Name: kidney regeneration in adult zebrafish by gentamicin induced injury
Uploaded: 2015-08-03T14:30:00.000+00:00
Duration: 6 min 25 s
Description: Watch this Scientific Journal Video about Kidney Regeneration in Adult Zebrafish by Gentamicin Induced Injury at JoVE.com

This work was supported by NIH grant F32DK091998 to CNK; NIH grant RO1DK041071 and Harvard Stem Cell Institute grant D001229 to IAD. The authors thank Neil Hukriede for the lhx1ain situ probe.

Declaración de Ética: NOTA: Todos los experimentos se llevaron a cabo de conformidad con las directrices del Hospital General de Massachusetts para el uso de animales en la investigación. 1. Preparación previa <ol><li> Determinar cuántos adultos de pez cebra 6-12 meses de edad para herir. Plan de lesionar 10-20% más pescado que se necesitará. Con el fin de minimizar la variabilidad, el uso edad igualada peces hermanos fueron criados en el mismo tanque de modo que son aproximadamente del mismo tamaño y tienen menos variabilidad genética. Hembras son mucho más fáciles de inyectar en comparación con el pez macho, debido a la mayor capacidad del abdomen. Sin embargo, los machos y las hembras dan resultados similares. </li><li> Al hacer el experimento por primera vez, trazar una curva dosis para determinar la dosis de gentamicina apropiado para una cepa específica de pescado. Pruebe cada nuevo lote de gentamicina antes de su uso en los experimentos ya que la pureza de gentamicina varía de lote a lote. Una dosificación adecuada debe determinarse mediante la evaluación expressiel de los marcadores de lesiones tales como lhx1a. (Ver Figura 1 A - D). </li><li> Prepare la solución de gentamicina. Solución madre de gentamicina puede ser almacenada en -20 ° C y se descongeló para su uso posterior. Por ejemplo, para un / kg dosis 80 mg (80 mg de gentamicina / kg de peso corporal) en un pez de pesaje 0,5 g, preparar 2 mg / ml de gentamicina en solución salina tamponada con fosfato como una solución de trabajo conveniente. Esto proporciona una 20 l de gentamicina para la inyección intraperitoneal en el pescado. NOTA: En general, 80 a 120 mg / kg de lograr buenos resultados, pero dosis tan bajas como 40 mg / kg puede ser apropiado. La gentamicina también se puede comprar en solución para minimizar la exposición peligrosa para el personal de laboratorio. NOTA: La gentamicina en dosis altas puede ser tóxico. Use guantes y máscara al pesar en polvo. </li><li> Preparar 100 ml de 0,016% de agua tricaína para anestesiar a los peces. Preparar un balance de 25x tricaína (4 g / 1 L) disuelto en agua estéril milliQ ajustar el pH a 7 y se almacena a 4 ° C hasta su uso. Diluir 4 ml en 100 ml de agua de pescado para una concentración de trabajo de 0,016%. NOTA: tricaína es un irritante de anestesia y la piel, use guantes al manipular. </li><li> Haz un pez cucharada de una pipeta de transferencia de plástico mediante la reducción de la bombilla en forma de cuchara y cortar 2 ranuras en la parte inferior para drenar el agua. Jeringas de 1 ml de carga (con 10 l gradaciones) con la solución de gentamicina preparado y adjuntar una aguja G 30½. Eliminar las burbujas de aire. Gire la aguja de la jeringa para asegurarse de que la punta en ángulo de la aguja está de espaldas y las marcas de jeringas están mirando hacia adelante y legible. Para las inyecciones de control, preparar una jeringa y aguja con PBS estéril. </li><li> Preparar una escala con una superficie limpia o pesar barco, así como toallas de papel para el secado de pescado y para la celebración de los peces para la inyección. </li><li> Preparar pequeños contenedores de medio litro con tapas individuales para la celebración de los peces para la inyección de observación post durante la noche. Las pequeñas jaulas de apareamiento de plástico transparentes son útiles para este propósito. Estascontenedores no deben ser blanco - idealmente deben ser claros para que los elencos epiteliales blancas derramadas por los peces se pueden observar en un banco de copa negro. Llene cada recipiente con agua suficiente pescado para los peces para nadar cómodamente. </li></ol> 2. La inyección intraperitoneal de Gentamicina <ol><li> Anestesiar a los peces en solución tricaína 0,016% en agua los peces. Espere a que la tasa de ventilación de enmalle para frenar y para que los peces ya no responder al tacto. </li><li> Recoja los peces usando la pala de pescado, pasando de la cabeza hacia la cola con el fin de evitar lesiones en las branquias o aletas. Coloque el pescado en toallas de papel para absorber el exceso de agua girando el pescado con cuidado en su lado y sacuda el exceso de agua de la primicia. </li><li> Recoja los peces de nuevo y lo coloca en el barco pesando en la escala cero y pesar el pescado. Redondea a la 0.25 g más cercano, y calcular la cantidad apropiada de gentamicina para inyectar. NOTA: Para un 0.5 g de pescado utiliza una inyección de 20 l al 80 mg / kg dosis. </li><li> Recoja los peces de nuevo y colóquela sobre una toalla de papel plegado seco. Si la inyección con la mano derecha, mantenga la toalla de papel en la mano izquierda y coloque la cabeza del pez señala a la izquierda, con el vientre de fácil acceso. </li><li> Mantenga la jeringa con la aguja en un ángulo de 45 ° a la piel del vientre, anterior a la cloaca. Empuje la aguja debajo de la piel, a continuación, disminuir el ángulo y deslice la aguja hacia adelante bajo la piel, evitando los órganos internos. Presione el émbolo de la cantidad adecuada, hacer una pausa para asegurarse de que ningún líquido está saliendo alrededor de la aguja, y luego retire la aguja. Si la celebración de los peces es un problema constante, apoyando los codos contra el torso para estabilizar ellos. NOTA: Use guantes al manipular la solución de gentamicina. </li><li> Caída de cada pez en un recipiente individual. Observar a los peces y asegurar su recuperación de la anestesia. Mantenga el pescado a 28,5 ° C durante la noche. NOTA: Si el pescado no revive inmediatamente, use un trans plásticofer pipeta para regar agua a través de sus branquias de revivirlo. </li><li> Usar la misma jeringa y la aguja cuando la inyección de pescado múltiple con la misma dosis de gentamicina. Deseche la jeringa y aguja usada en el recipiente previsto apropiado. </li></ol> 3. Publicar Observación La inyección de Lesiones <ol><li> Al día siguiente (después de la lesión 1 día), coloque el pescado se inyecta en su contenedor sobre una superficie oscura. Yesos blancos de tejido epitelial muerta excretado por los peces heridos deben ser visibles. (Ver Figura 1E - H). Si no hay moldes, ya sea el pescado no estaba lesionado por la inyección de gentamicina (ya sea la dosis era demasiado baja, o algunos de la gentamicina se filtró durante el proceso de inyección) o el pescado estaba gravemente herido que resulta en un bloqueo completo de los uréteres y de cloaca con el tejido desprendido. NOTA: Si el pescado no puede borrar los moldes de su cuerpo por lo general va a morir dentro de 2-3 días y por lo tanto inutilizable durante más tiempoensayos. Si no hay moldes son visibles, la eutanasia a los peces en el agua tricaína por inmersión durante al menos 10 minutos después detiene el movimiento de enmalle. </li><li> Si votas tejidos blancos son visibles, establezca el pescado a un lado y continuar revisando los otros peces. Una dosis adecuada de gentamicina resultará en el 80-90% de los peces de ser utilizable. Aunar los peces heridos y luego los divide en diferentes grupos de tratamiento si se desea. </li></ol> 4. Cuidado de pescado Recuperación <ol><li> Mantenga pez en el agua limpia y el medio ambiente con poca gente. El agua sucia y el hacinamiento dará lugar a infecciones y la muerte no intencional. Trate de mantener no más de 6 peces / 500 ml de agua peces y cambiar el agua diariamente si es posible. NOTA: Este es un volumen mínimo para la conservación de espacio o si el investigador desea para llevar a cabo experimentos utilizando los tratamientos con fármacos caros. </li><li> No alimentar a los peces hasta 3 días después de la lesión. Recuperación de peces no comen en un primer momento, y cualquier alimento en el tanque se descomponen y promover bacterias growth. A partir de 3 días después de la lesión, alimentar a una pequeña cantidad una vez al día. </li></ol> 5. Análisis de Lesionados Riñones <ol><li> Para la hibridación in situ para la expresión de ARNm lhx1a, los riñones se pueden cosechar a partir de peces en puntos de tiempo deseados. La eutanasia pez en el agua tricaína en hielo al menos 10 minutos hasta que el movimiento de enmalle se detiene, luego retire la cabeza con una hoja de afeitar y abrir la cavidad del cuerpo y eliminar los órganos internos con fórceps. </li><li> Deje el riñón en su lugar (órgano pigmentada unido a la pared dorsal del cuerpo) y fijar el pescado en 4% de paraformaldehído en PBS durante la noche. Diseccionar el riñón 15 y continuar con el estándar de hibridación in situ. <ol><li> Brevemente, lavar los riñones en PBST (tampón fosfato salino 0,5% de Tween 20), se permeabilizaron con proteinasa K en PBST (10 g / ml) durante 1 hora a temperatura ambiente. Post-fijar con paraformaldehído al 4%, y se lavó de nuevo con PBST. </li><li> Entonces pre-hibridar los riñones durante la noche a 68; ° C en tampón de hibridación (50% formamida, 5x SSC, 50 mg / ml de heparina, 500 mg / ml tRNA, 0,1% de Tween 20, pH 6,0). </li><li> Incubar las muestras con la sonda marcada con digoxigenina en tampón de hibridación, a continuación, lavar durante 5-10 min cada uno con 100%, 75%, 50%, 25% tampón de hibridación a 68 ° C, luego se trasladó a temperatura ambiente y lavar dos veces durante 30 min con 2x SSC con 0,1% de Tween 20, y lavar dos veces durante 30 min con 0,2 x SSC con 0,1% de Tween 20. </li><li> Equilibrar la muestra 3x 5 min en el MAB (0,1 M de ácido maleico, NaCl 0,15 M, pH 7,5) y se bloquearon durante la noche a 4 ° C en el MAB con 10% de suero de cabra. Entonces incubar las muestras durante la noche en anti-digoxigenina-AP Fab 1: 5000 en MAB. </li><li> Lavar el 5x muestra para 1 hr en MAB, y equilibrar 3x durante 15 min en NTMT (0,1 M Tris pH 9,5, 0,05 M MgCl 2, M NaCl 0,01, 50 l de Tween 20). </li><li> Luego tratar los riñones con NBT / BCIP para detectar la señal. Fijar los riñones con paraformaldehído al 4% y se incubaron en dimetilformamida a remove exceso de NBT / BCIP, blanqueada durante la noche en agua desionizada. Lavar los riñones y luego fotografiar en SAFT con un 50% de glicerol bajo un cubreobjetos. </li></ol></li></ol>

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The authors have no competing financial interests to disclose.

El pez cebra es ideal para el estudio de la regeneración de órganos adultos incluyendo el adulto riñón mesonéfrico 6. Estudios recientes han aprovechado de marcadores moleculares y nuevas líneas reportero transgénicos para caracterizar mejor los pasos que se dan durante la regeneración de nefronas y lo que las células podrían ser responsables 7,12. Observación de desechos urinarios se ha utilizado durante más de un siglo para diagnosticar la enfermedad renal en humanos 16. Aq...

El riñón es esencial para la homeostasis de fluidos, la regulación de la presión arterial y la filtración de los residuos del cuerpo. Aunque los mamíferos son capaces de reparar nefronas existentes después de la lesión utilizando células epiteliales diferenciadas 1-4, que parecen carecer de una piscina de células madre reservadas 5 y son incapaces de formar nuevas nefronas de novo. En contraste a los mamíferos, peces adultos son capaces de formar nuevas nefronas largo de la vida adulta para apoyar el crecimiento de los peces y en respuesta a la lesión 6,7. El pez cebra, Danio rerio, es un organismo modelo de gran valor para el estudio de la regeneración de órganos 8-10 y tiene el potencial para proporcionar información de gran alcance en las aplicaciones de la ingeniería de la reparación de los riñones humanos. La Tg (Lhx1a: EGFP) transgén 11 se ha demostrado para etiquetar un conjunto de células progenitoras nefrona en el riñón adultos de pez cebra 12, sin embargo los mecanismos que controlan la respuesta de lhx1a + células a una lesión rEmain claro. La gentamicina es un antibiótico aminoglucósido ampliamente utilizado con efectos nefrotóxicos y ototóxicos conocidos en humanos 13. La inyección intraperitoneal de gentamicina es un método establecido de inducir lesión renal aguda en peces 6. Esta lesión en imita pescado la pérdida de epitelio tubular y la cicatrización de los glomérulos que se produce en los seres humanos después de la gentamicina sobredosis 14. La inducción de lesiones en el pez cebra por inyección de gentamicina es una manera conveniente de inducir una respuesta regeneración síncrono fuerte, con muchas nuevas nefronas producidos y de proceder simultáneamente a través de etapas de la formación, la proliferación y la diferenciación. Este protocolo detalla nuestro método de lesión robusta y reproducible en el pez cebra riñón adulto mediante la utilización de un proceso de selección visual no invasiva para reducir al mínimo los valores atípicos. Aprovechamos el hecho de que las lesiones con gentamicina conduce a la muerte del tejido renal epitelial y formatoión de cilindros tubulares renales, que luego se acumulan en masas en los conductos mesonéfricos y cloaca. Estos son pasados ​​por el pescado y se pueden observar visualmente en el agua. Esto nos permite a la pantalla para los peces con una fuerte lesión no letal, que luego pueden ser agrupados para su posterior experimentación. Reducir al mínimo el número de peces o no lesionado que mueren antes de llegar al punto final del experimento conduce a más uniforme y la recopilación de datos eficiente y análisis. Además, no se requieren dispositivos especiales o reactivos, por lo que este método rentable y adecuado para su uso en un entorno académico o la enseñanza. Usando este método, aquí mostramos las crecientes efectos de la dosis de gentamicina en la regeneración del riñón, así como el efecto del aumento de temperatura.

<table><tbody><tr><td>gentamicin sodium sulfate</td><td>Sigma</td><td>G1264</td><td>TOXIC, purity varies from batch to batch</td></tr><tr><td>plastic transfer pipets</td><td>Fisher</td><td>13-711-7M</td><td></td></tr><tr><td>1 ml Norm-Ject syringes</td><td>Electron Microscopy Sciences</td><td>72520</td><td>green plastic syringes, ordinary 1 ml syringes are OK, but harder to read accurately</td></tr><tr><td>30 G1/2 needles</td><td>Becton Dickinson</td><td>305106</td><td></td></tr><tr><td>ethyl 3-aminobenzoaate methanesulfonate salt (tricaine)</td><td>Sigma</td><td>A5040</td><td>IRRITANT</td></tr><tr><td>16% paraformaldehyde</td><td>Electron Microscopy Sciences</td><td>15710</td><td>Make 4% in 1x PBS for working solution</td></tr></tbody></table>

kidney regeneration in adult zebrafish by gentamicin induced injury

The kidney is essential for fluid homeostasis, blood pressure regulation and filtration of waste from the body. The fundamental unit of kidney function is the nephron. Mammals are able to repair existing nephrons after injury, but lose the ability to form new nephrons soon after birth. In contrast to mammals, adult fish produce new nephrons (neonephrogenesis) throughout their lives in response to growth requirements or injury. Recently, lhx1a has been shown to mark nephron progenitor cells in the adult zebrafish kidney, however mechanisms controlling the formation of new nephrons after injury remain unknown. Here we show our method for robust and reproducible injury in the adult zebrafish kidney by intraperitoneal (i.p.) injection of gentamicin, which uses a noninvasive visual screening process to select for fish with strong but nonlethal injury. Using this method, we can determine optimal gentamicin dosages for injury and go on to demonstrate the effect of higher temperatures on kidney regeneration in zebrafish.

Lesión gentamicina puede ser confirmada visualmente por la observación de moldes epiteliales renales en el agua (Figura 1). Diferentes dosis de gentamicina se utilizaron para herir a adultos de pez cebra TUAB tipo salvaje, lo que resulta en un número creciente de lhx1a + agregados celulares en el riñón regeneración (Figura 1A - D). Yesos blancos pueden verse fácilmente en el agua 1 día después de la lesión (Figura 1E - H).</st...

Watch this Scientific Journal Video about Kidney Regeneration in Adult Zebrafish by Gentamicin Induced Injury at JoVE.com

Kidney Regeneration in Adult Zebrafish by Gentamicin Induced Injury

Here we present a reliable method to study adult kidney regeneration by inducing acute kidney injury by gentamicin injection. We show that injury is dependent on gentamicin dosage and environmental temperature using in situ hybridization to label lhx1a+ developing new nephrons.

La regeneración renal en adultos de pez cebra por gentamicina lesión inducida

The kidney is essential for fluid homeostasis, blood pressure regulation and filtration of waste from the body. The fundamental unit of kidney function is ...

kidney-regeneration-in-adult-zebrafish-by-gentamicin-induced-injury

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10.8K vistas.  Massachusetts General Hospital. Here we present a reliable method to study adult kidney regeneration by inducing acute kidney injury by gentamicin injection. We show that injury is dependent on gentamicin dosage and environmental temperature using in situ hybridization to label lhx1a+ developing new nephrons. 

Video: Kidney Regeneration in Adult Zebrafish by Gentamicin Induced Injury

Precise Cellular Ablation Approach for Modeling Acute Kidney Injury in Developing Zebrafish

Acute Kidney Injury (AKI) is a common medical condition with a high mortality rate. With the repair abilities of the kidney, it is possible to restore adequate kidney function after supportive treatment. However, a better understanding of how nephron cell death and repair occur on the cellular level is required to minimize cell death and to enhance the regenerative process. The zebrafish pronephros is a good model system to accomplish this goal because it contains anatomical segments that are similar to the mammalian nephron. Previously, the most common model used to study kidney injury in fish was the pharmacological gentamicin model. However, this model does not allow for precise spatiotemporal control of injury, and hence it is difficult to study cellular and molecular processes involved in kidney repair. To overcome this limitation, this work presents a method through which, in contrast to the gentamicin approach, a specific Green Fuorescent Protein (GFP)-expressing nephron segment can be photoablated using a violet laser light (405 nm). This novel model of AKI provides many advantages that other methods of epithelial injury lack. Its main advantages are the ability to &#34;dial&#34; the level of injury and the precise spatiotemporal control in the robust in vivo animal model. This new method has the potential to significantly advance the level of understanding of kidney injury and repair mechanisms.

This work presents a new in vivo model of segmental kidney injury using kidney GFP transgenic zebrafish. The model allows for the induction of the targeted ablation of kidney epithelial cells to show the cellular mechanisms of nephron injury and repair.

Enfoque preciso de la ablación celular para modelar lesiones renales agudas en el desarrollo de pez cebra

Acute Kidney Injury (AKI) is a common medical condition with a high mortality rate. With the repair abilities of the kidney, it is possible to restore ...

Investigación

Biología del Desarrollo

Stab Wound Injury of the Zebrafish Adult Telencephalon: A Method to Investigate Vertebrate Brain Neurogenesis and Regeneration

Adult zebrafish have an amazing capacity to regenerate their central nervous system after injury. To investigate the cellular response and the molecular mechanisms involved in zebrafish adult central nervous system (CNS) regeneration and repair, we developed a zebrafish model of adult telencephalic injury.
In this approach, we manually generate an injury by pushing an insulin syringe needle into the zebrafish adult telencephalon. At different post injury days, fish are sacrificed, their brains are dissected out and stained by immunohistochemistry and/or in situ hybridization (ISH) with appropriate markers to observe cell proliferation, gliogenesis, and neurogenesis. The contralateral unlesioned hemisphere serves as an internal control. This method combined for example with RNA deep sequencing can help to screen for new genes with a role in zebrafish adult telencephalon neurogenesis, regeneration, and repair.

To shed light on the cellular and molecular mechanisms of zebrafish adult neurogenesis and regeneration, we developed a protocol for invasive surgery causing mechanical injuries in the zebrafish adult telencephalon and subsequent monitoring of changes in the stabbed hemisphere by immunohistochemistry or in situ hybridization.

Puñalada de Lesiones de la herida del pez cebra Telencéfalo adultos: un método para investigar Vertebrados Cerebro neurogénesis y Regeneración

Adult zebrafish have an amazing capacity to regenerate their central nervous system after injury. To investigate the cellular response and the molecular ...

Neurociencias

Intravenous Microinjections of Zebrafish Larvae to Study Acute Kidney Injury

In this video article we describe a zebrafish model of AKI using gentamicin as the nephrotoxicant.  The technique consists of intravenous microinjections on 2 dpf zebrafish. This technique represents an efficient and rapid method to deliver soluble substances into the bloodstream of zebrafish larvae, allowing for the injection of 15-20 fish per hour. In addition to AKI studies, this microinjection technique can also be used for other types of experimental studies such as angiography.  We provide a detailed protocol of the technique from equipment required to visual measures of decreased kidney function. In addition, we also demonstrate the process of fixation, whole mount immunohistochemistry with a kidney tubule marker, plastic embedding and sectioning of the larval zebrafish. We demonstrate that zebrafish larvae injected with gentamicin show morphological features consistent with AKI: edema, loss of cell polarity in proximal tubular epithelial cells, and morphological disruption of the tubule.

We describe a technique of microinjecting the aminoglycoside, gentamicin, into 2 days post-fetilization (dpf) zebrafish larvae to induce acute kidney injury (AKI). We also describe a method for whole mount immunohistochemistry, plastic embedding and sectioning of zebrafish larvae to visualize the AKI mediated damage.

Microinyecciones intravenosa de larvas de pez cebra para el Estudio de la lesión renal aguda

In this video article we describe a zebrafish model of AKI using gentamicin as the nephrotoxicant.  The technique consists of intravenous microinjections ...

Biología

Stab Wound Injury Model of the Adult Optic Tectum Using Zebrafish and Medaka for the Comparative Analysis of Regenerative Capacity

While zebrafish have a superior capacity to regenerate their central nervous system (CNS), medaka has a lower CNS regenerative capacity. A brain injury model was developed in the adult optic tectum of zebrafish and medaka and comparative histological and molecular analyses were performed to elucidate the molecular mechanisms regulating the high regenerative capacity of this tissue across these fish species. Here a stab wound injury model is presented for the adult optic tectum using a needle and histological analyses for proliferation and differentiation of the neural stem cells (NSCs). A needle was manually inserted into the central region of the optic tectum, and then the fish were intracardially perfused, and their brains were dissected. These tissues were then cryosectioned and evaluated using immunostaining against the appropriate NSC proliferation and differentiation markers. This tectum injury model provides robust and reproducible results in both zebrafish and medaka, allowing for comparing NSC responses after injury. This method is available for small teleosts, including zebrafish, medaka, and African killifish, and enables us to compare their regenerative capacity and investigate unique molecular mechanisms.

A mechanical brain injury model in the adult zebrafish is described to investigate the molecular mechanisms regulating their high regenerative capacity. The method explains to create a stab wound injury in the optic tectum of multiple species of small fish to evaluate the regenerative responses using fluorescent immunostaining.

Modelo de lesión por puñalada del Tectum óptico adulto utilizando pez cebra y medaka para el análisis comparativo de la capacidad regenerativa

While zebrafish have a superior capacity to regenerate their central nervous system (CNS), medaka has a lower CNS regenerative capacity. A brain injury ...

Transplantation of Cells Directly into the Kidney of Adult Zebrafish

Regenerative medicine based on the transplantation of stem or progenitor cells into damaged tissues has the potential to treat a wide range of chronic diseases1. However, most organs are not easily accessible, necessitating the need to develop surgical methods to gain access to these structures. In this video article, we describe a method for transplanting cells directly into the kidney of adult zebrafish, a popular model to study regeneration and disease2. Recipient fish are pre-conditioned by irradiation to suppress the immune rejection of the injected cells3. We demonstrate how the head kidney can be exposed by a lateral incision in the flank of the fish, followed by the injection of cells directly in to the organ. Using fluorescently labeled whole kidney marrow cells comprising a mixed population of renal and hematopoietic precursors, we show that nephron progenitors can engraft and differentiate into new renal tissue - the gold standard of any cell-based regenerative therapy. This technique can be adapted to deliver purified stem or progenitor cells and/or small molecules to the kidney as well as other internal organs and further enhances the zebrafish as a versatile model to study regenerative medicine.

Cell transplantation is an essential technique for studying tissue regeneration and for developing cell-based therapies of disease. We demonstrate here a microsurgical technique that permits the transplantation of genetically labeled cells directly into the kidney of adult zebrafish fish.

El trasplante de células directamente en el riñón de adultos de pez cebra

Regenerative medicine based on the transplantation of stem or progenitor cells into damaged tissues has the potential to treat a wide range of chronic ...

Dissection of the Adult Zebrafish Kidney

Researchers working in the burgeoning field of adult stem cell biology seek to understand the signals that regulate the behavior and function of stem cells during normal homeostasis and disease states. The understanding of adult stem cells has broad reaching implications for the future of regenerative medicine1. For example, better knowledge about adult stem cell biology can facilitate the design of therapeutic strategies in which organs are triggered to heal themselves or even the creation of methods for growing organs in vitro that can be transplanted into humans1. The zebrafish has become a powerful animal model for the study of vertebrate cell biology2. There has been extensive documentation and analysis of embryonic development in the zebrafish3. Only recently have scientists sought to document adult anatomy and surgical dissection techniques4, as there has been a progressive movement within the zebrafish community to broaden the applications of this research organism to adult studies. For example, there are expanding interests in using zebrafish to investigate the biology of adult stem cell populations and make sophisticated adult models of diseases such as cancer5. Historically, isolation of the zebrafish adult kidney has been instrumental for studying hematopoiesis, as the kidney is the anatomical location of blood cell production in fish6,7. The kidney is composed of nephron functional units found in arborized arrangements, surrounded by hematopoietic tissue that is dispersed throughout the intervening spaces. The hematopoietic component consists of hematopoietic stem cells (HSCs) and their progeny that inhabit the kidney until they terminally differentiate8. In addition, it is now appreciated that a group of renal stem/progenitor cells (RPCs) also inhabit the zebrafish kidney organ and enable both kidney regeneration and growth, as observed in other fish species9-11. In light of this new discovery, the zebrafish kidney is one organ that houses the location of two exciting opportunities for adult stem cell biology studies. It is clear that many outstanding questions could be well served with this experimental system. To encourage expansion of this field, it is beneficial to document detailed methods of visualizing and then isolating the adult zebrafish kidney organ. This protocol details our procedure for dissection of the adult kidney from both unfixed and fixed animals.
Dissection of the kidney organ can be used to isolate and characterize hematopoietic and renal stem cells and their offspring using established techniques such as histology, fluorescence activated cell sorting (FACS)11,12, expression profiling13,14, and transplantation11,15. 
We hope that dissemination of this protocol will provide researchers with the knowledge to implement broader use of zebrafish studies that ultimately can be translated for human application.

The zebrafish kidney is home to both renal and hematopoietic adult stem/progenitor cells, and represents an outstanding opportunity to study these cell types and their progeny in a vertebrate model organism. Here, we demonstrate a detailed dissection procedure that enables the researcher to identify and surgically remove the adult zebrafish kidney, which can be used for applications such as cell isolation, transplantation, and expression studies of kidney and/or blood cell populations.

La disección del riñón adultos de pez cebra

Researchers working in the burgeoning field of adult stem cell biology seek to understand the signals that regulate the behavior and function of stem ...

Laser Ablation of the Zebrafish Pronephros to Study Renal Epithelial Regeneration

Acute kidney injury (AKI) is characterized by high mortality rates from deterioration of renal function over a period of hours or days that culminates in renal failure1. AKI can be caused by a number of factors including ischemia, drug-based toxicity, or obstructive injury1. This results in an inability to maintain fluid and electrolyte homeostasis. While AKI has been observed for decades, effective clinical therapies have yet to be developed. Intriguingly, some patients with AKI recover renal functions over time, a mysterious phenomenon that has been only rudimentally characterized1,2. Research using mammalian models of AKI has shown that ischemic or nephrotoxin-injured kidneys experience epithelial cell death in nephron tubules1,2, the functional units of the kidney that are made up of a series of specialized regions (segments) of epithelial cell types3. Within nephrons, epithelial cell death is highest in proximal tubule cells. There is evidence that suggests cell destruction is followed by dedifferentiation, proliferation, and migration of surrounding epithelial cells, which can regenerate the nephron entirely1,2. However, there are many unanswered questions about the mechanisms of renal epithelial regeneration, ranging from the signals that modulate these events to reasons for the wide variation of abilities among humans to regenerate injured kidneys.
The larval zebrafish provides an excellent model to study kidney epithelial regeneration as its pronephric kidney is comprised of nephrons that are conserved with higher vertebrates including mammals4,5. The nephrons of zebrafish larvae can be visualized with fluorescence techniques because of the relative transparency of the young zebrafish6. This provides a unique opportunity to image cell and molecular changes in real-time, in contrast to mammalian models where nephrons are inaccessible because the kidneys are structurally complex systems internalized within the animal. Recent studies have employed the aminoglycoside gentamicin as a toxic causative agent for study of AKI and subsequent renal failure: gentamicin and other antibiotics have been shown to cause AKI in humans, and researchers have formulated methods to use this agent to trigger kidney damage in zebrafish7,8. However, the effects of aminoglycoside toxicity in zebrafish larvae are catastrophic and lethal, which presents a difficulty when studying epithelial regeneration and function over time. Our method presents the use of targeted cell ablation as a novel tool for the study of epithelial injury in zebrafish. Laser ablation gives researchers the ability to induce cell death in a limited population of cells. Varying areas of cells can be targeted based on morphological location, function, or even expression of a particular cellular phenotype. Thus, laser ablation will increase the specificity of what researchers can study, and can be a powerful new approach to shed light on the mechanisms of renal epithelial regeneration. This protocol can be broadly applied to target cell populations in other organs in the zebrafish embryo to study injury and regeneration in any number of contexts of interest.

Acute kidney injury (AKI) in humans is a common clinical problem caused by damage to the epithelial cells that comprise kidney nephrons, and AKI is associated with high mortality rates of 50-70%1. Following epithelial cell destruction, nephrons have a limited ability to regenerate, though the mechanisms and limitations that guide this phenomenon remain poorly understood. In this video article, we describe our technique for targeted laser ablation of kidney nephron cells in the zebrafish embryo kidney, or pronephros. Our new method can be used to complement nephrotoxicity-induced models of AKI and gain a high-resolution understanding of the cell and molecular alterations that are associated with epithelial regeneration in the kidney nephron.

La ablación con láser de la pronefros pez cebra para el Estudio de la regeneración epitelial renal

Acute kidney injury (AKI) is characterized by high mortality rates from deterioration of renal function over a period of hours or days that culminates in ...

An Assay for Lateral Line Regeneration in Adult Zebrafish

Due to the clinical importance of hearing and balance disorders in man, model organisms such as the zebrafish have been used to study lateral line development and regeneration. The zebrafish is particularly attractive for such studies because of its rapid development time and its high regenerative capacity. To date, zebrafish studies of lateral line regeneration have mainly utilized fish of the embryonic and larval stages because of the lower number of neuromasts at these stages. This has made quantitative analysis of lateral line regeneration/and or development easier in the earlier developmental stages. Because many zebrafish models of neurological and non-neurological diseases are studied in the adult fish and not in the embryo/larvae, we focused on developing a quantitative lateral line regenerative assay in adult zebrafish so that an assay was available that could be applied to current adult zebrafish disease models. Building on previous studies by Van Trump et al.17&#160;that described procedures for ablation of hair cells in adult Mexican blind cave fish and zebrafish (Danio rerio), our assay was designed to allow quantitative comparison between control and experimental groups. This was accomplished by developing a regenerative neuromast standard curve based on the percent of neuromast reappearance over a 24 hr time period following gentamicin-induced necrosis of hair cells in a defined region of the lateral line. The assay was also designed to allow extension of the analysis to the individual hair cell level when a higher level of resolution is required.

Because many zebrafish models of neurological and non-neurological diseases are studied in the adult fish rather than the embryo/larvae, we developed a quantitative lateral line regenerative assay that can be applied to adult zebrafish disease models. The assay involved resolution at the 1) neuromast and 2) individual hair cell levels.

Un ensayo para la línea lateral de regeneración en adultos de pez cebra

Due to the clinical importance of hearing and balance disorders in man, model organisms such as the zebrafish have been used to study lateral line ...

Analysis of Nephron Composition and Function in the Adult Zebrafish Kidney

The zebrafish model has emerged as a relevant system to study kidney development, regeneration and disease. Both the embryonic and adult zebrafish kidneys are composed of functional units known as nephrons, which are highly conserved with other vertebrates, including mammals. Research in zebrafish has recently demonstrated that two distinctive phenomena transpire after adult nephrons incur damage: first, there is robust regeneration within existing nephrons that replaces the destroyed tubule epithelial cells; second, entirely new nephrons are produced from renal progenitors in a process known as neonephrogenesis. In contrast, humans and other mammals seem to have only a limited ability for nephron epithelial regeneration. To date, the mechanisms responsible for these kidney regeneration phenomena remain poorly understood. Since adult zebrafish kidneys undergo both nephron epithelial regeneration and neonephrogenesis, they provide an outstanding experimental paradigm to study these events. Further, there is a wide range of genetic and pharmacological tools available in the zebrafish model that can be used to delineate the cellular and molecular mechanisms that regulate renal regeneration. One essential aspect of such research is the evaluation of nephron structure and function. This protocol describes a set of labeling techniques that can be used to gauge renal composition and test nephron functionality in the adult zebrafish kidney. Thus, these methods are widely applicable to the future phenotypic characterization of adult zebrafish kidney injury paradigms, which include but are not limited to, nephrotoxicant exposure regimes or genetic methods of targeted cell death such as the nitroreductase mediated cell ablation technique. Further, these methods could be used to study genetic perturbations in adult kidney formation and could also be applied to assess renal status during chronic disease modeling.

The zebrafish adult kidney is an excellent system for renal regeneration and disease studies. An essential aspect of such research is the assessment of nephron structure and function. This protocol describes several methodologies that can be implemented to assess nephron tubule composition and to evaluate renal reabsorption.

Análisis de Composición Nephron y función en el riñón adultos de pez cebra

The zebrafish model has emerged as a relevant system to study kidney development, regeneration and disease. Both the embryonic and adult zebrafish kidneys ...

Acute Kidney Injury Model Induced by Cisplatin in Adult Zebrafish

Cisplatin is commonly used as chemotherapy. Although it has positive effects in cancer-treated individuals, cisplatin can easily accumulate in the kidney due to its low molecular weight. Such accumulation causes the death of tubular cells and can induce the development of Acute Kidney Injury (AKI), which is characterized by a quick decrease in kidney function, tissue damage, and immune cells infiltration. If administered in specific doses cisplatin can be a useful tool as an AKI inducer in animal models. The zebrafish has appeared as an interesting model to study renal function, kidney regeneration, and injury, as renal structures conserve functional similarities with mammals. Adult zebrafish injected with cisplatin shows decreased survival, kidney cell death, and increased inflammation markers after 24 h post-injection (hpi). In this model, immune cells infiltration and cell death can be assessed by flow cytometry and TUNEL assay. This protocol describes the procedures to induce AKI in adult zebrafish by intraperitoneal cisplatin injection and subsequently demonstrates how to collect the renal tissue for flow cytometry processing and cell death TUNEL assay. These techniques will be useful to understand the effects of cisplatin as a nephrotoxic agent and will contribute to the expansion of AKI models in adult zebrafish. This model can also be used to study kidney regeneration, in the search for compounds that treat or prevent kidney damage and to study inflammation in AKI. Moreover, the methods used in this protocol will improve the characterization of tissue damage and inflammation, which are therapeutic targets in kidney-associated comorbidities.

This protocol describes the procedures to induce Acute Kidney Injury (AKI) in adult zebrafish using cisplatin as a nephrotoxic agent. We detailed the steps to evaluate the reproducibility of the technique and two techniques to analyze inflammation and cell death in the renal tissue, flow cytometry and TUNEL, respectively.

Modelo de lesión renal aguda inducida por cisplatino en pez cebra adulto

Cisplatin is commonly used as chemotherapy. Although it has positive effects in cancer-treated individuals, cisplatin can easily accumulate in the kidney ...