Os autores gostariam de agradecer Glen Slocum (Medical College of Wisconsin) e Colleen A. Lavin (Nikon Instruments, Inc.) para a assistência técnica excelente, com experimentos de microscopia. Gregory Blass é reconhecida pela revisão crítica do manuscrito. Esta pesquisa foi apoiada pelos Institutos Nacionais de Saúde HL108880 concessão e American Diabetes Association conceder 1-15-BS-172 (para AS), eo Ben J. Lipps Research Fellowship da Sociedade Americana de Nefrologia (para DVI).

O uso de animais e bem-estar devem aderir ao Manual do NIH para o Cuidado e Uso de Animais de Laboratório seguindo protocolos revistos e aprovados pelo Comitê de Cuidado e Uso do animal Institucional (IACUC). 1. Lave rim <ol><li> Use 8 a 12 semanas de idade de ratos macho (sugerido é uma estirpe Sprague Dawley, no entanto outras estirpes de idade e sexo diferente pode ser utilizado com modificações apropriadas). </li><li> Anestesiar o animal de acordo com o procedimento permitido pelo protocolo IACUC; monitorar a profundidade da anestesia e inspecionar o animal. A descrição detalhada da cirurgia a ser realizada em 1,3-1,8 pode ser encontrado em Ilatovskaya et al 7. </li><li> Após anestesia adequada, colocar o animal em uma mesa cirúrgica, com temperatura controlada, fazer uma incisão na linha média do abdome (até 3 polegadas de comprimento), e descobrir a veia cava ea aorta. </li><li> Insira ligadura ao redor dos celíacos e artérias mesentérica superior e da abdominalaorta acima daqueles; não ligadura. </li><li> Blunt dissecar a aorta abdominal abaixo das artérias renais, e coloque duas ligaduras em torno dele, mas não ligar, em seguida, apertar a aorta acima das ligaduras e amarre o fio inferior. </li><li> Cateterize aorta com uma tubagem de polietileno PE50 (ligado a uma bomba seringa cheia com PBS) abaixo da braçadeira e fixar o cateter com a segunda ligadura; remover o grampo, ligar a bomba, e ligar a aorta e artérias mesentérica com doença celíaca. Rapidamente fazer incisão na veia renal para aliviar a pressão. </li><li> Infundir a aorta com o PBS pré-arrefecida durante 2 ou 3 minutos a um caudal de 6 ml / min. </li><li> Pare de perfusão, especiais de consumo e 7 decapsulate os rins, e colocá-los no gelo na solução PBS. Eutanásia do animal de acordo com o protocolo aprovado pelo IACUC. </li></ol> 2. Isolamento do Rato Os glomérulos <ol><li> Preparar 30 ml de solução fresca de 5% de BSA em meio RPMI 1640. </li><li> Usando uma lâmina de barbear e tesouras,isolar o córtex de ambos os rins, e em seguida, mediu até ficar homogénea. Este procedimento foi descrito anteriormente 7. </li><li> Empurrar o tecido moído durante o passo anterior através de um peneiro de aço inoxidável de malha 100 (pré-embebidos em solução de BSA a 5% / RPMI) utilizando uma espátula. Recolhe-se o fluxo de passagem e por força da gravidade permitir o fluxo de passagem para passar através de um peneiro de 140 mesh. </li><li> Filtra-se o fluxo através recolhidos a partir de 140 utilizando um peneiro de malha pré-embebido peneira de malha 200, descarta-se o filtrado e lava-se a parte superior do crivo com 10 - 15 ml da solução de BSA / RPMI preparado para recolher os glomérulos que sedimentam sobre o crivo. </li><li> Coloque a solução de BSA / RPMI contendo glomérulos sobre gelo em um tubo de 15 ml e deixar o sedimento glomérulos na parte inferior do tubo de até 20 min. O concentrado glomérulos no fundo do tubo irá ser claramente visto. Remover o excesso de solução, deixando cerca de 2 ml no tubo. </li></ol> 3. Single-channel patch-clamp Electrophysiology <ol><li> Prepare 5 x 5 milímetros chips de vidro capa de revestimento com 70.000 MW - 150.000 poli-ʟ-lisina, e deixe secar. Use cerca de 30 ml de solução estéril filtrado 0,01% em água por tampa de vidro. </li><li> Aquecer as soluções experimentais para a TA e encher a câmara de patch-clamp e pipeta. Para a monitorização canais trpC, usar uma solução de banho, em mM: 126 de NaCl, 1 CaCl2, 10 de HEPES, 2 de MgCl2, 10 de glucose, pH 7,4; pipeta: 126 de NaCl, CaCl2 1,5, HEPES 10, glucose 10; pH 7,4. <ol><li> Adicionar inibidores para a solução da pipeta para bloquear a actividade de canais endógenos, os quais não são relevantes para os estudos (recomendados são: 100 uM de ácido niflúmico ou DIDS (para bloquear Ca 2+ -activated canais Cl -), 10 mM de TEA (para inibir a grande condutância Ca 2 + - K + dependente do canal), 10 nM iberiotoxin (para bloquear os canais de K + -activated Ca2 +), 10 mM nicardipina (para bloquear N-tipo Ca2 + canais)) diretamente antes do experimento patch-clamp. </li></ol></li><li> Misture suavemente a solução contendo glomérulos, e em seguida, aplicar cerca de 50 l de la à tampa revestida chips de vidro poli-ʟ lisina. Deixe os glomérulos anexar por aproximadamente 5 min. </li><li> Mover os chips de vidro com glomérulos para a câmara de patch-clamp pré-cheia com a solução de banho; perfundir a câmara a um caudal de 3 ml / min durante 1 min para assegurar a remoção dos glomérulos não acopladas. </li><li> Realizar um experimento patch-clamp convencional em um modo ligado à célula 7. Com uma pipeta de vidro (7-10 mohms resistência pipeta) formar uma vedação de alta resistência entre uma pipeta e uma membrana podócitos por aplicação de sucção suave (uma pipeta ligada a um podócitos na superfície do glomérulo isolado é mostrado na Figura 2, em a esquerda). </li><li> Para as medições ligado de células, de baixo passam as correntes em 300 Hz por um filtro de oito pólos Bessel. </li><li> VOCÊse isolado glomérulos em experimentos de patch-clamp para até 4-6 horas. Mantenha a fração estoque glomérulos no gelo. </li></ol> 4. Raciometrico confocal de fluorescência As medições da concentração de cálcio intracelular no Podócitos <ol><li> Colocar 500 mL da fracção de glomérulos (descrito em 2.5) em 0,5 ml de tubo cónico e adicionar corantes cálcio Fura Red, AM e Fluo-4, AM. Utilizar 2 mM e 1 mM concentrações de banco de Fura Red, AM e Fluo-4, AM, respectivamente (armazenar a -20 ° C, dissolveu-se em DMSO) e 2,5 ul de usar cada corante por 500 ul de fracção de glomérulos. Imediatamente após a adição dos corantes cobrir o tubo com folha de alumínio. </li><li> Colocar os tubos num agitador rotativo durante pelo menos 20 minutos até 1 hora à TA. Nota: Os agentes farmacológicos podem ser adicionados durante este passo. </li><li> Prepare lamelas de vidro, cubra-os com poli-ʟ-lisina e permitir a secagem usando set placa aquecida a 70 ° C. </li><li> Uma vez que o carregamento de corantes de cálcio é complete, aplicar 100 ul de solução contendo os glomérulos para as lamelas revestidas com poli-ʟ-lisina e deixá-los aderir à superfície durante cerca de 5 min. Montar as lamelas anexado-glomérulos em uma câmara de imagem, e perfundir com a solução de banho (contendo (em mM): 145 NaCl, 4,5 de KCl, 2 de MgCl2, 10 HEPES, pH 7,35) com um caudal de 3 ml / min para remover os glomérulos acopladas e os corantes restantes. </li><li> Definir o microscópio confocal de varrimento a laser a um comprimento de onda de excitação de 488 nm e filtros de emissão (525/25 e 650/25 nm para Fluo-4 e Fura Red, respectivamente). Definir o software de imagem para uma freqüência e resolução desejada. </li><li> Encontre o glomérulos em claro e, em seguida, ligue a detecção do sinal de fluorescência. Ajustar a intensidade do laser para cada corante para evitar a saturação do sinal. Escolha o plano focal com as podocytes que estão diretamente ligados ao vidro. Isto minimiza o efeito causado pela contracção de um glomérulo em resposta à drogaaplicação. Verifique novamente se o glomérulo de escolha é bem ligado ao vidro; </li><li> Comece imaginando o plano focal de escolha (levar 512 x 512 imagens com a freqüência ajustada em 4 segundos para visualizar rápido Ca 2+ alterações transitórias. Use um / NA 1.4 ou lente objetiva semelhante 60X imagem de alta resolução para), aplicar medicamentos de interesse, e registar a resposta. <ol><li> Escolher um plano focal desejado (o mais próximo da superfície do vidro quanto possível, para assegurar a imagiologia de podócitos na superfície do glomérulo). Verifique a intensidade da fluorescência na Fluo4 e canais FuraRed, e certifique-se que o glomérulo é claramente visto em campo claro. </li><li> Iniciar a imagiologia. Antes da aplicação de todas as drogas, ficha, pelo menos, 1 min de linha de base de fluorescência para assegurar que o sinal é estável (não há picos repentinos ou desvanecimento do sinal). </li><li> Aplicar drogas desejadas com a ajuda de uma micropipeta; ter cuidado e garantir a droga foi capaz de difundir bem e alcançar o glomerulus. Misturar a solução de banho suavemente se necessário, monitorar o plano focal selecionado e verifique se ele não se moveu fora de foco por causa da aplicação de drogas. </li><li> Registar as alterações na intensidade de fluorescência para os sinais Fluo4 e FuraRed. Certifique-se a gravação é longo o suficiente, esperando até que o sinal atinge o nível de patamar ou atinge um pico e, em seguida, retorna à linha de base. Execute mudança solução ou adição de quaisquer outros medicamentos, se necessário. </li><li> Parar a gravação e salvar o arquivo no formato nativo do software. </li></ol></li></ol> 5. Análise de Imagem para as medições de cálcio <ol><li> Realizar análise de imagem com o software open ImageJ equipado com o plug-in utilitário ND que permite importar imagens no formato nativo ND2. </li><li> Importar a seqüência de imagens; certifique-se de dividir os canais e usar um modo hyperstack em tons de cinza. </li><li> Siga o → Ferramentas → caminho Analisar gerente ROI no software ImageJ para opena uma janela do gerenciador de ROI. Seleccione várias regiões de interesse (podocytes) usando uma ferramenta de seleção oval eo &quot;(t) Adicionar&quot; função no Gerenciador de ROI. Como o último ROI, selecione a área no fundo; salvar as ROIs selecionadas (Mais → Salvar). </li><li> Realce a janela que contém o canal selecionado para análises. Use a mais → função multi Medida no Gerenciador de ROI, marque a caixa &quot;uma linha por fatia&quot; no diálogo que aparece, e em seguida, clique em OK. Os resultados serão mostrados no formato que pode ser configurado na janela de resultados: entrar nas opções de menu Resultados → Definir Medidas, selecione &quot;valor de cinza média&quot; e calcular os valores de intensidade de pixel para cada ROI, que será exibido na janela de resultados em colunas separadas para cada ROI. </li><li> Copie os valores de intensidade de ROI medidos para cada canal (Fluo-4 e Fura Red) em software de análise de dados preferido; valores de intensidade fundo subtrair de cada datum ponto. </li><li> Para cada ponto de tempo calcular o rácio de intensidades do Fluo-4 aos canais Fura Red. Gráfico de dispersão / linha mudanças em tempo ponto de Ca 2+ transitória para cada ROI. Calcule os valores médios / SE para glomérulos selecionados. Nota: coluna Time deve ser definido de acordo com a frequência de imagem selecionada em 4.5. </li></ol> 6. Os cálculos de concentração intracelular de cálcio usando fluorescência de Fluo-4 Sinal <ol><li> Retirar uma amostra de glomérulos e executar o protocolo experimental acima para o passo 4.7. Depois de gravar a fluorescência de fundo adicionar Ionomicina (concentração final na câmara do banho deve ser de 10 uM) à solução de banho, e aumento da intensidade de fluorescência ficha. Uma vez que a intensidade atinge o seu máximo e começa a decadência, adicione MnCl2 (concentração final deve ser de 5 mm) para extinguir a fluorescência 8. </li><li> Analisar os dados obtidos em 6.1. Copie os Fluo-4 valores de intensidade de sinal ROI obtidos de acordocom o protocolo descrito em 5,1-5,5 por software de análise preferido. </li><li> Calcular a concentração intracelular de cálcio (em nM) na podócitos correspondente ao ROI através de uma fórmula: <img alt="Equação 1" src="/files/ftp_upload/52850/52850equation1.jpg" /> K onde d é uma dissociação pré-determinada constante para Fluo-4 (345 nM), F é a intensidade no ponto de tempo que está a calcular a concentração de cálcio para (linha de base), e F e F max min são os valores de intensidade na ponto de carga máxima de cálcio (após aplicação ionomicina) e após a extinção da fluorescência do (com MnCl 2), respectivamente (ver Figura 3). </li></ol>

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A abordagem aqui descrita permite a análise de manuseamento de cálcio pelos podócitos dos glomérulos de roedores. Esta técnica permite a aplicação de patch-clamp eletrofisiologia canal único e fluorescência confocal ratiometric imagem. No entanto, ambas as abordagens podem ser utilizadas separadamente, por conta própria. O protocolo proposto tem várias etapas relativamente simples, incluindo rubor 1) rim; 2) isolamento dos glomérulos por peneiração diferencial; 3) realização de patch-clamp experimentos e...

Rins manter o equilíbrio homeostático para várias substâncias e regular o volume de sangue de uma forma que determina a pressão arterial total. Distúrbios no filtração renal, reabsorção ou secreção de causar ou acompanham os estados patológicos, que variam de hiper ou hipotensão para a doença renal terminal que eventualmente exige transplante renal. A unidade de filtração renal (glomérulo) consiste em três camadas - o endotélio capilar, membrana basal e uma camada de célula única de células epiteliais - podócitos, que desempenham um papel importante na manutenção da integridade e função de um diafragma de fenda. Disfunção no filtro de permeabilidade selectiva glomerular provoca perda urinária de macromoléculas, tais como proteinúria. Vários agentes podem afectar a estrutura das podócitos e os seus processos de pé, que determinam a integridade da barreira de filtração glomérulos. Os podócitos estão envolvidos na manutenção do glomfunção de filtração eruli. Foi estabelecido que a manipulação imprópria de cálcio pelo podócitos leva a lesão celular e desempenha um papel importante na progressão de várias formas de nefropatias 2,3. Portanto, o desenvolvimento de um modelo que permite a medição direta da variação da concentração de cálcio intracelulares será fundamental para estudos de função podocyte. Isolado glomérulos foram anteriormente utilizados em um numerosos estudos, incluindo medição do coeficiente de reflexão albumina muda 4 e avaliação das correntes celulares integral na de células inteiras eletrofisiológicos medições de patch-clamp 5,6. No presente artigo descrevemos o protocolo que permite ao pesquisador para medir as mudanças de concentração de cálcio intracelular em resposta aos pedidos de agentes farmacológicos, estimar os níveis basais de cálcio dentro das células, e avaliar indivíduo atividade canais de cálcio. Medições da concentração de cálcio Ratometric e patch-clamp electrophysiology foram usadas para determinar as alterações na concentração intracelular de cálcio no seio da actividade podócitos e canal, respectivamente.

<table><tbody><tr><td>Fluo4 AM</td><td>Life Technologies</td><td>F14217</td><td>500 &amp;micro;l in DMSO</td></tr><tr><td>FuraRed AM</td><td>Life Technologies</td><td>F-3020</td><td></td></tr><tr><td>Poly-&amp;#671;-lysine</td><td>Sigma-Aldrich</td><td>P4707</td><td></td></tr><tr><td>Pluronic acid</td><td>Sigma-Aldrich</td><td>F-68</td><td>solution</td></tr><tr><td>Ionomycin</td><td>Sigma-Aldrich</td><td>I3909-1ML</td><td></td></tr><tr><td>Tube rotator</td><td>Miltenyi Biotec GmbH</td><td>130-090-753</td><td>Germany</td></tr><tr><td>Nikon confocal microscope (inverted)</td><td>Nikon</td><td>Nikon A1R</td><td>Laser exitation 488 nm. Emission filters 500-550 nm and 570-620 nm</td></tr><tr><td>Objective</td><td>Nikon</td><td>Plan Apo 60x/NA 1.4 Oil</td><td></td></tr><tr><td>Cover Glass</td><td>Thermo Scientific</td><td>6661B52</td><td></td></tr><tr><td>High vacuum grease</td><td>Dow Corning</td><td></td><td>Silicone Compound</td></tr><tr><td>Software</td><td>Nikon</td><td>Nikon NIS-Elements</td><td></td></tr><tr><td>Recording/perfusion chamber</td><td>Warner Instruments</td><td>RC-26</td><td></td></tr><tr><td>Patch clamp amplifier</td><td>Molecular Devices</td><td>MultiClamp 700B</td><td></td></tr><tr><td>Data acquisition system</td><td>Molecular Devices</td><td>Digidata 1440A</td><td>Axon Digidata&amp;reg; System</td></tr><tr><td>Low pass filter</td><td>Warner Instruments</td><td>LPF-8</td><td>8 pole Bessel</td></tr><tr><td>Borosilicate glass capillaries</td><td>World Precision Instruments</td><td>1B150F-4</td><td></td></tr><tr><td>Micropipette puller</td><td>Sutter Instrument Co</td><td>P-97</td><td>Flaming/Brown type micropipette puller</td></tr><tr><td>Microforge</td><td>Narishige</td><td>MF-830</td><td>Japan</td></tr><tr><td>Motorized micromanipulator</td><td>Sutter Instrument Co</td><td>MP-225</td><td></td></tr><tr><td>Inverted microscope</td><td>Nikon</td><td>Eclipse Ti</td><td></td></tr><tr><td>Microvibration isolation table</td><td>TMC</td><td></td><td>equipped with Faraday cage</td></tr><tr><td>Multichannel valve perfusion system</td><td>AutoMake Scientific</td><td>Valve Bank II</td><td></td></tr><tr><td>Recording/perfusion chamber</td><td>Warner Instruments</td><td>RC-26</td><td></td></tr><tr><td>Software</td><td>Molecular Devices</td><td>pClamp 10.2</td><td></td></tr><tr><td>Nicardipine</td><td>Sigma-Aldrich</td><td>N7510</td><td></td></tr><tr><td>Iberiotoxin</td><td>Sigma</td><td>I5904-5UG</td><td></td></tr><tr><td>Niflumic acid</td><td>Sigma-Aldrich</td><td>N0630</td><td></td></tr><tr><td>DIDS</td><td>Sigma-Aldrich</td><td>D3514-25MG</td><td></td></tr><tr><td>TEA chloride</td><td>Tocris</td><td>T2265</td><td></td></tr><tr><td>RPMI 1640</td><td>Life Technologies</td><td>11835030</td><td>without antibiotics</td></tr><tr><td>BSA</td><td>Sigma-Aldrich</td><td>A8327</td><td>30% albumin solution</td></tr><tr><td>Temperature controlled surgical table</td><td>MCW core</td><td></td><td>for rodents</td></tr><tr><td>Steel sieves:</td><td></td><td></td><td>#100 (150 &amp;mu;m), 140 (106 &amp;mu;m)</td></tr><tr><td>Gilson, Inc SIEVE 3 SS FH NO200</td><td>Fisher Sci</td><td>50-871-316</td><td></td></tr><tr><td>Gilson, Inc SIEVE 3 SS FH NO270</td><td>Fisher Sci</td><td>50-871-318</td><td></td></tr><tr><td>Gilson, Inc SIEVE 3 SS FH NO400</td><td>Fisher Sci</td><td>50-871-320</td><td></td></tr><tr><td>mesh 200</td><td>Sigma-Aldrich</td><td>s4145</td><td>screen for CD-1</td></tr><tr><td>Binocular microscope</td><td>Nikon</td><td>Eclipse TS100</td><td></td></tr><tr><td>Binocular microscope</td><td>Nikon</td><td>SMZ745</td><td></td></tr><tr><td>Syringe pump-based perfusion system</td><td>Harvard Apparatus</td><td></td><td></td></tr><tr><td>Polyethylene tubing</td><td>Sigma-Aldrich</td><td>PE50</td><td></td></tr><tr><td>Isofluorane anesthesia</td><td><a href="http://www.vetequip.com/" target="_blank"><img src="/img/new_window.png" /> VetEquip, Inc.</a></td><td>911103</td><td></td></tr><tr><td>Other basic reagents</td><td>Sigma-Aldrich</td><td></td><td></td></tr></tbody></table>

single-channel analysis and calcium imaging in the podocytes of the freshly isolated glomeruli

Podocytes (renal glomerular epithelial cells) are known to regulate glomerular permeability and maintain glomerular structure; a key role for these cells in the pathogenesis of various renal diseases has been established since podocyte injury leads to proteinuria and foot process effacement. It was previously reported that various endogenous agents may cause a dramatic overload in intracellular Ca2+ concentration in podocytes, presumably leading to albuminuria, and this likely occurs via calcium-conducting ion channels. Therefore, it appeared important to study calcium handling in the podocytes both under normal conditions and in various pathological states. However, available experimental approaches have remained somewhat limited to cultured and transfected cells. Although they represent a good basic model for such studies, they are essentially extracted from the native environment of the glomerulus. Here we describe the methodology of studying podocytes as a part of the freshly isolated whole glomerulus. This preparation retains the functional potential of the podocytes, which are still attached to the capillaries; therefore, podocytes remain in the environment that conserves the major parts of the glomeruli filtration apparatus. The present manuscript elaborates on two experimental approaches that allow 1) real-time detection of calcium concentration changes with the help of ratiometric confocal fluorescence microscopy, and 2) the recording of the single ion channels activity in the podocytes of the freshly isolated glomeruli. These methodologies utilize the advantages of the native environment of the glomerulus that enable researchers to resolve acute changes in the intracellular calcium handling in response to applications of various agents, measure basal concentration of calcium within the cells (for instance, to evaluate disease progression), and assess and manipulate calcium conductance at the level of single ion channels.

Aqui abordamos o problema de medir alterações agudas nos níveis de cálcio nos podocytes. A Figura 1 mostra uma representação esquemática do protocolo experimental projetado para realizar alta resolução de imagem confocal de fluorescência ao vivo e gravações de atividade de canais iônicos individuais nos podocytes do recém- glomérulos isolados de roedores. Resumidamente, após o rato é anestesiados, os rins devem ser lavados com PBS para limpá-las de sangue. Em seguida, os rins são exci...

Watch this Scientific Journal Video about Single-channel Analysis and Calcium Imaging in the Podocytes of the Freshly Isolated Glomeruli at JoVE.com

Single-channel Analysis and Calcium Imaging in the Podocytes of the Freshly Isolated Glomeruli

Changes in the intracellular calcium levels in the podocytes are one of the most important means to control the filtration function of glomeruli. Here we explain a high-throughput approach that allows detection of real-time calcium handling and single ion channels activity in the podocytes of the freshly isolated glomeruli.

Single-channel Análise e cálcio Criação de imagem do Podócitos do recentemente isolados glomérulos

Podocytes (renal glomerular epithelial cells) are known to regulate glomerular permeability and maintain glomerular structure; a key role for these cells ...

single-channel-analysis-calcium-imaging-podocytes-freshly-isolated

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

10.9K visualizações.  Medical College of Wisconsin. Changes in the intracellular calcium levels in the podocytes are one of the most important means to control the filtration function of glomeruli. Here we explain a high-throughput approach that allows detection of real-time calcium handling and single ion channels activity in the podocytes of the freshly isolated glomeruli. 

Vídeo: Single-channel Analysis and Calcium Imaging in the Podocytes of the Freshly Isolated Glomeruli

Immunohistochemical and Calcium Imaging Methods in Wholemount Rat Retina

In this paper we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of wholemount retinas for immunohistochemistry and, 2) calcium imaging for the study of voltage gated calcium channel (VGCC) mediated calcium signaling in retinal ganglion cells. The calcium imaging method we describe circumvents issues concerning non-specific loading of displaced amacrine cells in the ganglion cell layer.

Immunohistochemistry protocols are used to study the localization of a specific protein in the retina. Calcium imaging techniques are employed to study calcium dynamics in retinal ganglion cells and their axons.

Os métodos de imagem de imuno-histoquímica e cálcio em Wholemount Rat Retina

In this paper we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of wholemount retinas for ...

Neurociência

An Efficient Sieving Method to Isolate Intact Glomeruli from Adult Rat Kidney

Preservation of glomerular structure and function is pivotal in the prevention of glomerulonephritis, a category of kidney disease characterized by proteinuria which can eventually lead to chronic and end-stage renal disease. The glomerulus is a complex apparatus responsible for the filtration of plasma from the body. In disease, structural integrity is lost and allows for the abnormal leakage of plasma contents into the urine. A method to isolate and examine glomeruli in culture is critical for the study of these diseases. In this protocol, an efficient method of retrieving intact glomeruli from adult rat kidneys while conserving structural and morphological characteristics is described. This process is capable of generating high yields of glomeruli per kidney with minimal contamination from other nephron segments. With these glomeruli, injury conditions can be mimicked by incubating them with a variety of chemical toxins, including protamine sulfate, which causes foot process effacement and proteinuria in animal models. Degree of injury can be assessed using transmission electron microscopy, immunofluorescence staining, and western blotting. Nephrin and Wilms Tumor 1 (WT1) levels can also be assessed from these cultures. Due to the ease and flexibility of this protocol, the isolated glomeruli can be utilized as described or in a way that best suits the needs of the researcher to help better study glomerular health and structure in diseased states.

The main focus of this protocol is to efficiently isolate viable primary glomeruli cultures with minimal contaminants for use in a variety of downstream applications. The isolated glomeruli retain structural relationships between component cell types and can be cultured ex vivo for a short time.

Um método eficiente de crivagem para isolar intactos glomérulos do rim de rato adulto

Preservation of glomerular structure and function is pivotal in the prevention of glomerulonephritis, a category of kidney disease characterized by ...

Medicina

One-channel Cell-attached Patch-clamp Recording

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate depends on properties intrinsic to each channel protein as well as the mechanism by which it is generated and controlled and represents an important area of current research. Information about the operational dynamics of ion channel proteins can be obtained by observing long stretches of current produced by a single molecule. Described here is a protocol for obtaining one-channel cell-attached patch-clamp current recordings for a ligand gated ion channel, the NMDA receptor, expressed heterologously in HEK293 cells or natively in cortical neurons. Also provided are instructions on how to adapt the method to other ion channels of interest by presenting the example of the mechano-sensitive channel PIEZO1. This method can provide data regarding the channel&#8217;s conductance properties and the temporal sequence of open-closed conformations that make up the channel&#8217;s activation mechanism, thus helping to understand their functions in health and disease.

Described here is a procedure for obtaining long stretches of current recording from one ion channel with the cell-attached patch-clamp technique. This method allows for observing, in real time, the pattern of open-close channel conformations that underlie the biological signal. These data inform about channel properties in undisturbed biological membranes.

Um canal de gravação de patch-clamp anexado-Cell

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate ...

Biologia

Cytosolic Calcium Measurements in Renal Epithelial Cells by Flow Cytometry

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial function, cell death, cell metabolism and cell migration to name but a few. Cytosolic calcium is normally maintained at low nanomolar concentrations; rather it is found in high micromolar to millimolar concentrations in the endoplasmic reticulum, mitochondrial matrix and the extracellular compartment. Upon stimulation, a transient increase in cytosolic calcium serves to signal downstream events. Detecting changes in cytosolic calcium is normally performed using a live cell imaging set up with calcium binding dyes that exhibit either an increase in fluorescence intensity or a shift in the emission wavelength upon calcium binding. However, a live cell imaging set up is not freely accessible to all researchers. Alternative detection methods have been optimized for immunological cells with flow cytometry and for non-immunological adherent cells with a fluorescence microplate reader. Here, we describe an optimized, simple method for detecting changes in epithelial cells with flow cytometry using a single wavelength calcium binding dye. Adherent renal proximal tubule epithelial cells, which are normally difficult to load with dyes, were loaded with a fluorescent cell permeable calcium binding dye in the presence of probenecid, brought into suspension and calcium signals were monitored before and after addition of thapsigargin, tunicamycin and ionomycin.

Calcium is involved in numerous physiological and pathophysiological signaling pathways. Live cell imaging requires specialized equipment and can be time consuming. A quick, simple method using a flow cytometer to determine relative changes in cytosolic calcium in adherent epithelial cells brought into suspension was optimized.

Medidas cálcio citosólico em células epiteliais renais por citometria de fluxo

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial ...

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, electrical excitability and cell proliferation. The diversity and specificity of Ca2+ signaling derives from mechanisms by which Ca2+ signals are generated to act over different time and spatial scales, ranging from cell-wide oscillations and waves occurring over the periods of minutes to local transient Ca2+ microdomains (Ca2+ puffs) lasting milliseconds. Recent advances in electron multiplied CCD (EMCCD) cameras now allow for imaging of local Ca2+ signals with a 128 x 128 pixel spatial resolution at rates of &#62;500 frames sec-1 (fps). This approach is highly parallel and enables the simultaneous monitoring of hundreds of channels or puff sites in a single experiment. However, the vast amounts of data generated (ca. 1 Gb per min) render visual identification and analysis of local Ca2+ events impracticable. Here we describe and demonstrate the procedures for the acquisition, detection, and analysis of local IP3-mediated Ca2+ signals in intact mammalian cells loaded with Ca2+ indicators using both wide-field epi-fluorescence (WF) and total internal reflection fluorescence (TIRF) microscopy. Furthermore, we describe an algorithm developed within the open-source software environment Python that automates the identification and analysis of these local Ca2+ signals. The algorithm localizes sites of Ca2+ release with sub-pixel resolution; allows user review of data; and outputs time sequences of fluorescence ratio signals together with amplitude and kinetic data in an Excel-compatible table.

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Here we present techniques for imaging local IP3-mediated Ca2+ events using fluorescence microscopy in intact mammalian cells loaded with Ca2+ indicators together with an algorithm that automates identification and analysis of these events.

Imagem local Ca<sup&gt; 2+</sup&gt; Sinais em células de mamíferos em cultura

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, ...

Implementing Patch Clamp and Live Fluorescence Microscopy to Monitor Functional Properties of Freshly Isolated PKD Epithelium

Cyst initiation and expansion during polycystic kidney disease is a complex process characterized by abnormalities in tubular cell proliferation, luminal fluid accumulation and extracellular matrix formation. Activity of ion channels and intracellular calcium signaling are key physiologic parameters which determine functions of tubular epithelium. We developed a method suitable for real-time observation of ion channels activity with patch-clamp technique and registration of intracellular Ca2+ level in epithelial monolayers freshly isolated from renal cysts. PCK rats, a genetic model of autosomal recessive polycystic kidney disease (ARPKD), were used here for ex vivo analysis of ion channels and calcium flux. Described here is a detailed step-by-step procedure designed to isolate cystic monolayers and non-dilated tubules from PCK or normal Sprague Dawley (SD) rats, and monitor single channel activity and intracellular Ca2+ dynamics. This method does not require enzymatic processing and allows analysis in a native setting of freshly isolated epithelial monolayer. Moreover, this technique is very sensitive to intracellular calcium changes and generates high resolution images for precise measurements. Finally, isolated cystic epithelium can be further used for staining with antibodies or dyes, preparation of primary cultures and purification for various biochemical assays.

Ion channels expressed in renal tubular epithelium play a significant role in the pathology of polycystic kidney disease. Here we describe experimental protocols used to perform patch-clamp analysis and intracellular calcium level measurements in cystic epithelium freshly isolated from rodent kidneys.

Implementando Grampo patch e ao vivo microscopia de fluorescência para monitorar propriedades funcionais de recentemente isolados PKD Epithelium

Cyst initiation and expansion during polycystic kidney disease is a complex process characterized by abnormalities in tubular cell proliferation, luminal ...

In Vivo Calcium Imaging of Neuronal Ensembles in Networks of Primary Sensory Neurons in Intact Dorsal Root Ganglia

Ca2+ imaging can be used as a proxy for cellular activity, including action potentials and various signaling mechanisms involving Ca2+ entry into the cytoplasm or the release of intracellular Ca2+ stores. Pirt-GCaMP3-based Ca2+ imaging of primary sensory neurons of the dorsal root ganglion (DRG) in mice offers the advantage of simultaneous measurement of a large number of cells. Up to 1,800 neurons can be monitored, allowing neuronal networks and somatosensory processes to be studied as an ensemble in their normal physiological context at a populational level in vivo. The large number of neurons monitored allows the detection of activity patterns that would be challenging to detect using other methods. Stimuli can be applied to the mouse hindpaw, allowing the direct effects of stimuli on the DRG neuron ensemble to be studied. The number of neurons producing Ca2+ transients as well as the amplitude of Ca2+ transients indicates sensitivity to specific sensory modalities. The diameter of neurons provides evidence of activated fiber types (non-noxious mechano vs. noxious pain fibers, A&#946;, A&#948;, and C fibers). Neurons expressing specific receptors can be genetically labeled with td-Tomato and specific Cre recombinases together with Pirt-GCaMP. Therefore, Pirt-GCaMP3 Ca2+ imaging of DRG provides a powerful tool and model for the analysis of specific sensory modalities and neuron subtypes acting as an ensemble at the populational level to study pain, itch, touch, and other somatosensory signals.

In Vivo Calcium Imaging of Neuronal Ensembles in Networks of Primary Sensory Neurons in Intact Dorsal Root Ganglia

This protocol describes the surgical exposure of the dorsal root ganglion (DRG) followed by GCaMP3 (genetically-encoded Ca2+ indicator; Green Fluorescent Protein-Calmodulin-M13 Protein 3) Ca2+ imaging of the neuronal ensembles using Pirt-GCaMP3 mice while applying a variety of stimuli to the ipsilateral hind paw.

In vivo Imagem com cálcio de conjuntos neuronais em redes de neurônios sensoriais primários em gânglios intactos da raiz dorsal

Ca2+ imaging can be used as a proxy for cellular activity, including action potentials and various signaling mechanisms involving Ca2+ entry into the ...

Imaging Calcium Dynamics in Subpopulations of Mouse Pancreatic Islet Cells

Pancreatic islet hormones regulate blood glucose homeostasis. Changes in blood glucose induce oscillations of cytosolic calcium in pancreatic islet cells that trigger secretion of three main hormones: insulin (from &#946;-cells), glucagon (&#945;-cells) and somatostatin (&#948;-cells). &#946;-Cells, which make up the majority of islet cells and are electrically coupled to each other, respond to the glucose stimulus as one single entity. The excitability of the minor subpopulations, &#945;-cells and &#948;-cells (making up around 20% (30%) and 4% (10%) of the total rodent1 (human2) islet cell numbers, respectively) is less predictable and is therefore of special interest.
Calcium sensors are delivered into the peripheral layer of cells within the isolated islet. The islet or a group of islets is then immobilized and imaged using a fluorescence microscope. The choice of the imaging mode is between higher throughput (wide-field) and better spatial resolution (confocal). Conventionally, laser scanning confocal microscopy is used for imaging tissue, as it provides the best separation of the signal between the neighboring cells. A wide-field system can be utilized too, if the contaminating signal from the dominating population of &#946;-cells is minimized.
Once calcium dynamics in response to specific stimuli have been recorded, data are expressed in numerical form as fluorescence intensity vs. time, normalized to the initial fluorescence and baseline-corrected, to remove the effects linked to bleaching of the fluorophore. Changes in the spike frequency or partial area under the curve (pAUC) are computed vs. time, to quantify the observed effects. pAUC is more sensitive and quite robust whereas spiking frequency provides more information on the mechanism of calcium increase.
Minor cell subpopulations can be identified using functional responses to marker compounds, such as adrenaline and ghrelin, that induce changes in cytosolic calcium in a specific populations of islet cells.

Here, we present a protocol for imaging and quantifying calcium dynamics in heterogeneous cell populations, such as pancreatic islet cells. Fluorescent reporters are delivered into the peripheral layer of cells within the islet, which is then immobilized and imaged, and per-cell analysis of the dynamics of fluorescence intensity is performed.

Dinâmica de cálcio de imagem em subpopulações de células de ilhotas pancreáticas do rato

Pancreatic islet hormones regulate blood glucose homeostasis. Changes in blood glucose induce oscillations of cytosolic calcium in pancreatic islet cells ...

<meta charset="utf8"></head><body>Imagem de cálcio de célula única para estudos de ativação de canal

Single-Cell Calcium Imaging for Studying the Activation of Calcium Ion Channels

Single cell Ca2+ imaging is essential for the study of Ca2+ channels activated by various stimulations like temperature, voltage, native compound and chemicals et al. It primarily relies on microscopy imaging technology and the related Ca2+ indicator Fura-2/AM (AM is the abbreviation for Acetoxymethyl ester). Inside the cells, Fura-2/AM is hydrolyzed by esterases into Fura-2, which can reversibly bind with free cytoplasmic Ca2+. The maximum excitation wavelength shifts from 380nm to 340nm (when saturated with Ca2+) upon binding. The emitted fluorescence intensity is quantitatively related to the concentration of bound Ca2+. By measuring the 340/380 ratio, the Ca2+ concentration in the cytoplasm can be determined, eliminating errors caused by variations in the loading efficiency of the fluorescent probe among different samples. This technology allows for real-time, quantitative, and simultaneous monitoring of Ca2+ changes in multiple cells. The results are stored in &#8220;.XLSX&#8221; format for subsequent analysis, which is fast and generates intuitive change curves, greatly improving the detection efficiency. From different experimental perspectives, this article lists the use of this technology to detect Ca2+ signals in cells with endogenous or overexpressed channel proteins. Meantime, different methods for activating cells were also showed and compared. The aim is to provide readers with a clearer understanding of the usage and applications of single cell Ca2+ imaging.

<meta charset="utf8"></head><body>Imagem de cálcio de célula única para estudar a ativação de canais de íons de cálcio

This article presents a method for real-time, quantitative monitoring of calcium ion (Ca2+) concentrations in cells using single-cell Ca2+ imaging with the Fura-2/AM dye. This technique enables efficient dye loading and accurate calculation of Ca2+ levels through fluorescence intensity ratios, making it a simple and rapid approach for research applications.

Imagem de cálcio de célula única para estudar a ativação de canais de íons de cálcio

Single cell Ca2+ imaging is essential for the study of Ca2+ channels activated by various stimulations like temperature, voltage, native compound and ...

Single-channel Análise e cálcio Criação de imagem do Podócitos do recentemente isolados glomérulos

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