Los autores desean agradecer a Glen Slocum (Colegio Médico de Wisconsin) y Colleen A. Lavin (Nikon Instruments, Inc.) para una excelente asistencia técnica con los experimentos de microscopía. Gregory Blass es reconocido por corrección crítica del manuscrito. Esta investigación fue financiada por los Institutos Nacionales de Salud de subvención HL108880 y la Asociación Americana de Diabetes conceder 1-15-BS-172 (AS), y el Ben J. Lipps de Becas de Investigación de la Sociedad Americana de Nefrología (DVI).

El uso de animales y el bienestar deben adherirse a la Guía del NIH para el Cuidado y Uso de Animales de Laboratorio siguiendo los protocolos revisados ​​y aprobados por el Cuidado y Uso de Animales Comité Institucional (IACUC). Flush 1. Riñón <ol><li> Emplear de 8 a 12 semanas de edad rata macho (sugerida es una cepa Sprague Dawley, sin embargo otras cepas de diferente edad y el género se pueden utilizar con los cambios apropiados). </li><li> Anestesiar al animal de acuerdo con el procedimiento permitido por protocolo de IACUC; controlar la profundidad de la anestesia e inspeccionar el animal. La descripción detallada de la cirugía que se realiza en 01.03 a 01.08 se pueden encontrar en Ilatovskaya et al 7. </li><li> Después de la anestesia adecuada, coloque el animal en una mesa quirúrgica con control de temperatura, hacer una incisión en la línea media del abdomen (hasta 3 pulgadas de largo), y descubrir la vena cava y la aorta. </li><li> Inserte ligadura alrededor de los celíacos y mesentéricos superiores arterias y el abdomenaorta por encima de aquellos; no ligar. </li><li> Blunt disección de la aorta abdominal por debajo de las arterias renales, y coloque dos ligaduras alrededor de ella, pero no ligar, a continuación, sujetar la aorta por encima de las ligaduras y atar el hilo inferior. </li><li> Cateterizar la aorta con un tubo de PE50 de polietileno (que se adjunta a una bomba de jeringa llena con PBS) por debajo de la abrazadera y fijar el catéter con la segunda ligadura; quitar la pinza, encienda la bomba, y ligar la aorta y arterias mesentérica con celíacos. Hacer rápidamente incisión en la vena renal para aliviar la presión. </li><li> Infundir la aorta con el PBS pre-enfriado para 2 o 3 min a una velocidad de 6 ml / min. </li><li> Deje de perfusión, los impuestos especiales y decapsulate 7 los riñones, y los puso en el hielo en la solución de PBS. La eutanasia del animal según el protocolo aprobado por el IACUC. </li></ol> 2. Aislamiento de la rata Los glomérulos <ol><li> Preparar 30 ml de solución fresca de 5% de BSA en medio RPMI 1640. </li><li> Utilizando una hoja de afeitar y tijeras,aislar la corteza de ambos riñones, y luego picar hasta homogeneidad. Este procedimiento ha sido descrito anteriormente 7. </li><li> Empujar el tejido picada durante el paso anterior a través de un tamiz de acero inoxidable de malla 100 (pre-empapado en 5% de solución de BSA / RPMI) usando una espátula. Recoger el flujo a través y por la fuerza de gravedad permiten que el flujo a través pase a través de un tamiz de 140 mesh. </li><li> Filtrar el flujo a través recogido de la malla 140 usando un tamiz de 200 tamiz de malla pre-empapado, desechar el filtrado, y lavar la parte superior del tamiz con 10 - 15 ml de la solución de BSA / RPMI preparado para recoger los glomérulos que el sedimento en el tamiz. </li><li> Ponga la solución de BSA / RPMI que contiene glomérulos en el hielo en un tubo de 15 ml y dejar que el sedimento glomérulos en la parte inferior del tubo para un máximo de 20 min. El concentrado glomérulos en la parte inferior del tubo se puede ver claramente. Eliminar el exceso de solución, dejando aproximadamente 2 ml en el tubo. </li></ol> 3. monocanal Patch-clamp Electrophysiology <ol><li> Preparar 5 x 5 mm chips de vidrio cubierta por revestimiento con 70.000 MW - 150000 poli-ʟ-lisina, y dejar secar. Utilice aproximadamente 30 l de solución filtrada estéril 0,01% en agua por cubierta de vidrio. </li><li> Calentar las soluciones experimentales a temperatura ambiente y llenar la cámara de patch-clamp y la pipeta. Para el monitoreo canales TRPC, utilice una solución de baño, en mM: 126 NaCl, CaCl2 1, 10 HEPES, 2 MgCl2, 10 glucosa, pH 7,4; pipeta: 126 NaCl, 1,5 CaCl 2, 10 HEPES, 10 glucosa; pH 7,4. <ol><li> Añadir inhibidores a la solución de la pipeta para bloquear la actividad de los canales endógenos, que no son relevantes para los estudios (recomendados son: 100 mM de ácido niflúmico, o DIDS (para bloquear Ca 2 + activados por Cl - canales), TEA 10 mM (para inhibir la de gran conductancia Ca 2 + - K + dependientes de canal), 10 nM iberiotoxina (para bloquear los Ca2 + activados por canales de K +), 10 mM nicardipina (para bloquear de tipo N Ca2 + canales)) directamente antes del experimento de patch-clamp. </li></ol></li><li> Mezclar suavemente la solución que contiene los glomérulos, y luego aplicar aproximadamente 50 l de a la cubierta revestida virutas de cristal poli-ʟ-lisina. Deje que los glomérulos conceden durante aproximadamente 5 minutos. </li><li> Mover los chips de vidrio con glomérulos a la cámara de patch-clamp pre-llenado con la solución del baño; perfundir la cámara a una velocidad de 3 ml / min durante 1 min para asegurar la eliminación de los glomérulos no fijadas. </li><li> Llevar a cabo un experimento de patch-clamp convencional en un modo celular conectado a 7. Con una pipeta de vidrio (7 - 10 de mO resistencia pipeta) formar un sello de alta resistencia entre una pipeta y una membrana de podocitos mediante la aplicación de una succión suave (una pipeta unida a una podocitos en la superficie del glomérulo aislado se muestra en la Figura 2, en la izquierda). </li><li> Para las mediciones de células inscritos, de paso bajo las corrientes de 300 Hz por un filtro de ocho polos de Bessel. </li><li> Use aisló glomérulos en experimentos de patch-clamp para un máximo de 4-6 horas. Mantenga la fracción de stock glomérulos en hielo. </li></ol> 4. Ratiometric confocal de fluorescencia mediciones de la concentración de calcio intracelular en los podocitos <ol><li> Coloque 500 l de la fracción de glomérulos (descrito en 2.5) en 0,5 ml tubo cónico y añadir colorantes calcio Fura Red, AM y Fluo-4, de AM. Utilice 2 mM y 1 mM concentraciones stock de Fura Red, AM y Fluo-4 AM, respectivamente (almacenar a -20 ° C, se disolvió en DMSO) y el uso de 2,5 l de cada colorante por unos 500 l de fracción glomérulos. Inmediatamente después de la adición de los colorantes cubrir el tubo con papel de aluminio. </li><li> Colocar los tubos en un agitador giratorio durante al menos 20 min hasta 1 h a RT. Nota: Los agentes farmacológicos pueden ser añadidos durante este paso. </li><li> Preparar cubreobjetos de vidrio, cubrirlos con poli-ʟ-lisina y permitir el secado utilizando conjunto placa calentada a 70 ° C. </li><li> Una vez que la carga de colorantes de calcio es complete, aplicar 100 l de los glomérulos que contienen solución a los cubreobjetos recubiertos de poli-ʟ-lisina y dejar que ellos se adhieren a la superficie durante aproximadamente 5 min. Montar los cubreobjetos glomérulos conectado a una cámara de formación de imágenes, y perfundir con la solución del baño (que contiene (en mM): 145 NaCl, 4,5 KCl, 2 MgCl 2, 10 HEPES, pH 7,35) a una velocidad de 3 ml / min para eliminar los glomérulos no unidas y los tintes restantes. </li><li> Ajuste el microscopio de barrido confocal láser a una longitud de onda de excitación de 488 nm y filtros de emisión (525/25 y 650/25 nm para Fluo-4 y Fura Red, respectivamente). Ajuste el software de imágenes a una frecuencia y resolución deseada. </li><li> Encuentra los glomérulos en campo claro y luego girar en la detección de la señal de fluorescencia. Ajuste de la intensidad del láser para cada tinte para evitar la saturación de la señal. Elija el plano focal con los podocitos que están directamente conectados a la copa. Esto minimiza el efecto causado por la contracción de un glomérulo en respuesta a drogassolicitud. Haga doble verificación que el glomérulo de elección es bien unido al cristal; </li><li> Comience imágenes del plano focal de elección (tomar 512 x 512 imágenes con la frecuencia establecida en 4 segundos para visualizar Ca 2+ rápida cambios transitorios. Use un NA 1.4 o / lente de objetivo similar 60X imagen de alta resolución para), aplicar fármacos de interés, y registrar la respuesta. <ol><li> Seleccionar un plano focal deseada (tan cerca de la superficie del vidrio como sea posible, para asegurar la obtención de imágenes de los podocitos en la superficie del glomérulo). Compruebe la intensidad de la fluorescencia en el Fluo4 y canales FuraRed, y asegúrese de que el glomérulo se ve claramente en campo claro. </li><li> Iniciar la formación de imágenes. Antes de la aplicación de cualquier droga, ficha al menos 1 min de la fluorescencia de referencia para asegurarse de que la señal es estable (no hay picos repentinos o desvanecimiento de la señal). </li><li> Aplicar medicamentos deseados con la ayuda de una micropipeta; tener cuidado y asegurarse de la droga era capaz de difundir bien y llegar a la glomerulus. Mezcle la solución del baño suavemente, si es necesario, controlar el plano focal seleccionada y comprobar que no se movió fuera de foco debido a la aplicación del fármaco. </li><li> Registre los cambios en la intensidad de fluorescencia de las señales Fluo4 y FuraRed. Asegúrese de que la grabación es lo suficientemente largo, por la espera hasta que la señal alcanza el nivel de meseta o alcanza un pico y luego vuelve a la línea de base. Realizar el cambio o adición de solución de otros medicamentos si es necesario. </li><li> Detener la grabación y guardar el archivo en el formato nativo del software. </li></ol></li></ol> 5. Análisis de imagen para las mediciones de calcio <ol><li> Realizar análisis de imágenes con el software ImageJ abierta equipada con el plugin Utilidad ND que permite la importación de imágenes en el formato nativo ND2. </li><li> Importe la secuencia de imágenes; asegúrese de dividir los canales y utilizar un modo de escala de grises HyperStack. </li><li> Siga el camino → Herramientas → Analizar gerente ROI en el software ImageJ para opluma una ventana del gestor ROI. Seleccione varias regiones de interés (podocitos) utilizando una herramienta de selección ovalada y el &quot;Añadir (t)&quot; función en el Administrador de retorno de la inversión. Como el último ROI, seleccione el área en el fondo; guardar la ROI seleccionados (Más → Guardar). </li><li> Resalte la ventana que contiene el canal seleccionado para los análisis. Utilice la función de Más → Medida múltiples en el Administrador de retorno de la inversión, marque la casilla de &quot;una fila por rebanada&quot; en el diálogo que aparece, y luego haga clic en Aceptar. Los resultados se muestran en el formato que se puede configurar en la ventana de resultados: entrar en la opción de menú Resultados → Set mediciones, seleccione &quot;valor de gris medio&quot; y calcular los valores de intensidad de píxel para cada ROI, que se muestra en el ventana Resultados en columnas separadas para cada ROI. </li><li> Copiar los valores de intensidad medidos de ROI para cada canal (Fluo-4 y Fura Red) en el software de análisis de datos preferido; valores de intensidad fondo restar de cada datun punto. </li><li> Para cada punto de tiempo de calcular la relación de intensidades de la Fluo-4 a los canales de Fura rojas. Gráfico de dispersión / línea cambios en tiempo punto de Ca 2+ transitorio para cada ROI. Calcular los valores medios / SE para glomérulos seleccionados. Nota: la columna Tiempo debe ajustarse de acuerdo a la frecuencia de imagen seleccionado en el 4,5. </li></ol> 6. intracelulares cálculos de concentración de calcio Fluo-4 Uso de la señal de fluorescencia <ol><li> Tomar una muestra de los glomérulos y realizar el protocolo experimental hasta el paso 4.7. Después de la grabación de la fluorescencia de fondo añadir ionomicina (concentración final en la cámara de baño debe ser de 10 M) a la solución del baño, y el aumento de la intensidad de fluorescencia registro. Una vez que la intensidad alcanza su máximo y la decadencia se inicia, agregue MnCl2 (concentración final debe ser de 5 mM) para apagar la fluorescencia 8. </li><li> Analizar los datos obtenidos en 6.1. Copie los Fluo-4 valores de intensidad de señal de retorno de la inversión obtenidos de acuerdocon el protocolo descrito en 5.1 a 5.5 por software de análisis preferido. </li><li> Calcular la concentración de calcio intracelular (en nM) en el podocito correspondiente a la ROI mediante una fórmula: <img alt="Ecuación 1" src="/files/ftp_upload/52850/52850equation1.jpg" /> donde K d es una disociación predeterminado constante de Fluo-4 (345 nm), F es la intensidad en el punto de tiempo que se está calculando la concentración de calcio para la (línea de base), y F min y F max son los valores de intensidad de la punto de carga máxima de calcio (después de la aplicación ionomicina) y después de la extinción de la fluorescencia (con MnCl 2), respectivamente (véase la Figura 3). </li></ol>

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Los autores no tienen nada que revelar.

El enfoque descrito aquí permite el análisis de manejo del calcio por los podocitos de los glomérulos de roedores. Esta técnica permite la aplicación de patch-clamp electrofisiología solo canal y de fluorescencia de imagen confocal radiométrica. Sin embargo, ambos enfoques pueden ser utilizados por separado, por su propia cuenta. El protocolo propuesto tiene varios pasos relativamente simples, incluyendo ras 1) del riñón; 2) el aislamiento de los glomérulos por tamizado diferencial; 3) la realización de patch...

Los riñones mantienen el equilibrio homeostático para diversas sustancias y regulan el volumen de sangre en una forma que determina la presión de la sangre total. Las perturbaciones en la filtración renal, reabsorción o plomo secreción o acompañar a los estados patológicos, que van desde la hiper o hipotensión que terminan enfermedad renal que eventualmente requiere un trasplante de riñón. La unidad de filtración renal (glomérulo) consiste en tres capas - el endotelio capilar, la membrana basal y una capa de una sola célula de las células epiteliales - podocitos, que desempeñan un papel importante en el mantenimiento de la integridad y la función 1-rendija de diafragma. Disfunción en el filtro glomerular de permeabilidad selectiva provoca la pérdida urinaria de macromoléculas, tales como proteinuria. Varios agentes pueden afectar a la estructura de los podocitos y sus procesos de pie, que determinan la integridad de la barrera de filtración glomérulos. Los podocitos están involucrados en el mantenimiento de la Glomfunción de filtración eruli. Se ha establecido que el manejo inadecuado de calcio por el podocitos conduce a la lesión de las células y juega un papel importante en la progresión de diversas formas de nefropatías 2,3. Por lo tanto, el desarrollo de un modelo que permite la medición directa de los cambios de concentración de calcio intracelular será fundamental para el estudio de la función de podocitos. Glomérulos aislados se utilizaron previamente en un numerosos estudios incluyendo la medición del coeficiente de reflexión albúmina cambia 4 y la evaluación de las corrientes celulares integrales en las mediciones electrofisiológicas de patch-clamp de células enteras 5,6. En el presente trabajo se describe el protocolo que permite al investigador para medir cambios en la concentración de calcio intracelular en respuesta a las solicitudes de los agentes farmacológicos, estimar los niveles basales de calcio dentro de las células, y evaluar la actividad de los canales de calcio individual. Mediciones de la concentración de calcio y Ratometric electrop patch-clamphysiology se utilizaron para determinar los cambios en la concentración de calcio intracelular dentro de la actividad de podocitos y el canal, respectivamente.

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			<td>Fluo4 AM</td>
			<td>Life Technologies</td>
			<td>F14217</td>
			<td>500&#181;l in DMSO</td>
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		<tr>
			<td>FuraRed AM</td>
			<td>Life Technologies</td>
			<td>F-3020</td>
			<td></td>
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			<td>Poly-L-lysine</td>
			<td>Sigma-Aldrich</td>
			<td>P4707</td>
			<td></td>
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		<tr>
			<td>Pluronic acid</td>
			<td>Sigma-Aldrich</td>
			<td>F-68&#160;</td>
			<td>solution</td>
		</tr>
		<tr>
			<td>Ionomycin</td>
			<td>Sigma-Aldrich</td>
			<td>I3909-1ML</td>
			<td></td>
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			<td>Tube rotator</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>130-090-753</td>
			<td>Germany</td>
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		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon confocal microscope (inverted)</td>
			<td>Nikon</td>
			<td>Nikon A1R</td>
			<td>&#160;Laser exitation 488nm. Emission filters 500-550nm and 570-620nm</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&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Recording/perfusion chamber</td>
			<td>Warner Instruments</td>
			<td>RC-26</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></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&#174; 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>
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			<td>Iberiotoxin</td>
			<td>Sigma</td>
			<td>I5904-5UG</td>
			<td></td>
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		<tr>
			<td>Niflumic acid</td>
			<td>Sigma-Aldrich</td>
			<td>N0630</td>
			<td></td>
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			<td>DIDS</td>
			<td>Sigma-Aldrich</td>
			<td>D3514-25MG</td>
			<td></td>
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			<td>TEA chloride</td>
			<td>Tocris</td>
			<td>T2265</td>
			<td></td>
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		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
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		<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&#160;</td>
			<td>MCW core</td>
			<td></td>
			<td>for rodents</td>
		</tr>
		<tr>
			<td>Steel sieves:</td>
			<td></td>
			<td></td>
			<td>#100 (150 &#956;m), 140 (106 &#956;m)</td>
		</tr>
		<tr>
			<td>Gilson, Inc&#160; SIEVE 3 SS FH NO200</td>
			<td>Fisher Sci</td>
			<td>50-871-316</td>
			<td></td>
		</tr>
		<tr>
			<td>Gilson, Inc&#160; SIEVE 3 SS FH NO270</td>
			<td>Fisher Sci</td>
			<td>50-871-318</td>
			<td></td>
		</tr>
		<tr>
			<td>Gilson, Inc&#160; SIEVE 3 SS FH NO400</td>
			<td>Fisher Sci</td>
			<td>50-871-320</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;mesh 200&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>s4145</td>
			<td>screen for CD-1</td>
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		<tr>
			<td>Binocular microscope</td>
			<td>Nikon</td>
			<td>Eclipse TS100</td>
			<td></td>
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		<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>
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			<td>polyethylene tubing</td>
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			<td>PE50</td>
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			<td>Isofluorane anesthesia</td>
			<td><a href="http://www.vetequip.com/">http://www.vetequip.com/</a></td>
			<td>911103</td>
			<td></td>
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			<td></td>
			<td></td>
			<td></td>
			<td></td>
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		<tr>
			<td>Other basic reagents</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td></td>
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</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.

Aquí abordamos el problema de la medición de los cambios agudos en los niveles de calcio en los podocitos. La figura 1 muestra una representación esquemática del protocolo experimental diseñado para realizar alta resolución de imagen confocal de fluorescencia en vivo y grabaciones de actividades individuales de los canales iónicos en los podocitos del recién glomérulos roedores aislados. En pocas palabras, después de que se anestesió la rata, los riñones deben lavarse con PBS para borrarlos ...

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.

Análisis y calcio monocanal Imaging en los podocitos de los glomérulos recién aisladas

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

Research

JoVE Journal

Biology

10.9K vistas.  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. 

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

Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT

Brown adipose tissue (BAT) differs from white adipose tissue (WAT) by its discrete location and a brown-red color due to rich vascularization and high density of mitochondria. BAT plays a major role in energy expenditure and non-shivering thermogenesis in newborn mammals as well as the adults 1. BAT-mediated thermogenesis is highly regulated by the sympathetic nervous system, predominantly via &beta; adrenergic receptor 2, 3. Recent studies have shown that BAT activities in human adults are negatively correlated with body mass index (BMI) and other diabetic parameters 4-6. BAT has thus been proposed as a potential target for anti-obesity/anti-diabetes therapy focusing on modulation of energy balance 6-8. While several cold challenge-based positron emission tomography (PET) methods are established for detecting human BAT 9-13, there is essentially no standardized protocol for imaging and quantification of BAT in small animal models such as mice. Here we describe a robust PET/CT imaging method for functional assessment of BAT in mice. Briefly, adult C57BL/6J mice were cold treated under fasting conditions for a duration of 4 hours before they received one dose of 18F-Fluorodeoxyglucose (FDG). The mice were remained in the cold for one additional hour post FDG injection, and then scanned with a small animal-dedicated micro-PET/CT system. The acquired PET images were co-registered with the CT images for anatomical references and analyzed for FDG uptake in the interscapular BAT area to present BAT activity. This standardized cold-treatment and imaging protocol has been validated through testing BAT activities during pharmacological interventions, for example, the suppressed BAT activation by the treatment of &beta;-adrenoceptor antagonist propranolol 14, 15, or the enhanced BAT activation by &beta;3 agonist BRL37344 16. The method described here can be applied to screen for drugs/compounds that modulate BAT activity, or to identify genes/pathways that are involved in BAT development and regulation in various preclinical and basic studies.

Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT

A method of functional imaging of mouse brown adipose tissue (BAT) is described in which cold-stimulated uptake of 18F-Fluorodeoxyglucose (FDG) in BAT is non-invasively assessed with a standardized micro-PET/CT protocol. This method is robust and sensitive to detect differences in BAT activities in mouse models.

Imagen funcional de grasa marrón en ratones con FDG micro-PET/CT

Brown adipose tissue (BAT) differs from white adipose  tissue (WAT) by its discrete location and a brown-red color due to rich  vascularization and high ...

Investigación

Biología

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes - macrophages and dendritic cells to 'non-professional' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Here we describe an effective method for studying dynamics of apoptotic cell clearance in vivo. This method employs live Drosophila embryos as a powerful model for monitoring phagocytosis of apoptotic cells using specific labeling of apoptotic cells and phagocytes.

Imágenes de Apoptotic Liquidación de células vivas durante<em&gt; Drosophila</em&gt; Embriogénesis

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

Assessment of Calcium Sparks in Intact Skeletal Muscle Fibers

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in the striated muscle fibers that appear as highly localized Ca2+ release events mediated by ryanodine receptor (RyR) Ca2+ release channels on the sarcoplasmic reticulum (SR) membrane. Proper assessment of muscle Ca2+ sparks could provide information on the intracellular Ca2+&#160;handling properties of healthy and diseased striated muscles. Although Ca2+ sparks events are commonly seen in resting cardiomyocytes, they are rarely observed in resting skeletal muscle fibers; thus there is a need for methods to generate and analyze sparks in skeletal muscle fibers.
Detailed here is an experimental protocol for measuring Ca2+ sparks in isolated flexor digitorm brevis (FDB) muscle fibers using fluorescent Ca2+ indictors and laser scanning confocal microscopy. In this approach, isolated FDB fibers are exposed to transient hypoosmotic stress followed by a return to isotonic physiological solution. Under these conditions, a robust Ca2+ sparks response is detected adjacent to the sarcolemmal membrane in young healthy FDB muscle fibers. Altered Ca2+ sparks response is detected in dystrophic or aged skeletal muscle fibers. This approach has recently demonstrated that membrane-delimited signaling involving cross-talk between inositol (1,4,5)-triphosphate receptor (IP3R) and RyR contributes to Ca2+ spark activation in skeletal muscle. In summary, our studies using osmotic stress induced Ca2+ sparks showed that this intracellular response reflects a muscle signaling mechanism in physiology and aging/disease states, including mouse models of muscle dystrophy (mdx mice) or amyotrophic lateral sclerosis (ALS model).

Described here is a method to directly measure calcium sparks, the elementary units of Ca2+ release from sarcoplasmic reticulum&#160;in intact skeletal muscle fibers. This method utilizes osmotic-stress-mediated triggering of Ca2+ release from ryanodine receptor in isolated muscle fibers. The dynamics and homeostatic capacity of intracellular Ca2+ signaling can be employed to assess muscle function in health and disease.

Evaluación de calcio en Sparks intactas las fibras musculares esqueléticas

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in ...

Quantifying Single Microvessel Permeability in Isolated Blood-perfused Rat Lung Preparation

The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with real time imaging, it becomes feasible to determine permeability changes in individual pulmonary microvessels. Herein we describe steps to isolate rat lungs and perfuse them with autologous blood. Then, we outline steps to infuse fluorophores or agents via a microcatheter into a small lung region. Using these procedures described, we determined permeability increases in rat lung microvessels in response to infusions of bacterial lipopolysaccharide. The data revealed that lipopolysaccharide increased fluid leak across both venular and capillary microvessel segments. Thus, this method makes it possible to compare permeability responses among vascular segments and thus, define any heterogeneity in the response. While commonly used methods to define lung permeability require postprocessing of lung tissue samples, the use of real time imaging obviates this requirement as evident from the present method. Thus, the isolated lung preparation combined with real time imaging offers several advantages over traditional methods to determine lung microvascular permeability, yet is a straightforward method to develop and implement.

The isolated blood-perfused lung preparation makes it feasible to visualize microvessel networks on the lung surface. Here we describe an approach to quantify permeability of single microvessels in isolated lungs using real time fluorescence imaging.

La cuantificación individual Microvessel Permeabilidad en aislado de rata Blood-perfundido Preparación de pulmón

The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with ...

Measuring the Osmotic Water Permeability Coefficient (Pf) of Spherical Cells: Isolated Plant Protoplasts as an Example

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is a simple and very efficient method for the determination of the osmotic water permeability coefficient (Pf) in plant protoplasts, applicable in principle also to other spherical cells such as frog oocytes. The first step of the assay is the isolation of protoplasts from the plant tissue of interest by enzymatic digestion into a chamber with an appropriate isotonic solution. The second step consists of an osmotic challenge assay: protoplasts immobilized on the bottom of the chamber are submitted to a constant perfusion starting with an isotonic solution and followed by a hypotonic solution. The cell swelling is video recorded. In the third step, the images are processed offline to yield volume changes, and the time course of the volume changes is correlated with the time course of the change in osmolarity of the chamber perfusion medium, using a curve fitting procedure written in Matlab (the &#8216;PfFit&#8217;), to yield Pf.

Measuring the Osmotic Water Permeability Coefficient Pf of Spherical Cells: Isolated Plant Protoplasts as an Example

Measuring the osmotic water permeability coefficient (Pf) of cells can help understand the regulatory mechanisms of aquaporins (AQPs). Pf determination in spherical plant cell protoplasts presented here involves protoplasts isolation and numerical analysis of their initial rate of volume change as a result of an osmotic challenge during constant bath perfusion.

Medición de la osmótica de agua Coeficiente de Permeabilidad (P<sub&gt; F</sub&gt;) De células esféricas: protoplastos de plantas aisladas como ejemplo

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is ...

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.

La implementación de Patch Clamp y vivo microscopía de fluorescencia para supervisar propiedades funcionales de recién aisladas PKD Epitelio

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

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

-Alta resolución de imagen de lapso de tiempo y análisis automatizado de los microtúbulos en la dinámica de estar umbilical humana Las células endoteliales de vena

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

The Mouse Isolated Perfused Kidney Technique

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. This is a prerequisite for studying the physiology of the isolated organ and for many innovative applications that may be possible in the future, including perfusion decellularization for kidney bioengineering or the administration of anti-rejection or genome-editing drugs in high doses to prime the kidney for transplantation. During the time of the perfusion, the kidney can be manipulated, renal function can be assessed, and various pharmaceuticals administered. After the procedure, the kidney can be transplanted or processed for molecular biology, biochemical analysis, or microscopy.
This paper describes the perfusate and the surgical technique needed for the ex vivo perfusion of mouse kidneys. Details of the perfusion apparatus are given and data are presented showing the viability of the kidney&#39;s preparation: renal blood flow, vascular resistance, and urine data as functional, transmission electron micrographs of different nephron segments as morphological readouts, and western blots of transport proteins of different nephron segments as molecular readout.

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. The buffers and surgical technique are described in detail.

La Técnica del riñón perfundido aislado del ratón

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. This is a ...

Analysis of Beta-cell Function Using Single-cell Resolution Calcium Imaging in Zebrafish Islets

Pancreatic beta-cells respond to increasing blood glucose concentrations by secreting the hormone insulin. The dysfunction of beta-cells leads to hyperglycemia and severe, life-threatening consequences. Understanding how the beta-cells operate under physiological conditions and what genetic and environmental factors might cause their dysfunction could lead to better treatment options for diabetic patients. The ability to measure calcium levels in beta-cells serves as an important indicator of beta-cell function, as the influx of calcium ions triggers insulin release. Here we describe a protocol for monitoring the glucose-stimulated calcium influx in zebrafish beta-cells by using GCaMP6s, a genetically encoded sensor of calcium. The method allows monitoring the intracellular calcium dynamics with single-cell resolution in ex vivo mounted islets. The glucose-responsiveness of beta-cells within the same islet can be captured simultaneously under different glucose concentrations, which suggests the presence of functional heterogeneity among zebrafish beta-cells. Furthermore, the technique provides high temporal and spatial resolution, which reveals the oscillatory nature of the calcium influx upon glucose stimulation. Our approach opens the doors to use the zebrafish as a model to investigate the contribution of genetic and environmental factors to beta-cell function and dysfunction.

Beta-cell functionality is important for blood-glucose homeostasis, which is evaluated at single-cell resolution using a genetically encoded reporter for calcium influx.

Análisis de la función de célula Beta usando proyección de imagen de resolución sola célula calcio en islotes de pez cebra

Pancreatic beta-cells respond to increasing blood glucose concentrations by secreting the hormone insulin. The dysfunction of beta-cells leads to ...

Simultaneous Measurements of Intracellular Calcium and Membrane Potential in Freshly Isolated and Intact Mouse Cerebral Endothelium

Cerebral arteries and their respective microcirculation deliver oxygen and nutrients to the brain via blood flow regulation. Endothelial cells line the lumen of blood vessels and command changes in vascular diameter as needed to meet the metabolic demand of neurons. Primary endothelial-dependent signaling pathways of hyperpolarization of membrane potential (Vm) and nitric oxide typically operate in parallel to mediate vasodilation and thereby increase blood flow. Although integral to coordinating vasodilation over several millimeters of vascular length, components of endothelium-derived hyperpolarization (EDH) have been historically difficult to measure. These components of EDH entail intracellular Ca2+ [Ca2+]i increases and subsequent activation of small- and intermediate conductance Ca2+-activated K+ (SKCa/IKCa) channels.
Here, we present a simplified illustration of the isolation of fresh endothelium from mouse cerebral arteries; simultaneous measurements of endothelial [Ca2+]i and Vm using Fura-2 photometry and intracellular sharp electrodes, respectively; and a continuous superfusion of salt solutions and pharmacological agents under physiological conditions (pH 7.4, 37 &#176;C). Posterior cerebral arteries from the Circle of Willis are removed free of the posterior communicating and the basilar arteries. Enzymatic digestion of cleaned posterior cerebral arterial segments and subsequent trituration facilitates removal of adventitia, perivascular nerves, and smooth muscle cells. Resulting posterior cerebral arterial endothelial &#34;tubes&#34; are then secured under a microscope and examined using a camera, photomultiplier tube, and one to two electrometers while under continuous superfusion. Collectively, this method can simultaneously measure changes in endothelial [Ca2+]i and Vm in discrete cellular locations, in addition to the spreading of EDH through gap junctions up to millimeter distances along the intact endothelium. This method is expected to yield a high-throughput analysis of the cerebral endothelial functions underlying mechanisms of blood flow regulation in the normal and diseased brain.

Demonstrated here are protocols for (1) freshly isolating intact cerebral endothelial "tubes" and (2) simultaneous measurements of endothelial calcium and membrane potential during endothelium-derived hyperpolarization. Further, these methods allow for pharmacological tuning of endothelial cell calcium and electrical signaling as individual or interactive experimental variables.

Medidas simultáneas de calcio intracelular y el potencial de membrana en el endotelio Cerebral de ratón recién aislado e intacto

Cerebral arteries and their respective microcirculation deliver oxygen and nutrients to the brain via blood flow regulation. Endothelial cells line the ...