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1.
Nat Commun ; 11(1): 4320, 2020 08 28.
Artigo em Inglês | MEDLINE | ID: mdl-32859916

RESUMO

In autosomal dominant polycystic kidney disease (ADPKD) multiple bilateral renal cysts gradually enlarge, leading to a decline in renal function. Transepithelial chloride secretion through cystic fibrosis transmembrane conductance regulator (CFTR) and TMEM16A (anoctamin 1) are known to drive cyst enlargement. Here we demonstrate that loss of Pkd1 increased expression of TMEM16A and CFTR and Cl- secretion in murine kidneys, with TMEM16A essentially contributing to cyst growth. Upregulated TMEM16A enhanced intracellular Ca2+ signaling and proliferation of Pkd1-deficient renal epithelial cells. In contrast, increase in Ca2+ signaling, cell proliferation and CFTR expression was not observed in Pkd1/Tmem16a double knockout mice. Knockout of Tmem16a or inhibition of TMEM16A in vivo by the FDA-approved drugs niclosamide and benzbromarone, as well as the TMEM16A-specific inhibitor Ani9 largely reduced cyst enlargement and abnormal cyst cell proliferation. The present data establish a therapeutic concept for the treatment of ADPKD.


Assuntos
Anoctamina-1/genética , Anoctamina-1/metabolismo , Cistos/metabolismo , Rim Policístico Autossômico Dominante/metabolismo , Canais de Cátion TRPP/genética , Canais de Cátion TRPP/metabolismo , Animais , Anoctamina-1/efeitos dos fármacos , Benzobromarona/farmacologia , Canais de Cálcio , Proliferação de Células , Cloretos/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística , Cistos/tratamento farmacológico , Cistos/genética , Modelos Animais de Doenças , Cães , Células Epiteliais/metabolismo , Humanos , Rim/metabolismo , Rim/patologia , Células Madin Darby de Rim Canino , Camundongos , Camundongos Knockout , Néfrons/metabolismo , Niclosamida/farmacologia , Rim Policístico Autossômico Dominante/tratamento farmacológico , Rim Policístico Autossômico Dominante/genética
2.
Proc Biol Sci ; 287(1931): 20200970, 2020 07 29.
Artigo em Inglês | MEDLINE | ID: mdl-32673558

RESUMO

Ocean warming impacts the fitness of marine ectothermic species, leading to poleward range shifts, re-shuffling of communities, and changes in ecosystem services. While the detrimental effects of summer heat waves have been widely studied, little is known about the impacts of winter warming on marine species in temperate regions. Many species benefit from low winter temperature-induced reductions in metabolism, as these permit conservation of energy reserves that are needed to support reproduction in spring. Here, we used a unique outdoor mesocosm system to expose a coastal predator-prey system, the sea star Asterias and the blue mussel Mytilus, to different winter warming scenarios under near-natural conditions. We found that the body condition of mussels decreased in a linear fashion with increasing temperature. Sea star growth also decreased with increasing temperature, which was a function of unaltered predation rates and decreased mussel body condition. Asterias relative digestive gland mass strongly declined over the studied temperature interval (ca twofold). This could have severe implications for reproductive capacity in the following spring, as digestive glands provide reserve compounds to maturing gonads. Thus, both predator and prey suffered from a mismatch of energy acquisition versus consumption in warmer winter scenarios, with pronounced consequences for food web energy transfer in future oceans.


Assuntos
Bivalves/fisiologia , Mudança Climática , Comportamento Predatório/fisiologia , Água do Mar/química , Estrelas-do-Mar/fisiologia , Animais , Ecossistema , Cadeia Alimentar , Oceanos e Mares , Estações do Ano , Inanição , Temperatura
3.
J Mol Med (Berl) ; 98(5): 659-671, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32185407

RESUMO

Polycystic kidney disease (PKD) leads to continuous decline of renal function by growth of renal cysts. Enhanced proliferation and transepithelial chloride secretion through cystic fibrosis transmembrane conductance regulator (CFTR) and Ca2+-activated TMEM16A Cl- channels is thought to cause an increase in cyst volume. Recent work shows the pro-proliferative role of the Ca2+ activated Cl- channel TMEM16A (anoctamin 1), and demonstrates the essential contribution of TMEM16A to CFTR-dependent Cl- secretion. The present data demonstrate an increase in intracellular Ca2+ ([Ca2+]i) signals and Cl- secretion by TMEM16A, in renal collecting duct principle cells from dog (MDCK) and mouse (M1) as well as primary tubular epithelial cells from PKD1-/- knockout mice. M1 organoids proliferated, increased expression of TMEM16A, and secreted Cl- upon knockdown of endogenous polycystin 1 or 2 (PKD1,2), by retroviral transfection with shPKD1 and shPKD2, respectively. Knockdown of PKD1 or PKD2 increased basal intracellular Ca2+ levels and enhanced purinergic Ca2+ release from endoplasmic reticulum. In contrast, ryanodine receptors were found not to be expressed in mouse renal epithelial cells and caffeine had no effects on [Ca2+]i. Ca2+ signals, proliferation, and Cl- secretion were largely reduced by knockdown or blockade of TMEM16A. TMEM16A may be therefore important for enhanced Ca2+ release from IP3-sensitive Ca2+ stores in polycystic kidney disease. KEY MESSAGES: • ADPKD leads to continuous decline of renal function by growth of renal cysts. • Knockdown of PKD1 or PKD2 increases TMEM16A expression. • TMEM16A enhanced intracellular Ca2+ signals, Cl- secretion, and proliferation. • TMEM16A contributes to cyst growth in ADPKD.

4.
J Am Soc Nephrol ; 30(2): 228-242, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30606785

RESUMO

BACKGROUND: Transepithelial chloride- secretion, through the chloride channels cystic fibrosis transmembrane conductance regulator (CFTR) and TMEM16A (anoctamin 1), drives cyst enlargement in polycystic kidney disease (PKD). Polycystic kidneys are hypoxic, and oxidative stress activates TMEM16A. However, mechanisms for channel activation in PKD remain obscure. METHODS: Using tissue samples from patients with autosomal dominant PKD, embryonic kidney cultures, and an MDCK in vitro cyst model, we assessed peroxidation of plasma membrane phospholipids in human and mouse polycystic kidneys. We also used electrophysiologic Ussing chamber and patch clamp experiments to analyze activation of TMEM16A and growth of renal cysts. RESULTS: Peroxidation of phospholipids in human and mouse kidneys as well as MDCK cysts in vitro is probably due to enhanced levels of reactive oxygen species. Lipid peroxidation correlated with increased cyst volume as shown in renal cultures and MDCK cysts in three-dimensional cultures. Reactive oxygen species and lipid peroxidation strongly activated TMEM16A, leading to depletion of calcium ion stores and store-operated calcium influx. Activation of TMEM16A- and CFTR-dependent chloride secretion strongly augmented cyst growth. Exposure to scavengers of reactive oxygen species, such as glutathione, coenzyme Q10, or idebenone (a synthetic coenzyme Q10 homolog), as well as inhibition of oxidative lipid damage by ferrostatin-1 largely reduced activation of TMEM16A. Inhibition of TMEM16A reduced proliferation and fluid secretion in vitro. CONCLUSIONS: These findings indicate that activation of TMEM16A by lipid peroxidation drives growth of renal cysts. We propose direct inhibition of TMEM16A or inhibition of lipid peroxidation as potentially powerful therapeutic approaches to delay cyst development in PKD.


Assuntos
Anoctamina-1/genética , Proliferação de Células/efeitos dos fármacos , Peroxidação de Lipídeos/fisiologia , Rim Policístico Autossômico Dominante/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Animais , Biópsia por Agulha , Proliferação de Células/genética , Células Cultivadas , Humanos , Imuno-Histoquímica , Técnicas In Vitro , Camundongos , Estresse Oxidativo , Rim Policístico Autossômico Dominante/patologia , Sensibilidade e Especificidade
5.
ChemMedChem ; 14(1): 94-99, 2019 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-30380199

RESUMO

Prolyl hydroxylation domain (PHD) enzymes catalyze the hydroxylation of the transcription factor hypoxia-inducible factor (HIF) and serve as cellular oxygen sensors. HIF and the PHD enzymes regulate numerous potentially tissue-protective target genes which can adapt cells to metabolic and ischemic stress. We describe a fluorescent PHD inhibitor (1-chloro-4-hydroxybenzo[g]isoquinoline-3-carbonyl)glycine which is suited to fluorescence-based detection assays and for monitoring PHD inhibitors in biological systems. In cell-based assays, application of the fluorescent PHD inhibitor allowed co-localization with a cellular PHD enzyme and led to live cell imaging of processes involved in cellular oxygen sensing.


Assuntos
Benzilisoquinolinas/farmacologia , Corantes Fluorescentes/farmacologia , Imagem Molecular/métodos , Imagem Óptica/métodos , Prolil Hidroxilases/metabolismo , Inibidores de Prolil-Hidrolase/farmacologia , Benzilisoquinolinas/síntese química , Benzilisoquinolinas/química , Biocatálise/efeitos dos fármacos , Relação Dose-Resposta a Droga , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/química , Células HeLa , Humanos , Estrutura Molecular , Inibidores de Prolil-Hidrolase/síntese química , Inibidores de Prolil-Hidrolase/química , Relação Estrutura-Atividade
6.
Kidney Int ; 91(3): 616-627, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-27927598

RESUMO

Chronic kidney disease (CKD) is associated with increased risk and worse prognosis of cardiovascular disease, including peripheral artery disease. An impaired angiogenic response to ischemia may contribute to poor outcomes of peripheral artery disease in patients with CKD. Hypoxia inducible factors (HIF) are master regulators of angiogenesis and therefore represent a promising target for therapeutic intervention. To test this we induced hind-limb ischemia in rats with CKD caused by 5/6 nephrectomy and administered two different treatments known to stabilize HIF protein in vivo: carbon monoxide and a pharmacological inhibitor of prolyl hydroxylation 2-(1-chloro-4- hydroxyisoquinoline-3-carboxamido) acetate (ICA). Expression levels of pro-angiogenic HIF target genes (Vegf, Vegf-r1, Vegf-r2, Ho-1) were measured by qRT-PCR. Capillary density was measured by CD31 immunofluorescence staining and HIF expression was evaluated by immunohistochemistry. Capillary density in ischemic skeletal muscle was significantly lower in CKD animals compared to sham controls. Rats with CKD showed significantly lower expression of HIF and all measured pro-angiogenic HIF target genes, including VEGF. Both HIF stabilizing treatments rescued HIF target gene expression in animals with CKD and led to significantly higher ischemia-induced capillary sprouting compared to untreated controls. ICA was effective regardless of whether it was administered before or after induction of ischemia and led to a HIF expression in skeletal muscle. Thus, impaired ischemia-induced angiogenesis in rats with CKD can be improved by HIF stabilization, even if started after onset of ischemia.


Assuntos
Capilares/efeitos dos fármacos , Monóxido de Carbono/farmacologia , Glicina/análogos & derivados , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Isquemia/tratamento farmacológico , Isoquinolinas/farmacologia , Músculo Esquelético/irrigação sanguínea , Neovascularização Fisiológica/efeitos dos fármacos , Insuficiência Renal Crônica/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Capilares/metabolismo , Capilares/fisiopatologia , Linhagem Celular , Modelos Animais de Doenças , Regulação da Expressão Gênica , Glicina/farmacologia , Heme Oxigenase (Desciclizante)/genética , Heme Oxigenase (Desciclizante)/metabolismo , Membro Posterior , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Isquemia/genética , Isquemia/metabolismo , Isquemia/fisiopatologia , Masculino , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Estabilidade Proteica , Ratos Sprague-Dawley , Insuficiência Renal Crônica/genética , Insuficiência Renal Crônica/fisiopatologia , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo
7.
Am J Pathol ; 174(5): 1663-74, 2009 May.
Artigo em Inglês | MEDLINE | ID: mdl-19349364

RESUMO

Hypoxia-inducible transcription factors (HIFs) play important roles in the response of the kidney to systemic and regional hypoxia. Degradation of HIFs is mediated by three oxygen-dependent HIF-prolyl hydroxylases (PHDs), which have partially overlapping characteristics. Although PHD inhibitors, which can induce HIFs in the presence of oxygen, are already in clinical development, little is known about the expression and regulation of these enzymes in the kidney. Therefore, we investigated the expression levels of the three PHDs in both isolated tubular cells and rat kidneys. All three PHDs were present in the kidney and were expressed predominantly in three different cell populations: (a) in distal convoluted tubules and collecting ducts (PHD1,2,3), (b) in glomerular podocytes (PHD1,3), and (c) in interstitial fibroblasts (PHD1,3). Higher levels of PHDs were found in tubular segments of the inner medulla where oxygen tensions are known to be physiologically low. PHD expression levels were unchanged in HIF-positive tubular and interstitial cells after induction by systemic hypoxia. In rat models of acute renal injury, changes in PHD expression levels were variable; while cisplatin and ischemia/reperfusion led to significant decreases in PHD2 and 3 expression levels, no changes were seen in a model of contrast media-induced nephropathy. These results implicate the non-uniform expression of HIF-regulating enzymes that modify the hypoxic response in the kidney under both regional and temporal conditions.


Assuntos
Lesão Renal Aguda/enzimologia , Proteínas de Ligação a DNA/metabolismo , Proteínas de Homeodomínio/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Proteínas Imediatamente Precoces/metabolismo , Rim/enzimologia , Pró-Colágeno-Prolina Dioxigenase/metabolismo , Animais , Antineoplásicos/toxicidade , Western Blotting , Cisplatino/toxicidade , Meios de Contraste/farmacologia , Proteínas de Ligação a DNA/genética , Fibroblastos/efeitos dos fármacos , Fibroblastos/enzimologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Proteínas de Homeodomínio/genética , Prolina Dioxigenases do Fator Induzível por Hipóxia , Proteínas Imediatamente Precoces/genética , Técnicas Imunoenzimáticas , Isquemia/metabolismo , Isquemia/patologia , Rim/efeitos dos fármacos , Rim/lesões , Medula Renal/efeitos dos fármacos , Medula Renal/enzimologia , Túbulos Renais/efeitos dos fármacos , Túbulos Renais/enzimologia , Túbulos Renais Coletores/efeitos dos fármacos , Túbulos Renais Coletores/enzimologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Oxigênio/metabolismo , Podócitos/efeitos dos fármacos , Podócitos/enzimologia , Pró-Colágeno-Prolina Dioxigenase/genética , Prognóstico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa
8.
EMBO J ; 28(5): 490-9, 2009 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-19153608

RESUMO

Ca(2+) is an important signalling molecule that regulates multiple cellular processes, including apoptosis. Although Ca(2+) influx through transient receptor potential (TRP) channels in the plasma membrane is known to trigger cell death, the function of intracellular TRP proteins in the regulation of Ca(2+)-dependent signalling pathways and apoptosis has remained elusive. Here, we show that TRPP2, the ion channel mutated in autosomal dominant polycystic kidney disease (ADPKD), protects cells from apoptosis by lowering the Ca(2+) concentration in the endoplasmic reticulum (ER). ER-resident TRPP2 counteracts the activity of the sarcoendoplasmic Ca(2+) ATPase by increasing the ER Ca(2+) permeability. This results in diminished cytosolic and mitochondrial Ca(2+) signals upon stimulation of inositol 1,4,5-trisphosphate receptors and reduces Ca(2+) release from the ER in response to apoptotic stimuli. Conversely, knockdown of TRPP2 in renal epithelial cells increases ER Ca(2+) release and augments sensitivity to apoptosis. Our findings indicate an important function of ER-resident TRPP2 in the modulation of intracellular Ca(2+) signalling, and provide a molecular mechanism for the increased apoptosis rates in ADPKD upon loss of TRPP2 channel function.


Assuntos
Apoptose/fisiologia , Cálcio/fisiologia , Retículo Endoplasmático/metabolismo , Ativação do Canal Iônico/fisiologia , Canais de Cátion TRPP/fisiologia , Animais , Sinalização do Cálcio/fisiologia , Linhagem Celular , Citosol/fisiologia , Cães , Feminino , Humanos , Receptores de Inositol 1,4,5-Trifosfato/fisiologia , Mitocôndrias/fisiologia , Oócitos/fisiologia , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/fisiologia , Xenopus
9.
J Am Soc Nephrol ; 20(1): 48-56, 2009 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-18945944

RESUMO

Autosomal dominant polycystic kidney disease (ADPKD) is a common hereditary disease associated with progressive renal failure. Although cyst growth and compression of surrounding tissue may account for some loss of renal tissue, the other factors contributing to the progressive renal failure in patients with ADPKD are incompletely understood. Here, we report that secreted frizzled-related protein 4 (sFRP4) is upregulated in human ADPKD and in four different animal models of PKD, suggesting that sFRP4 expression is triggered by a common mechanism that underlies cyst formation. Cyst fluid from ADPKD kidneys activated the sFRP4 promoter and induced production of sFRP4 protein in renal tubular epithelial cell lines. Antagonism of the vasopressin 2 receptor blocked both promoter activity and tubular sFRP4 expression. In addition, sFRP4 selectively influenced members of the canonical Wnt signaling cascade and promoted cystogenesis of the zebrafish pronephros. sFRP4 was detected in the urine of both patients and animals with PKD, suggesting that sFRP4 may be a potential biomarker for monitoring the progression of ADPKD. Taken together, these observations suggest a potential role for SFRP4 in the pathogenesis of ADPKD.


Assuntos
Rim/metabolismo , Rim Policístico Autossômico Dominante/etiologia , Proteínas Proto-Oncogênicas/fisiologia , Animais , Células Cultivadas , Líquido Cístico/fisiologia , Modelos Animais de Doenças , Humanos , Camundongos , Morfolinas/farmacologia , Néfrons/embriologia , Doenças Renais Policísticas/metabolismo , Rim Policístico Autossômico Dominante/metabolismo , Proteínas Proto-Oncogênicas/análise , Transdução de Sinais , Compostos de Espiro/farmacologia , Canais de Cátion TRPP/fisiologia , Fatores de Transcrição/fisiologia , Proteínas Wnt/fisiologia , Xenopus , Peixe-Zebra
10.
J Cell Biol ; 182(3): 437-47, 2008 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-18695040

RESUMO

The primary cilium has evolved as a multifunctional cellular compartment that decorates most vertebrate cells. Cilia sense mechanical stimuli in various organs, but the molecular mechanisms that convert the deflection of cilia into intracellular calcium transients have remained elusive. Polycystin-2 (TRPP2), an ion channel mutated in polycystic kidney disease, is required for cilia-mediated calcium transients but lacks mechanosensitive properties. We find here that TRPP2 utilizes TRPV4 to form a mechano- and thermosensitive molecular sensor in the cilium. Depletion of TRPV4 in renal epithelial cells abolishes flow-induced calcium transients, demonstrating that TRPV4, like TRPP2, is an essential component of the ciliary mechanosensor. Because TRPV4-deficient zebrafish and mice lack renal cysts, our findings challenge the concept that defective ciliary flow sensing constitutes the fundamental mechanism of cystogenesis.


Assuntos
Canais de Cátion TRPP/metabolismo , Canais de Cátion TRPV/metabolismo , Animais , Sinalização do Cálcio , Linhagem Celular , Cílios/metabolismo , Cistos/metabolismo , Células Epiteliais/metabolismo , Humanos , Oócitos/metabolismo , Ligação Proteica , Transporte Proteico , Temperatura
11.
J Immunol ; 180(7): 4697-705, 2008 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-18354193

RESUMO

Dendritic cells (DC) play a key role in linking innate and adaptive immunity. In inflamed tissues, where DC become activated, oxygen tensions are usually low. Although hypoxia is increasingly recognized as an important determinant of cellular functions, the consequences of hypoxia and the role of one of the key players in hypoxic gene regulation, the transcription factor hypoxia inducible factor 1alpha (HIF-1alpha), are largely unknown. Thus, we investigated the effects of hypoxia and HIF-1alpha on murine DC activation and function in the presence or absence of an exogenous inflammatory stimulus. Hypoxia alone did not activate murine DC, but hypoxia combined with LPS led to marked increases in expression of costimulatory molecules, proinflammatory cytokine synthesis, and induction of allogeneic lymphocyte proliferation compared with LPS alone. This DC activation was accompanied by accumulation of HIF-1alpha protein levels, induction of glycolytic HIF target genes, and enhanced glycolytic activity. Using RNA interference techniques, knockdown of HIF-1alpha significantly reduced glucose use in DC, inhibited maturation, and led to an impaired capability to stimulate allogeneic T cells. Alltogether, our data indicate that HIF-1alpha and hypoxia play a crucial role for DC activation in inflammatory states, which is highly dependent on glycolysis even in the presence of oxygen.


Assuntos
Células Dendríticas/citologia , Células Dendríticas/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Lipopolissacarídeos/farmacologia , Animais , Diferenciação Celular , Hipóxia Celular/efeitos dos fármacos , Sobrevivência Celular , Células Cultivadas , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Glicólise , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Linfócitos/citologia , Linfócitos/efeitos dos fármacos , Linfócitos/imunologia , Camundongos , RNA Interferente Pequeno/genética , Regulação para Cima/efeitos dos fármacos
12.
J Am Soc Nephrol ; 19(3): 486-94, 2008 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-18256363

RESUMO

The contribution of hypoxia to cisplatin-induced renal tubular injury is controversial. Because the hypoxia-inducible factor (HIF) pathway is a master regulator of adaptation to hypoxia, we measured the effects of cisplatin on HIF accumulation in vitro and in vivo, and tested whether hypoxic preconditioning is protective against cisplatin-induced injury. We found that cisplatin did not stabilize HIF-1alpha protein in vitro or in vivo under normoxic conditions. However, hypoxic preconditioning of cisplatin-treated proximal tubular cells in culture reduced apoptosis in an HIF-1alpha-dependent fashion and increased cell proliferation as measured by BrdU incorporation. In vivo, rats preconditioned with carbon monoxide before cisplatin administration had significantly better renal function than rats kept in normoxic conditions throughout. Moreover, the histomorphological extent of renal damage and tubular apoptosis was reduced by the preconditional treatment. Therefore, development of pharmacologic agents to induce renal HIF might provide a new approach to ameliorate cisplatin-induced nephrotoxicity.


Assuntos
Antineoplásicos/efeitos adversos , Cisplatino/efeitos adversos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Hipóxia , Rim/efeitos dos fármacos , Lesão Renal Aguda/induzido quimicamente , Animais , Apoptose/efeitos dos fármacos , Monóxido de Carbono/farmacologia , Linhagem Celular , DNA/biossíntese , Expressão Gênica/efeitos dos fármacos , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Rim/metabolismo , Rim/patologia , Testes de Função Renal , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ratos , Ratos Sprague-Dawley
13.
EMBO J ; 24(4): 705-16, 2005 Feb 23.
Artigo em Inglês | MEDLINE | ID: mdl-15692563

RESUMO

The trafficking of ion channels to the plasma membrane is tightly controlled to ensure the proper regulation of intracellular ion homeostasis and signal transduction. Mutations of polycystin-2, a member of the TRP family of cation channels, cause autosomal dominant polycystic kidney disease, a disorder characterized by renal cysts and progressive renal failure. Polycystin-2 functions as a calcium-permeable nonselective cation channel; however, it is disputed whether polycystin-2 resides and acts at the plasma membrane or endoplasmic reticulum (ER). We show that the subcellular localization and function of polycystin-2 are directed by phosphofurin acidic cluster sorting protein (PACS)-1 and PACS-2, two adaptor proteins that recognize an acidic cluster in the carboxy-terminal domain of polycystin-2. Binding to these adaptor proteins is regulated by the phosphorylation of polycystin-2 by the protein kinase casein kinase 2, required for the routing of polycystin-2 between ER, Golgi and plasma membrane compartments. Our paradigm that polycystin-2 is sorted to and active at both ER and plasma membrane reconciles the previously incongruent views of its localization and function. Furthermore, PACS proteins may represent a novel molecular mechanism for ion channel trafficking, directing acidic cluster-containing ion channels to distinct subcellular compartments.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Transporte/metabolismo , Canais Iônicos/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Sequência de Aminoácidos , Proteínas de Transporte/genética , Linhagem Celular , Retículo Endoplasmático/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Canais Iônicos/química , Proteínas de Membrana/química , Proteínas de Membrana/genética , Dados de Sequência Molecular , Mutação/genética , Fosforilação , Ligação Proteica , Transporte Proteico , Alinhamento de Sequência , Canais de Cátion TRPP , Proteínas de Transporte Vesicular
14.
Biochem Biophys Res Commun ; 322(1): 177-85, 2004 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-15313189

RESUMO

Ca(2+) influx across the plasma membrane after stimulation of G protein-coupled receptors is important for many physiological functions. Here we studied the regulation of an inwardly rectifying whole cell current and its putative role in Ca(2+) entry in Xenopus oocytes. Expression of P2Y(1) or M1 receptors in Xenopus oocytes elicited a characteristic inwardly rectifying current without receptor stimulation. This current displayed distinct activation and inactivation kinetics and was highly Ca(2+)-dependent. After stimulation of endogenous G(q)-coupled receptors in water-injected cells similar currents were observed. We therefore speculated that the current could be activated via Ca(2+) store depletion induced by constitutive stimulation of the IP(3) cascade in cells overexpressing G(q)-coupled receptors. Receptor-independent Ca(2+) store depletion also induced the current. In conclusion, this current is activated after store depletion suggesting a role in Ca(2+) entry after stimulation of G(q)-coupled receptors. Finally, our data do not support the proposed ionotropic properties of the P2Y(1) receptor.


Assuntos
Canais de Cálcio/metabolismo , Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Potenciais da Membrana/fisiologia , Receptor Muscarínico M1/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores Purinérgicos P2/metabolismo , Animais , Células Cultivadas , Receptores de Inositol 1,4,5-Trifosfato , Receptores Acoplados a Proteínas-G/metabolismo , Receptores Purinérgicos P2Y1 , Proteínas Recombinantes/metabolismo , Xenopus laevis
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