation domain that promotes association with the membrane; and a multiple homology 2 domain of previously unknown function. To further examine MLP-1 in DCT-15 cells, we constructed several MLP-1 mutants: WT, a full-length wild-type protein; S3A, three substitutions in the effector domain to prevent phosphorylation; S3D mimicked constitutive phosphorylation by replacing three serines with aspartates; and GA replaced the myristoylation site glycine with alanine, so GA could not be myristoylated. Each mutant was tagged with either NH2-terminal 3XFLAG or COOH-terminal mCherry or V5. Transfection with MLP mutants modified ENaC activity in DCT-15 cells: activity was highest in S3A and lowest in S3D, and the activity after transfection with either construct was significantly different from WT. In Western blots, when transfected with 3XFLAG-tagged MLP-1 mutants, the expression of the full length of MLP-1 at 52 kDa increased in mutant S3A-MLP-1-transfected DCT-15 cells and decreased in S3D-MLP-1-transfected DCT-15 cells. Several lower molecular mass bands were also detected that correspond to potential presumptive calpain cleavage products. Confocal imaging shows that the different mutants localize in different subcellular compartments consistent with their preferred location in the membrane or in the cytosol. Activation of protein kinase C increases phosphorylation of endogenous MLP-1 and reduces ENaC activity. Our results suggest a complicated role for proteolytic processing in MLP-1 regulation of ENaC.


Assuntos
Proteínas de Ligação a Calmodulina/metabolismo , Canais Epiteliais de Sódio/metabolismo , Proteínas dos Microfilamentos/metabolismo , Néfrons/metabolismo , Animais , Proteínas de Ligação a Calmodulina/genética , Linhagem Celular , Membrana Celular/metabolismo , Camundongos , Proteínas dos Microfilamentos/genética , Fosfatidilinositóis/metabolismo , Fosforilação , Proteína Quinase C/metabolismo
6.
Am J Physiol Renal Physiol ; 319(2): F323-F334, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32628540

RESUMO

We investigated the regulation of Na+ and K+ excretion and the epithelial Na+ channel (ENaC) in mice lacking the gene for aldosterone synthase (AS) using clearance methods to assess excretion and electrophysiology and Western blot analysis to test for ENaC activity and processing. After 1 day of dietary Na+ restriction, AS-/- mice lost more Na+ in the urine than AS+/+ mice did. After 1 wk on this diet, both genotypes strongly reduced urinary Na+ excretion, but creatinine clearance decreased only in AS-/- mice. Only AS+/+ animals exhibited increased ENaC function, assessed as amiloride-sensitive whole cell currents in collecting ducts or cleavage of αENaC and γENaC in Western blots. To assess the role of aldosterone in the excretion of a K+ load, animals were fasted overnight and refed with high-K+ or low-K+ diets for 5 h. Both AS+/+ and AS-/- mice excreted a large amount of K+ during this period. In both phenotypes the excretion was benzamil sensitive, indicating increased K+ secretion coupled to ENaC-dependent Na+ reabsorption. However, the increase in plasma K+ under these conditions was much larger in AS-/- animals than in AS+/+ animals. In both groups, cleavage of αENaC and γENaC increased. However, Na+ current measured ex vivo in connecting tubules was enhanced only in AS+/+ mice. We conclude that in the absence of aldosterone, mice can conserve Na+ without ENaC activation but at the expense of diminished glomerular filtration rate. Excretion of a K+ load can be accomplished through aldosterone-independent upregulation of ENaC, but aldosterone is required to excrete the excess K+ without hyperkalemia.


Assuntos
Citocromo P-450 CYP11B2/metabolismo , Canais Epiteliais de Sódio/metabolismo , Potássio/metabolismo , Sódio na Dieta/metabolismo , Sódio/metabolismo , Animais , Canais Epiteliais de Sódio/genética , Túbulos Renais Coletores/metabolismo , Camundongos , Natriurese/fisiologia
7.
Neuron ; 107(1): 95-111.e6, 2020 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-32380032

RESUMO

Progressive synapse loss is an inevitable and insidious part of age-related neurodegenerative disease. Typically, synapse loss precedes symptoms of cognitive and motor decline. This suggests the existence of compensatory mechanisms that can temporarily counteract the effects of ongoing neurodegeneration. Here, we demonstrate that presynaptic homeostatic plasticity (PHP) is induced at degenerating neuromuscular junctions, mediated by an evolutionarily conserved activity of presynaptic ENaC channels in both Drosophila and mouse. To assess the consequence of eliminating PHP in a mouse model of ALS-like degeneration, we generated a motoneuron-specific deletion of Scnn1a, encoding the ENaC channel alpha subunit. We show that Scnn1a is essential for PHP without adversely affecting baseline neural function or lifespan. However, Scnn1a knockout in a degeneration-causing mutant background accelerated motoneuron loss and disease progression to twice the rate observed in littermate controls with intact PHP. We propose a model of neuroprotective homeostatic plasticity, extending organismal lifespan and health span.


Assuntos
Canais Epiteliais de Sódio/metabolismo , Homeostase/fisiologia , Plasticidade Neuronal/fisiologia , Neuroproteção/fisiologia , Terminações Pré-Sinápticas/metabolismo , Esclerose Amiotrófica Lateral/metabolismo , Esclerose Amiotrófica Lateral/patologia , Animais , Modelos Animais de Doenças , Progressão da Doença , Drosophila melanogaster , Camundongos , Camundongos Knockout , Junção Neuromuscular/metabolismo
8.
Elife ; 92020 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-32452762

RESUMO

Molecular mimicry is an evolutionary strategy adopted by viruses to exploit the host cellular machinery. We report that SARS-CoV-2 has evolved a unique S1/S2 cleavage site, absent in any previous coronavirus sequenced, resulting in the striking mimicry of an identical FURIN-cleavable peptide on the human epithelial sodium channel α-subunit (ENaC-α). Genetic alteration of ENaC-α causes aldosterone dysregulation in patients, highlighting that the FURIN site is critical for activation of ENaC. Single cell RNA-seq from 66 studies shows significant overlap between expression of ENaC-α and the viral receptor ACE2 in cell types linked to the cardiovascular-renal-pulmonary pathophysiology of COVID-19. Triangulating this cellular characterization with cleavage signatures of 178 proteases highlights proteolytic degeneracy wired into the SARS-CoV-2 lifecycle. Evolution of SARS-CoV-2 into a global pandemic may be driven in part by its targeted mimicry of ENaC-α, a protein critical for the homeostasis of airway surface liquid, whose misregulation is associated with respiratory conditions.


Assuntos
Betacoronavirus/metabolismo , Infecções por Coronavirus/virologia , Canais Epiteliais de Sódio/metabolismo , Mimetismo Molecular , Peptídeo Hidrolases/metabolismo , Pneumonia Viral/virologia , Proteínas do Envelope Viral/metabolismo , Proteínas Virais/metabolismo , Betacoronavirus/genética , Betacoronavirus/patogenicidade , Canais Epiteliais de Sódio/genética , Interações Hospedeiro-Patógeno , Humanos , Pandemias , Peptidil Dipeptidase A/genética , Peptidil Dipeptidase A/metabolismo , Proteólise , Especificidade por Substrato , Proteínas do Envelope Viral/genética , Proteínas Virais/genética
9.
Am J Physiol Renal Physiol ; 318(6): F1341-F1356, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32281415

RESUMO

We characterized mouse blood pressure and ion transport in the setting of commonly used rodent diets that drive K+ intake to the extremes of deficiency and excess. Male 129S2/Sv mice were fed either K+-deficient, control, high-K+ basic, or high-KCl diets for 10 days. Mice maintained on a K+-deficient diet exhibited no change in blood pressure, whereas K+-loaded mice developed an ~10-mmHg blood pressure increase. Following challenge with NaCl, K+-deficient mice developed a salt-sensitive 8 mmHg increase in blood pressure, whereas blood pressure was unchanged in mice fed high-K+ diets. Notably, 10 days of K+ depletion induced diabetes insipidus and upregulation of phosphorylated NaCl cotransporter, proximal Na+ transporters, and pendrin, likely contributing to the K+-deficient NaCl sensitivity. While the anionic content with high-K+ diets had distinct effects on transporter expression along the nephron, both K+ basic and KCl diets had a similar increase in blood pressure. The blood pressure elevation on high-K+ diets correlated with increased Na+-K+-2Cl- cotransporter and γ-epithelial Na+ channel expression and increased urinary response to furosemide and amiloride. We conclude that the dietary K+ maneuvers used here did not recapitulate the inverse effects of K+ on blood pressure observed in human epidemiological studies. This may be due to the extreme degree of K+ stress, the low-Na+-to-K+ ratio, the duration of treatment, and the development of other coinciding events, such as diabetes insipidus. These factors must be taken into consideration when studying the physiological effects of dietary K+ loading and depletion.


Assuntos
Pressão Arterial , Hipertensão/metabolismo , Túbulos Renais/metabolismo , Deficiência de Potássio/metabolismo , Potássio na Dieta/metabolismo , Cloreto de Sódio na Dieta/metabolismo , Ração Animal , Animais , Diabetes Insípido/etiologia , Diabetes Insípido/metabolismo , Diabetes Insípido/fisiopatologia , Canais Epiteliais de Sódio/metabolismo , Hipertensão/etiologia , Hipertensão/fisiopatologia , Transporte de Íons , Túbulos Renais/fisiopatologia , Masculino , Camundongos da Linhagem 129 , Natriurese , Fosforilação , Deficiência de Potássio/etiologia , Deficiência de Potássio/fisiopatologia , Potássio na Dieta/administração & dosagem , Potássio na Dieta/toxicidade , Simportadores de Cloreto de Sódio/metabolismo , Cloreto de Sódio na Dieta/toxicidade , Simportadores de Cloreto de Sódio-Potássio/metabolismo , Transportadores de Sulfato/metabolismo
10.
Am J Physiol Renal Physiol ; 318(5): F1220-F1228, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32281419

RESUMO

Consumption of a Western diet (WD) induces central aortic stiffening that contributes to the transmittance of pulsatile blood flow to end organs, including the kidney. Our recent work supports that endothelial epithelial Na+ channel (EnNaC) expression and activation enhances aortic endothelial cell stiffening through reductions in endothelial nitric oxide (NO) synthase (eNOS) and bioavailable NO that result in inflammatory and oxidant responses and perivascular fibrosis. However, the role that EnNaC activation has on endothelial responses in the renal circulation remains unknown. We hypothesized that cell-specific deletion of the α-subunit of EnNaC would prevent WD-induced central aortic stiffness and protect the kidney from endothelial dysfunction and vascular stiffening. Twenty-eight-week-old female αEnNaC knockout and wild-type mice were fed either mouse chow or WD containing excess fat (46%), sucrose, and fructose (17.5% each). WD feeding increased fat mass, indexes of vascular stiffening in the aorta and renal artery (in vivo pulse wave velocity and ultrasound), and renal endothelial cell stiffening (ex vivo atomic force microscopy). WD further impaired aortic endothelium-dependent relaxation and renal artery compliance (pressure myography) without changes in blood pressure. WD-induced renal arterial stiffening occurred in parallel to attenuated eNOS activation, increased oxidative stress, and aortic and renal perivascular fibrosis. αEnNaC deletion prevented these abnormalities and support a novel mechanism by which WD contributes to renal arterial stiffening that is endothelium and Na+ channel dependent. These results demonstrate that cell-specific EnNaC is important in propagating pulsatility into the renal circulation, generating oxidant stress, reduced bioavailable NO, and renal vessel wall fibrosis and stiffening.


Assuntos
Aorta/metabolismo , Dieta Ocidental/efeitos adversos , Canais Epiteliais de Sódio/metabolismo , Artéria Renal/fisiopatologia , Doenças Vasculares/metabolismo , Rigidez Vascular , Animais , Aorta/patologia , Aorta/fisiopatologia , Elasticidade , Canais Epiteliais de Sódio/deficiência , Canais Epiteliais de Sódio/genética , Feminino , Fibrose , Camundongos Endogâmicos C57BL , Camundongos Knockout , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Estresse Oxidativo , Artéria Renal/patologia , Transdução de Sinais , Doenças Vasculares/genética , Doenças Vasculares/patologia , Doenças Vasculares/fisiopatologia , Remodelação Vascular
11.
Nat Commun ; 11(1): 2012, 2020 04 24.
Artigo em Inglês | MEDLINE | ID: mdl-32332792

RESUMO

Idiopathic pulmonary fibrosis (IPF) is a chronic progressive interstitial lung disease characterized by patchy scarring of the distal lung with limited therapeutic options and poor prognosis. Here, we show that conditional deletion of the ubiquitin ligase Nedd4-2 (Nedd4l) in lung epithelial cells in adult mice produces chronic lung disease sharing key features with IPF including progressive fibrosis and bronchiolization with increased expression of Muc5b in peripheral airways, honeycombing and characteristic alterations in the lung proteome. NEDD4-2 is implicated in the regulation of the epithelial Na+ channel critical for proper airway surface hydration and mucus clearance and the regulation of TGFß signaling, which promotes fibrotic remodeling. Our data support a role of mucociliary dysfunction and aberrant epithelial pro-fibrotic response in the multifactorial disease pathogenesis. Further, treatment with the anti-fibrotic drug pirfenidone reduced pulmonary fibrosis in this model. This model may therefore aid studies of the pathogenesis and therapy of IPF.


Assuntos
Células Epiteliais/patologia , Fibrose Pulmonar Idiopática/genética , Pulmão/patologia , Ubiquitina-Proteína Ligases Nedd4/genética , Ubiquitina-Proteína Ligases Nedd4/metabolismo , Adulto , Idoso , Animais , Biópsia , Modelos Animais de Doenças , Canais Epiteliais de Sódio/metabolismo , Humanos , Fibrose Pulmonar Idiopática/tratamento farmacológico , Fibrose Pulmonar Idiopática/patologia , Pulmão/citologia , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Mucina-5B/metabolismo , Proteômica , Piridonas/administração & dosagem , Ubiquitinação
12.
Neuron ; 106(5): 816-829.e6, 2020 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-32229307

RESUMO

Sodium taste regulates salt intake. The amiloride-sensitive epithelial sodium channel (ENaC) is the Na+ sensor in taste cells mediating attraction to sodium salts. However, cells and intracellular signaling underlying sodium taste in taste buds remain long-standing enigmas. Here, we show that a subset of taste cells with ENaC activity fire action potentials in response to ENaC-mediated Na+ influx without changing the intracellular Ca2+ concentration and form a channel synapse with afferent neurons involving the voltage-gated neurotransmitter-release channel composed of calcium homeostasis modulator 1 (CALHM1) and CALHM3 (CALHM1/3). Genetic elimination of ENaC in CALHM1-expressing cells as well as global CALHM3 deletion abolished amiloride-sensitive neural responses and attenuated behavioral attraction to NaCl. Together, sodium taste is mediated by cells expressing ENaC and CALHM1/3, where oral Na+ entry elicits suprathreshold depolarization for action potentials driving voltage-dependent neurotransmission via the channel synapse. Thus, all steps in sodium taste signaling are voltage driven and independent of Ca2+ signals. This work also reveals ENaC-independent salt attraction.


Assuntos
Potenciais de Ação/fisiologia , Cálcio/metabolismo , Canais Epiteliais de Sódio/metabolismo , Sódio/metabolismo , Papilas Gustativas/citologia , Paladar/fisiologia , Potenciais de Ação/efeitos dos fármacos , Amilorida/farmacologia , Animais , Canais de Cálcio/metabolismo , Células Quimiorreceptoras/metabolismo , Células Quimiorreceptoras/fisiologia , Bloqueadores do Canal de Sódio Epitelial/farmacologia , Camundongos , Neurônios Aferentes/metabolismo , Técnicas de Patch-Clamp , Transdução de Sinais/efeitos dos fármacos , Transmissão Sináptica , Papilas Gustativas/metabolismo , Papilas Gustativas/fisiologia
13.
PLoS One ; 15(3): e0229756, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32126132

RESUMO

The aim of this work was to study the effect of a high sodium (HS) diet on blood pressure and renal function in male adult rats that have been treated with a dual Endothelin receptor antagonist (ERA) during their early postnatal period (day 1 to 20 of life). Male Sprague-Dawley rats were divided in four groups: CNS: control rats with normosodic diet; ERANS: ERA-treated rats with normosodic diet; CHS: control rats with high sodium diet; ERAHS: ERA-treated rats with HS diet. Systolic blood pressure (SBP) was recorded before and after the diet and 24-hour metabolic cage studies were performed. AQP2 and α-ENac expressions were measured by western blot and real time PCR in the renal medulla. Vasopressin (AVP) pathway was evaluated by measuring V2 receptor and adenylyl cyclase 6 (AC6) expression and cAMP production in the renal medulla. Pre-pro ET-1mRNA was also evaluated in the renal medulla. Only rats that had been treated with an ERA during their postnatal period increased their SBP after consumption of a HS diet, showing an impaired capacity to excrete sodium and water, i.e. developing salt sensitivity. This salt sensitivity would be mediated by an increase in renomedullary expression and activity of AQP2 and α-ENaC as a consequence of increased AC6 expression and cAMP production and/or a decreased ET-1 production in the renal medulla. The knowledge of the molecular mechanisms underlying the perinatal programming of salt sensitive hypertension will allow the development of reprogramming strategies in order to avoid this pathology.


Assuntos
Endotelinas/metabolismo , Hipertensão/etiologia , Medula Renal/crescimento & desenvolvimento , Receptores de Endotelina/metabolismo , Transdução de Sinais/fisiologia , Adulto , Animais , Animais Recém-Nascidos , Aquaporina 2/metabolismo , Pressão Sanguínea/efeitos dos fármacos , Pressão Sanguínea/fisiologia , Modelos Animais de Doenças , Antagonistas dos Receptores de Endotelina/farmacologia , Endotelinas/antagonistas & inibidores , Canais Epiteliais de Sódio/metabolismo , Humanos , Hipertensão/fisiopatologia , Recém-Nascido , Medula Renal/efeitos dos fármacos , Masculino , Ratos , Ratos Sprague-Dawley , Eliminação Renal/efeitos dos fármacos , Eliminação Renal/fisiologia , Transdução de Sinais/efeitos dos fármacos , Cloreto de Sódio na Dieta/efeitos adversos , Cloreto de Sódio na Dieta/metabolismo , Vasopressinas/metabolismo
14.
Am J Physiol Lung Cell Mol Physiol ; 318(4): L787-L800, 2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-32129084

RESUMO

Clinical studies have established that the capacity of removing excess fluid from alveoli is impaired in most patients with acute respiratory distress syndrome. Impaired alveolar fluid clearance (AFC) correlates with poor outcomes. Adenosine A2B receptor (A2BAR) has the lowest affinity with adenosine among four adenosine receptors. It is documented that A2BAR can activate adenylyl cyclase (AC) resulting in elevated cAMP. Based on the understanding that cAMP is a key regulator of epithelial sodium channel (ENaC), which is the limited step in sodium transport, we hypothesized that A2BAR signaling may affect AFC in acute lung injury (ALI) through regulating ENaC via cAMP, thus attenuating pulmonary edema. To address this, we utilized pharmacological approaches to determine the role of A2BAR in AFC in rats with endotoxin-induced lung injury and further focused on the mechanisms in vitro. We observed elevated pulmonary A2BAR level in rats with ALI and the similar upregulation in alveolar epithelial cells exposed to LPS. A2BAR stimulation significantly attenuated pulmonary edema during ALI, an effect that was associated with enhanced AFC and increased ENaC expression. The regulatory effects of A2BAR on ENaC-α expression were further verified in cultured alveolar epithelial type II (ATII) cells. More importantly, activation of A2BAR dramatically increased amiloride-sensitive Na+ currents in ATII cells. Moreover, we observed that A2BAR activation stimulated cAMP accumulation, whereas the cAMP inhibitor abolished the regulatory effect of A2BAR on ENaC-α expression, suggesting that A2BAR activation regulates ENaC-α expression via cAMP-dependent mechanism. Together, these findings suggest that signaling through alveolar epithelial A2BAR promotes alveolar fluid balance during endotoxin-induced ALI by regulating ENaC via cAMP pathway, raising the hopes for treatment of pulmonary edema due to ALI.


Assuntos
Lesão Pulmonar Aguda/metabolismo , Células Epiteliais Alveolares/metabolismo , AMP Cíclico/metabolismo , Alvéolos Pulmonares/metabolismo , Receptor A2B de Adenosina/metabolismo , Transdução de Sinais/fisiologia , Lesão Pulmonar Aguda/induzido quimicamente , Adenosina/metabolismo , Células Epiteliais Alveolares/efeitos dos fármacos , Animais , Endotoxinas/farmacologia , Canais Epiteliais de Sódio/metabolismo , Lipopolissacarídeos/farmacologia , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Masculino , Alvéolos Pulmonares/efeitos dos fármacos , Edema Pulmonar/induzido quimicamente , Edema Pulmonar/metabolismo , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos
15.
Am J Physiol Heart Circ Physiol ; 318(4): H1018-H1027, 2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-32167780

RESUMO

Preeclampsia is a pregnancy-related disorder characterized by hypertension, vascular dysfunction and an increase in circulating inflammatory factors including the cytokine, tumor necrosis factor-α (TNF-α). Studies have shown that placental ischemia is associated with 1) increased circulating TNF-α, 2) attenuated pressure-induced cerebral vascular tone, and 3) suppression of ß-epithelial Na+ channel (ßENaC) protein in cerebral vessels. In addition to its role in epithelial Na+ and water transport, ßENaC is an essential signaling element in transduction of pressure-induced (aka "myogenic") constriction, a critical mechanism of blood flow autoregulation. While cytokines inhibit expression of certain ENaC proteins in epithelial tissue, it is unknown if the increased circulating TNF-α associated with placental ischemia mediates the loss of cerebrovascular ßENaC and cerebral blood flow regulation. Therefore, the purpose of this study was to test the hypothesis that increasing plasma TNF-α in normal pregnant rats reduces cerebrovascular ßENaC expression and impairs cerebral blood flow (CBF) regulation. In vivo TNF-α infusion (200 ng/day, 5 days) inhibited cerebrovascular expression of ßENaC and impaired CBF regulation in pregnant rats. To determine the direct effects of TNF-α and underlying pathways mediating vascular smooth muscle cell ßENaC reduction, we exposed cultured VSMCs (A10 cell line) to TNF-α (1-100 ng/mL) for 16-24 h. TNF-α reduced ßENaC protein expression in a concentration-dependent fashion from 0.1 to 100 ng/mL, without affecting cell death. To assess the role of canonical MAPK signaling in this response, VSMCs were treated with p38MAPK or c-Jun kinase (JNK) inhibitors in the presence of TNF-α. We found that both p38MAPK and JNK blockade prevented TNF-α-mediated ßENaC protein suppression. These data provide evidence that disorders associated with increased circulating TNF-α could lead to impaired cerebrovascular regulation, possibly due to reduced ßENaC-mediated vascular function.NEW & NOTEWORTHY This manuscript identifies TNF-α as a possible placental-derived cytokine that could be involved in declining cerebrovascular health observed in preeclampsia. We found that infusion of TNF-α during pregnancy impaired cerebral blood flow control in rats at high arterial pressures. We further discovered that cerebrovascular ß-epithelial sodium channel (ßENaC) protein, a degenerin protein involved in mechanotransduction, was reduced by TNF-α in pregnant rats, indicating a potential link between impaired blood flow and this myogenic player. We next examined this effect in vitro using a rat vascular smooth muscle cell line. TNF-α reduced ßENaC through canonical MAPK-signaling pathways and was not dependent on cell death. This study demonstrates the pejorative effects of TNF-α on cerebrovascular function during pregnancy and warrants future investigations to study the role of cytokines on vascular function during pregnancy.


Assuntos
Circulação Cerebrovascular , Canais Epiteliais de Sódio/metabolismo , Músculo Liso Vascular/metabolismo , Pré-Eclâmpsia/etiologia , Fator de Necrose Tumoral alfa/sangue , Animais , Pressão Sanguínea , Linhagem Celular , Células Cultivadas , Artérias Cerebrais/efeitos dos fármacos , Artérias Cerebrais/metabolismo , Canais Epiteliais de Sódio/genética , Feminino , Homeostase , Sistema de Sinalização das MAP Quinases , Músculo Liso Vascular/efeitos dos fármacos , Gravidez , Inibidores de Proteínas Quinases/farmacologia , Ratos , Ratos Sprague-Dawley , Fator de Necrose Tumoral alfa/farmacologia
16.
Biochem Biophys Res Commun ; 525(3): 675-680, 2020 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-32139118

RESUMO

Glucocorticoids require the glucocorticoid receptor (GR), a type of ligand-dependent nuclear receptor to transmit their downstream effects. Upon glucocorticoid binding, GR associates with glucocorticoid response elements (GREs) and recruits other transcriptional coregulators to activate or repress target gene transcription. Many SET-domain family proteins have been demonstrated to contribute to GR-mediated transcriptional activity. However, whether histone H3K4-specific methyltransferase plays a cell-type-specific role in GR transcriptional regulation remains poorly understood. In this report, we examined MLL2 (KMT2D), a histone-lysine methyltransferase that catalyzes histone H3 lysine 4 methylation (H3K4me). Furthermore, we demonstrated that MLL2 specifically regulates the transcription of some GR target genes (e.g., ENACα and FLJ20371) in ARPE-19 cells, but has no effect in A549 cells. Mechanistically, co-immunoprecipitation assays revealed that MLL2 is associated with GR in a ligand-independent manner in APRE-19 cells. Moreover, chromatin immunoprecipitation analyses demonstrated that MLL2 could co-occupy glucocorticoid response elements (GREs) of GR target genes along with GR following Dex stimulation. Finally, the FAIRE-qPCR results illustrated that MLL2 is pivotal in establishing chromatin structure accessibility at the GREs of ARPE-19 specific genes in the presence of Dex. Taken together, our study determined that MLL2 regulates GR-mediated transcription in a cell-type-specific manner, and we provide a molecular mechanism to explain the specific role of MLL2 in regulating GR target gene expression in ARPE-19 cells.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Células Epiteliais/metabolismo , Canais Epiteliais de Sódio/metabolismo , Proteínas de Neoplasias/metabolismo , Receptores de Glucocorticoides/metabolismo , Epitélio Pigmentado da Retina/citologia , Transcrição Genética , Sítios de Ligação , Linhagem Celular , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina/efeitos dos fármacos , Dexametasona/farmacologia , Regulação da Expressão Gênica , Humanos
17.
J Vis Exp ; (157)2020 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-32202533

RESUMO

Studying posttranscriptional regulation is fundamental to understanding the modulation of a given messenger RNA (mRNA) and its impact on cell homeostasis and metabolism. Indeed, fluctuations in transcript expression could modify the translation efficiency and ultimately the cellular activity of a transcript. Several experimental approaches have been developed to investigate the half-life of mRNA although some of these methods have limitations that prevent the proper study of posttranscriptional modulation. A promoter induction system can express a gene of interest under the control of a synthetic tetracycline-regulated promoter. This method allows the half-life estimation of a given mRNA under any experimental condition without disturbing cell homeostasis. One major drawback of this method is the necessity to transfect cells, which limits the use of this technique in isolated primary cells that are highly resistant to conventional transfection techniques. Alveolar epithelial cells in primary culture have been used extensively to study the cellular and molecular biology of the alveolar epithelium. The unique characteristics and phenotype of primary alveolar cells make it essential to study the posttranscriptional modulations of genes of interest in these cells. Therefore, our aim was to develop a novel tool to investigate the posttranscriptional modulations of mRNAs of interest in alveolar epithelial cells in primary culture. We designed a fast and efficient transient transfection protocol to insert a transcriptionally controlled plasmid expression system into primary alveolar epithelial cells. This cloning strategy, using a viral epitope to tag the construct, allows for the easy discrimination of construct expression from that of endogenous mRNAs. Using a modified ΔΔ quantification cycle (Cq) method, the expression of the transcript can then be quantified at different time intervals to measure its half-life. Our data demonstrate the efficiency of this novel approach in studying posttranscriptional regulation in various pathophysiological conditions in primary alveolar epithelial cells.


Assuntos
Células Epiteliais Alveolares/metabolismo , Regulação da Expressão Gênica , Plasmídeos/genética , Estabilidade de RNA/genética , Transcrição Genética , Regiões 3' não Traduzidas/genética , Células Epiteliais Alveolares/efeitos dos fármacos , Animais , Células Cultivadas , Primers do DNA/metabolismo , Dactinomicina/farmacologia , Doxiciclina/farmacologia , Eletroporação , Canais Epiteliais de Sódio/genética , Canais Epiteliais de Sódio/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Inflamação/patologia , Cinética , Estabilidade de RNA/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ratos , Transcrição Genética/efeitos dos fármacos , Transfecção
18.
Am J Physiol Renal Physiol ; 318(5): F1113-F1121, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32174140

RESUMO

Ubiquitination of the epithelial Na+ channel (ENaC) in epithelial cells may influence trafficking and hormonal regulation of the channels. We assessed ENaC ubiquitination (ub-ENaC) in mouse and rat kidneys using affinity beads to capture ubiquitinated proteins from tissue homogenates and Western blot analysis with anti-ENaC antibodies. Ub-αENaC was observed primarily as a series of proteins of apparent molecular mass of 40-70 kDa, consistent with the addition of variable numbers of ubiquitin molecules primarily to the NH2-terminal cleaved fragment (~30 kDa) of the subunit. No significant Ub-ßENaC was detected, indicating that ubiquitination of this subunit is minimal. For γENaC, the protein eluted from the affinity beads had the same apparent molecular mass as the cleaved COOH-terminal fragment of the subunit (~65 kDa). This suggests that the ubiquitinated NH2 terminus remains attached to the COOH-terminal moiety during isolation through disulfide bonds. Consistent with this, under nonreducing conditions, eluates contained material with increased molecular mass (90-150 kDa). In mice with a Liddle syndrome mutation (ß566X) deleting a putative binding site for the ubiquitin ligase neural precursor cell expressed developmentally downregulated 4-2, the amount of ub-γENaC was reduced as expected. To assess aldosterone dependence of ubiquitination, we fed rats either control or low-Na+ diets for 7 days before kidney harvest. Na+ depletion increased the amounts of ub-αENaC and ub-γENaC by three- to fivefold, probably reflecting increased amounts of fully cleaved ENaC. We conclude that ubiquitination occurs after complete proteolytic processing of the subunits, contributing to retrieval and/or disposal of channels expressed at the cell surface. Diminished ubiquitination does not appear to be a major factor in aldosterone-dependent ENaC upregulation.


Assuntos
Canais Epiteliais de Sódio/metabolismo , Rim/metabolismo , Síndrome de Liddle/metabolismo , Ubiquitinação , Aldosterona/sangue , Animais , Modelos Animais de Doenças , Canais Epiteliais de Sódio/genética , Feminino , Síndrome de Liddle/genética , Masculino , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Mutação , Proteólise , Ratos Sprague-Dawley
19.
J Am Soc Nephrol ; 31(3): 483-499, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32054691

RESUMO

BACKGROUND: Aldosterone activates the intercalated cell mineralocorticoid receptor, which is enhanced with hypokalemia. Whether this receptor directly regulates the intercalated cell chloride/bicarbonate exchanger pendrin is unclear, as are potassium's role in this response and the receptor's effect on intercalated and principal cell function in the cortical collecting duct (CCD). METHODS: We measured CCD chloride absorption, transepithelial voltage, epithelial sodium channel activity, and pendrin abundance and subcellular distribution in wild-type and intercalated cell-specific mineralocorticoid receptor knockout mice. To determine if the receptor directly regulates pendrin, as well as the effect of serum aldosterone and potassium on this response, we measured pendrin label intensity and subcellular distribution in wild-type mice, knockout mice, and receptor-positive and receptor-negative intercalated cells from the same knockout mice. RESULTS: Ablation of the intercalated cell mineralocorticoid receptor in CCDs from aldosterone-treated mice reduced chloride absorption and epithelial sodium channel activity, despite principal cell mineralocorticoid receptor expression in the knockout mice. With high circulating aldosterone, intercalated cell mineralocorticoid receptor gene ablation directly reduced pendrin's relative abundance in the apical membrane region and pendrin abundance per cell whether serum potassium was high or low. Intercalated cell mineralocorticoid receptor ablation blunted, but did not eliminate, aldosterone's effect on pendrin total and apical abundance and subcellular distribution. CONCLUSIONS: With high circulating aldosterone, intercalated cell mineralocorticoid receptor ablation reduces chloride absorption in the CCD and indirectly reduces principal cell epithelial sodium channel abundance and function. This receptor directly regulates pendrin's total abundance and its relative abundance in the apical membrane region over a wide range in serum potassium concentration. Aldosterone regulates pendrin through mechanisms both dependent and independent of the IC MR receptor.


Assuntos
Aldosterona/metabolismo , Proteínas de Transporte de Ânions/fisiologia , Túbulos Renais Coletores/metabolismo , Potássio/sangue , Receptores de Mineralocorticoides/metabolismo , Transportadores de Sulfato/genética , Angiotensina II/farmacologia , Animais , Células Cultivadas , Antiportadores de Cloreto-Bicarbonato/metabolismo , Células Epiteliais/metabolismo , Canais Epiteliais de Sódio/metabolismo , Técnicas In Vitro , Transporte de Íons/fisiologia , Túbulos Renais Coletores/citologia , Camundongos , Camundongos Knockout , Sensibilidade e Especificidade , Transdução de Sinais/efeitos dos fármacos , Canais de Sódio/genética
20.
Am J Physiol Regul Integr Comp Physiol ; 318(2): R418-R427, 2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-31913682

RESUMO

Genes for the epithelial sodium channel (ENaC) subunits are expressed in a circadian manner, but whether this results in time-of-day differences in activity is not known. Recent data show that protein expression of ENaC subunits is higher in kidneys from female rats, yet females are more efficient in excreting an acute salt load. Thus, our in vivo study determined whether there is a time-of-day difference as well as a sex difference in the response to ENaC inhibition by benzamil. Our results showed that the natriuretic and diuretic responses to a single dose of benzamil were significantly greater in male compared with female rats whether given at the beginning of the inactive period [Zeitgeber time 0 (ZT0), 7 AM] or active period (ZT12, 7 PM). However, the response to benzamil was not significantly different between ZT0 and ZT12 dosing in either male or female rats. There was no difference in renal cortical α-ENaC protein abundance between ZT0 and ZT12 or males and females. Given previous reports of flow-induced stimulation of endothelin-1 (ET-1) production and sex differences in the renal endothelin system, we measured urinary ET-1 excretion to assess the effects of increased urine flow on intrarenal ET-1. ET-1 excretion was significantly increased following benzamil administration in both sexes, but this increase was significantly greater in females. These results support the hypothesis that ENaC activity is less prominent in maintaining Na+ balance in females independent of renal ET-1. Because ENaC subunit genes and protein expression vary by time of day and are greater in female rat kidneys, this suggests a clear disconnect between ENaC expression and channel activity.


Assuntos
Amilorida/análogos & derivados , Bloqueadores do Canal de Sódio Epitelial/farmacologia , Canais Epiteliais de Sódio/efeitos dos fármacos , Rim/efeitos dos fármacos , Natriurese/efeitos dos fármacos , Ciclos de Atividade , Amilorida/farmacologia , Animais , Endotelina-1/urina , Canais Epiteliais de Sódio/metabolismo , Feminino , Rim/metabolismo , Masculino , Ovariectomia , Ratos Sprague-Dawley , Eliminação Renal/efeitos dos fármacos , Fatores Sexuais , Fatores de Tempo , Urodinâmica/efeitos dos fármacos
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