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1.
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
2.
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
3.
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
4.
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
5.
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
6.
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
7.
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
8.
Am J Physiol Regul Integr Comp Physiol ; 318(2): R320-R328, 2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-31913688

RESUMO

The modifications of the hemodynamic system and hydromineral metabolism are physiological features characterizing a normal gestation. Thus, the ability to expand plasma volume without increasing the level of blood pressure is necessary for the correct perfusion of the placenta. The kidney is essential in this adaptation by reabsorbing avidly sodium and fluid. In this study, we observed that the H,K-ATPase type 2 (HKA2), an ion pump expressed in kidney and colon and already involved in the control of the K+ balance during gestation, is also required for the correct plasma volume expansion and to maintain normal blood pressure. Indeed, compared with WT pregnant mice that exhibit a 1.6-fold increase of their plasma volume, pregnant HKA2-null mice (HKA2KO) only modestly expand their extracellular volume (×1.2). The renal expression of the epithelial Na channel (ENaC) α- and γ-subunits and that of the pendrin are stimulated in gravid WT mice, whereas the Na/Cl- cotransporter (NCC) expression is downregulated. These modifications are all blunted in HKA2KO mice. This impeded renal adaptation to gestation is accompanied by the development of hypotension in the pregnant HKA2KO mice. Altogether, our results showed that the absence of the HKA2 during gestation leads to an "underfilled" situation and has established this transporter as a key player of the renal control of salt and potassium metabolism during gestation.


Assuntos
Pressão Sanguínea , ATPase Trocadora de Hidrogênio-Potássio/metabolismo , Rim/enzimologia , Volume Plasmático , Potássio/metabolismo , Sódio/metabolismo , Animais , Aquaporina 2/metabolismo , Colo/enzimologia , Canais Epiteliais de Sódio/genética , Canais Epiteliais de Sódio/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Idade Gestacional , ATPase Trocadora de Hidrogênio-Potássio/deficiência , ATPase Trocadora de Hidrogênio-Potássio/genética , Homeostase , Camundongos Endogâmicos C57BL , Camundongos Knockout , Gravidez , Membro 3 da Família 12 de Carreador de Soluto/genética , Membro 3 da Família 12 de Carreador de Soluto/metabolismo , Transportadores de Sulfato/genética , Transportadores de Sulfato/metabolismo
9.
Am J Physiol Cell Physiol ; 318(3): C570-C580, 2020 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-31913693

RESUMO

Cystic fibrosis (CF) lung disease persists and remains life-limiting for many patients. Elevated high-mobility group box-1 protein (HMGB-1) levels and epithelial sodium channel hyperactivity (ENaC) are hallmark features of the CF lung. The objective of this study was to better understand the pathogenic role of HMGB-1 signaling and ENaC in CF airway cells. We hypothesize that HMGB-1 links airway inflammation [via signaling to the receptor for advanced glycation end products (RAGE)] and airway surface liquid dehydration (via upregulation of ENaC) in the CF lung. We calculated equivalent short-current (Isc) and single-channel ENaC open probability (Po) in normal and CF human small airway epithelial cells (SAEC) in the presence and absence of human HMGB-1 peptide (0.5 µg/mL). In normal SAECs, HMGB-1 increased amiloride-sensitive Isc and elevated ENaC Po from 0.15 ± 0.03 to 0.28 ± 0.04 (P < 0.01). In CF SAECs, ENaC Po increased from 0.45 ± 0.06 to 0.73 ± 0.04 (P < 0.01). Pretreatment with 1 µM FPS-ZM1 (a RAGE inhibitor) attenuated all HMGB-1 effects on ENaC current in normal and CF SAECs. Confocal analysis of SAECs indicates that nuclear size and HMBG-1 localization can be impacted by ENaC dysfunction. Masson's trichrome labeling of mouse lung showed that intraperitoneally injected HMGB-1 significantly increased pulmonary fibrosis. Bronchoalveolar lavage fluid from HMGB-1-treated mice showed significant increases in IL-1ß, IL-10, IL-6, IL-27, IL-17A, IFN-ß, and granulocyte-macrophage colony-stimulating factor compared with vehicle-injected mice (P < 0.05). These studies put forth a new model in which HMGB-1 signaling to RAGE plays an important role in perpetuating ENaC dysfunction and inflammation in the CF lung.


Assuntos
Canais Epiteliais de Sódio/metabolismo , Proteína HMGB1/toxicidade , Mediadores da Inflamação/metabolismo , Receptor para Produtos Finais de Glicação Avançada/metabolismo , Mucosa Respiratória/metabolismo , Animais , Células Cultivadas , Feminino , Humanos , Inflamação/induzido quimicamente , Inflamação/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mucosa Respiratória/citologia , Mucosa Respiratória/efeitos dos fármacos
10.
Am J Physiol Lung Cell Mol Physiol ; 318(3): L518-L524, 2020 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-31994896

RESUMO

Airway mucus obstruction is a hallmark of chronic lung diseases such as cystic fibrosis, asthma, and COPD, and the development of more effective mucus-mobilizing therapies remains an important unmet need for patients with these muco-obstructive lung diseases. However, methods for sensitive visualization and quantitative assessment of immediate effects of therapeutic interventions on mucus clearance in vivo are lacking. In this study, we determined whether newly developed high-speed microscopic optical coherence tomography (mOCT) is sensitive to detect and compare in vivo effects of inhaled isotonic saline, hypertonic saline, and bicarbonate on mucus mobilization and clearance in Scnn1b-transgenic mice with muco-obstructive lung disease. In vivo mOCT imaging showed that inhaled isotonic saline-induced rapid mobilization of mucus that was mainly transported as chunks from the lower airways of Scnn1b-transgenic mice. Hypertonic saline mobilized a significantly greater amount of mucus that showed a more uniform distribution compared with isotonic saline. The addition of bicarbonate-to-isotonic saline had no effect on mucus mobilization, but also led to a more uniform mucus layer compared with treatment with isotonic saline alone. mOCT can detect differences in response to mucus-mobilizing interventions in vivo, and may thus support the development of more effective therapies for patients with muco-obstructive lung diseases.


Assuntos
Modelos Animais de Doenças , Canais Epiteliais de Sódio/fisiologia , Microscopia Intravital/métodos , Pneumopatias Obstrutivas/diagnóstico por imagem , Depuração Mucociliar , Muco/diagnóstico por imagem , Tomografia de Coerência Óptica/métodos , Animais , Humanos , Pneumopatias Obstrutivas/patologia , Pneumopatias Obstrutivas/terapia , Camundongos , Camundongos Transgênicos , Muco/fisiologia
11.
Am J Physiol Cell Physiol ; 318(1): C150-C162, 2020 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-31721612

RESUMO

Epithelial Na+ channels (ENaCs) are members of a family of cation channels that function as sensors of the extracellular environment. ENaCs are activated by specific proteases in the biosynthetic pathway and at the cell surface and remove embedded inhibitory tracts, which allows channels to transition to higher open-probability states. Resolved structures of ENaC and an acid-sensing ion channel revealed highly organized extracellular regions. Within the periphery of ENaC subunits are unique domains formed by antiparallel ß-strands containing the inhibitory tracts and protease cleavage sites. ENaCs are inhibited by Na+ binding to specific extracellular site(s), which promotes channel transition to a lower open-probability state. Specific inositol phospholipids and channel modification by Cys-palmitoylation enhance channel open probability. How these regulatory factors interact in a concerted manner to influence channel open probability is an important question that has not been resolved. These various factors are reviewed, and the impact of specific factors on human disorders is discussed.


Assuntos
Canais Epiteliais de Sódio/metabolismo , Ativação do Canal Iônico , Sódio/metabolismo , Animais , Canais Epiteliais de Sódio/química , Humanos , Potenciais da Membrana , Modelos Moleculares , Conformação Proteica , Transdução de Sinais , Relação Estrutura-Atividade
12.
Am J Physiol Renal Physiol ; 318(1): F1-F13, 2020 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-31657249

RESUMO

Renal Na+ reabsorption, facilitated by the epithelial Na+ channel (ENaC), is subject to multiple forms of control to ensure optimal body blood volume and pressure through altering both the ENaC population and activity at the cell surface. Here, the focus is on regulating the number of ENaCs present in the apical membrane domain through pathways of ENaC synthesis and targeting to the apical membrane as well as ENaC removal, recycling, and degradation. Finally, the mechanisms by which ENaC trafficking pathways are regulated are summarized.


Assuntos
Membrana Celular/metabolismo , Células Epiteliais/metabolismo , Canais Epiteliais de Sódio/metabolismo , Rim/metabolismo , Transporte Proteico/fisiologia , Animais , Humanos , Sódio/metabolismo
13.
Am J Physiol Renal Physiol ; 318(2): F402-F421, 2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-31841393

RESUMO

Hypokalemia increases ammonia excretion and decreases K+ excretion. The present study examined the role of the proximal tubule protein NBCe1-A in these responses. We studied mice with Na+-bicarbonate cotransporter electrogenic, isoform 1, splice variant A (NBCe1-A) deletion [knockout (KO) mice] and their wild-type (WT) littermates were provided either K+ control or K+-free diet. We also used tissue sections to determine the effect of extracellular ammonia on NaCl cotransporter (NCC) phosphorylation. The K+-free diet significantly increased proximal tubule NBCe1-A and ammonia excretion in WT mice, and NBCe1-A deletion blunted the ammonia excretion response. NBCe1-A deletion inhibited the ammoniagenic/ammonia recycling enzyme response in the cortical proximal tubule (PT), where NBCe1-A is present in WT mice. In the outer medulla, where NBCe1-A is not present, the PT ammonia metabolism response was accentuated by NBCe1-A deletion. KO mice developed more severe hypokalemia and had greater urinary K+ excretion during the K+-free diet than did WT mice. This was associated with blunting of the hypokalemia-induced change in NCC phosphorylation. NBCe1-A KO mice have systemic metabolic acidosis, but experimentally induced metabolic acidosis did not alter NCC phosphorylation. Although KO mice have impaired ammonia metabolism, experiments in tissue sections showed that lack of ammonia does impair NCC phosphorylation. Finally, urinary aldosterone was greater in KO mice than in WT mice, but neither expression of epithelial Na+ channel α-, ß-, and γ-subunits nor of H+-K+-ATPase α1- or α2-subunits correlated with changes in urinary K+. We conclude that NBCe1-A is critical for the effect of diet-induced hypokalemia to increase cortical proximal tubule ammonia generation and for the expected decrease in urinary K+ excretion.


Assuntos
Amônia/urina , Hipopotassemia/metabolismo , Túbulos Renais Proximais/metabolismo , Potássio na Dieta/sangue , Eliminação Renal , Simportadores de Sódio-Bicarbonato/metabolismo , Acidose/genética , Acidose/metabolismo , Acidose/fisiopatologia , Aldosterona/urina , Animais , Biomarcadores/sangue , Biomarcadores/urina , Modelos Animais de Doenças , Canais Epiteliais de Sódio/metabolismo , Glutamato-Amônia Ligase/metabolismo , ATPase Trocadora de Hidrogênio-Potássio/genética , ATPase Trocadora de Hidrogênio-Potássio/metabolismo , Hipopotassemia/genética , Hipopotassemia/fisiopatologia , Túbulos Renais Proximais/fisiopatologia , Camundongos Knockout , Fosforilação , Simportadores de Sódio-Bicarbonato/deficiência , Simportadores de Sódio-Bicarbonato/genética , Membro 3 da Família 12 de Carreador de Soluto/metabolismo
14.
Proc Natl Acad Sci U S A ; 117(1): 717-726, 2020 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-31871197

RESUMO

Mechanosensitive ion channels are crucial for normal cell function and facilitate physiological function, such as blood pressure regulation. So far little is known about the molecular mechanisms of how channels sense mechanical force. Canonical vertebrate epithelial Na+ channel (ENaC) formed by α-, ß-, and γ-subunits is a shear force (SF) sensor and a member of the ENaC/degenerin protein family. ENaC activity in epithelial cells contributes to electrolyte/fluid-homeostasis and blood pressure regulation. Furthermore, ENaC in endothelial cells mediates vascular responsiveness to regulate blood pressure. Here, we provide evidence that ENaC's ability to mediate SF responsiveness relies on the "force-from-filament" principle involving extracellular tethers and the extracellular matrix (ECM). Two glycosylated asparagines, respectively their N-glycans localized in the palm and knuckle domains of αENaC, were identified as potential tethers. Decreased SF-induced ENaC currents were observed following removal of the ECM/glycocalyx, replacement of these glycosylated asparagines, or removal of N-glycans. Endothelial-specific overexpression of αENaC in mice induced hypertension. In contrast, expression of αENaC lacking these glycosylated asparagines blunted this effect. In summary, glycosylated asparagines in the palm and knuckle domains of αENaC are important for SF sensing. In accordance with the force-from-filament principle, they may provide a connection to the ECM that facilitates vascular responsiveness contributing to blood pressure regulation.


Assuntos
Asparagina/metabolismo , Canais Epiteliais de Sódio/metabolismo , Matriz Extracelular/metabolismo , Domínios Proteicos/genética , Animais , Asparagina/química , Modelos Animais de Doenças , Células Endoteliais , Endotélio Vascular/citologia , Endotélio Vascular/patologia , Endotélio Vascular/fisiopatologia , Canais Epiteliais de Sódio/química , Canais Epiteliais de Sódio/genética , Feminino , Glicosilação , Células HEK293 , Humanos , Hipertensão/etiologia , Hipertensão/patologia , Hipertensão/fisiopatologia , Masculino , Camundongos , Camundongos Transgênicos , Mutagênese Sítio-Dirigida , Oócitos , Técnicas de Patch-Clamp , Mutação Puntual , Polissacarídeos/química , Estresse Mecânico , Xenopus laevis
15.
Am J Physiol Renal Physiol ; 318(3): F817-F825, 2020 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-31841392

RESUMO

We have previously shown that activation of (pro)renin receptor (PRR) induces epithelial Na+ channel (ENaC) activity in cultured collecting duct cells. Here, we examined the role of soluble PRR (sPRR), the cleavage product of PRR in ENaC regulation, and further tested its relevance to aldosterone signaling. In cultured mpkCCD cells, administration of recombinant histidine-tagged sPRR (sPRR-His) at 10 nM within minutes induced a significant and transient increase in the amiloride-sensitive short-circuit current as assessed using the Ussing chamber technique. The acute ENaC activation was blocked by the NADPH oxidase 1/4 inhibitor GKT137892 and siRNA against Nox4 but not the ß-catenin inhibitor ICG-001. In primary rat inner medullary collecting duct cells, administration of sPRR-His at 10 nM for 24 h induced protein expression of the α-subunit but not ß- or γ-subunits of ENaC, in parallel with upregulation of mRNA expression as well as promoter activity of the α-subunit. The transcriptional activation of α-ENaC was dependent on ß-catenin signaling. Consistent results obtained by epithelial volt ohmmeter measurement of equivalent current and Ussing chamber determination of short-circuit current showed that aldosterone-induced transepithelial Na+ transport was inhibited by the PRR decoy inhibitor PRO20 and PF-429242, an inhibitor of sPRR-generating enzyme site-1 protease, and the response was restored by the addition of sPRR-His. Medium sPRR was elevated by aldosterone and inhibited by PF-429242. Taken together, these results demonstrate that sPRR induces two phases of ENaC activation via distinct mechanisms and functions as a mediator of the natriferic action of aldosterone.


Assuntos
Aldosterona/metabolismo , Canais Epiteliais de Sódio , Túbulos Renais Coletores/citologia , Receptores de Superfície Celular/metabolismo , Transdução de Sinais/fisiologia , Animais , Transporte Biológico , Células Cultivadas , Fenômenos Eletrofisiológicos , Bloqueadores do Canal de Sódio Epitelial/administração & dosagem , Bloqueadores do Canal de Sódio Epitelial/farmacologia , Masculino , NADPH Oxidase 4/genética , NADPH Oxidase 4/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de Superfície Celular/genética , Sódio/metabolismo
17.
Pol Merkur Lekarski ; 47(281): 190-192, 2019 Nov 29.
Artigo em Polonês | MEDLINE | ID: mdl-31812974

RESUMO

Liddle syndrome is an uncommon genetic disorder featuring hypertension, hypokalemia, metabolic alcalosis, decreased rennin and aldosterone secretion. It is caused by a point mutation of a gene encoding one of the three subunits of the epithelial sodium channel (ENaC). Because of its rarity, the availability of the literature on the diagnosis of this syndrome is limited. A CASE REPORT: The 14 years old adolescent with resistant hypertension was analyzed genetically, because of the family history. The significance of it and biochemical findings in recognition of Liddle Syndrome was discussed. It has been concluded that performing a genetic test at the suspicion of monogenic background of hypertension allows for accurate and effective treatment.


Assuntos
Hipertensão , Síndrome de Liddle , Adolescente , Canais Epiteliais de Sódio , Humanos , Hipopotassemia
18.
Int J Mol Sci ; 21(1)2019 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-31861781

RESUMO

Preterm infants frequently suffer from respiratory distress syndrome (RDS), possibly due to lower expression of epithelial Na+ channels (ENaC). RDS incidence is sex-specific, affecting males almost twice as often. Despite the use of antenatal glucocorticoids (GCs), the sex difference persists. It is still controversial whether both sexes benefit equally from GCs. We previously showed that Na+ transport is higher in female compared with male fetal distal lung epithelial (FDLE) cells. Since GCs increase Na+ transport, we hypothesized that their stimulating effect might be sex-specific. We analyzed FDLE cells with Ussing chambers and RT-qPCR in the presence or absence of fetal serum. In serum-free medium, GCs increased the ENaC activity and mRNA expression, independent of sex. In contrast, GCs did not increase the Na+ transport in serum-supplemented media and abolished the otherwise observed sex difference. Inhibition of the GC receptor in the presence of serum did not equalize Na+ transport between male and female cells. The GC-induced surfactant protein mRNA expression was concentration and sex-specific. In conclusion, female and male FDLE cells exhibit no sex difference in response to GCs with regard to Na+ transport, and GR activity does not contribute to the higher Na+ transport in females.


Assuntos
Células Epiteliais Alveolares/efeitos dos fármacos , Canais Epiteliais de Sódio/metabolismo , Glucocorticoides/farmacologia , Células Epiteliais Alveolares/metabolismo , Animais , Animais Recém-Nascidos , Transporte Biológico/efeitos dos fármacos , Células Cultivadas , Feminino , Humanos , Masculino , Ratos , Síndrome do Desconforto Respiratório do Recém-Nascido/tratamento farmacológico , Síndrome do Desconforto Respiratório do Recém-Nascido/metabolismo , Caracteres Sexuais , Sódio/metabolismo
19.
Bull Exp Biol Med ; 168(2): 219-223, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31776946

RESUMO

We analyzed the expression of molecular targets of natriuretic action of prolactin in different layers of the kidney in the rat model of cholestasis of pregnancy. Sodium bicarbonate cotransporter NBCe1 was most sensitive to the conditions of cholestasis and cholestasis of pregnancy: the expression NBCe1 mRNA and protein in the renal outer medulla decreased in comparison with the normal. All forms of cholestasis affected the mRNA expression of sodium-potassium chloride co-transporter NCC, α-subunit of the ENaCα epithelial sodium channel, and Nedd4-2 ubiquitin ligase in different layers of the kidney. The obtained data suggest that prolactin provides fine tuning of various sodium transporters in different parts of the nephron under pathological conditions.


Assuntos
Colestase/patologia , Transporte de Íons/fisiologia , Medula Renal/metabolismo , Prolactina/metabolismo , Equilíbrio Hidroeletrolítico/fisiologia , Animais , Modelos Animais de Doenças , Canais Epiteliais de Sódio/biossíntese , Canais Epiteliais de Sódio/genética , Feminino , Ubiquitina-Proteína Ligases Nedd4/biossíntese , Ubiquitina-Proteína Ligases Nedd4/genética , Gravidez , RNA Mensageiro/biossíntese , Ratos , Simportadores de Sódio-Bicarbonato/biossíntese , Simportadores de Sódio-Bicarbonato/genética , Membro 3 da Família 12 de Carreador de Soluto/biossíntese , Membro 3 da Família 12 de Carreador de Soluto/genética
20.
Am J Physiol Renal Physiol ; 317(6): F1612-F1622, 2019 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-31566425

RESUMO

Low Na+ intake activates aldosterone signaling, which increases renal Na+ reabsorption through increased apical activity of the NaCl cotransporter (NCC) and the epithelial Na+ channel (ENaC). Na+ transporter proteins are excreted in urine as an integral part of cell-derived extracellular vesicles (uEVs). It was hypothesized that Na+ transport protein levels in uEVs from healthy humans reflect their physiological regulation by aldosterone. Urine and plasma samples from 10 healthy men (median age: 22.8 yr) were collected after 5 days on a low-Na+ (70 mmol/day) diet and 5 days on a high-Na+ (250 mmol/day) diet. uEVs were isolated by ultracentrifugation and analyzed by Western blot analysis for EV markers (CD9, CD63, and ALIX), transport proteins (Na+-K+-ATPase α1-subunit, NCC, ENaC α- and γ-subunits, and aquaporin 2), and the ENaC-cleaving protease prostasin. Plasma renin and aldosterone concentrations increased during the low-Na+ diet. uEV size and concentration were not different between diets by tunable resistive pulse sensing. EV markers ALIX and CD9 increased with the low-Na+ diet, whereas CD63 and aquaporin 2 excretion were unchanged. Full-length ENaC γ-subunits were generally not detectable in uEVs, whereas ENaC α-subunits, NCC, and phosphorylated NCC were consistently detected but not changed by Na+ intake. Prostasin increased with low Na+ in uEVs. uEV excretion of transporters was not correlated with blood pressure, urinary Na+ and K+ excretion, plasma renin, or aldosterone. In conclusion, apical Na+ transporter proteins and proteases were excreted in uEVs, and while the excretion rate and size of uEVs were not affected, EV markers and prostasin increased in response to the low-Na+ diet.


Assuntos
Canais Epiteliais de Sódio/metabolismo , Serina Endopeptidases/urina , Sódio na Dieta/farmacologia , Adenosina Trifosfatases/urina , Adulto , Albuminúria/urina , Creatinina/urina , Dieta Hipossódica , Eletrólitos/urina , Canais Epiteliais de Sódio/efeitos dos fármacos , Exossomos/metabolismo , Vesículas Extracelulares , Humanos , Rim/patologia , Masculino , Sistema Renina-Angiotensina , Membro 3 da Família 12 de Carreador de Soluto/efeitos dos fármacos , Membro 3 da Família 12 de Carreador de Soluto/metabolismo , Adulto Jovem
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