La investigación fue apoyada por la Subvención de Desarrollo de Científicos Investigadores del NHLBI (1K01HL135461-01) a JAG. Gracias a David Carmona-Berrio e Isabel Adarve-Rengifo por su asistencia técnica.

Los ratones fueron alojados y cuidados en la División de Cuidado de Animales del Centro Médico de la Universidad de Vanderbilt (VUMC) siguiendo las pautas de los Institutos Nacionales de Salud (NIH) y la Guía para el Cuidado y Uso de Animales de Laboratorio, Departamento de Salud y Servicios Humanos de los Estados Unidos. Todos los procedimientos con animales fueron aprobados por el Comité Institucional de Cuidado y Uso de Animales de VUMC antes de comenzar los experimentos.
1. Preparación y disección de animales
<ol><li>Encienda el germinador y la bomba de agua de la almohadilla térmica unos 30 minutos antes de comenzar la cirugía.</li>
	<li>Corte tubos de poliuretano de 0,5 mm de longitud con un bisturí afilado. Retire 0,2 mm de la circunferencia haciendo un corte longitudinal para hacer un manguito. NOTA: Esta es una parte crítica del procedimiento de estenosis de la arteria renal que requiere extrema precisión, atención a los detalles y paciencia. Trate de cortar varias piezas de tubos de poliuretano a la vez. Realice todo este procedimiento bajo el microscopio.</li>
	<li>Antes de continuar, póngase guantes estériles quirúrgicos y una máscara quirúrgica.</li>
	<li>Utilice ratones C57BL/6 de tipo salvaje (WT) de 6-8 semanas. Use un número igual de ratones machos y hembras para evitar cualquier sesgo sexual.</li>
	<li>Registre el peso de los ratones antes de realizar la cirugía. El peso ideal para realizar estenosis de la arteria renal con tubo de poliuretano es de 18-22 g. Manipule los ratones suavemente y no agite mientras inyecta la anestesia. Administrar analgesia preoperatoria (Ketofgen, 5 mg/kg de peso corporal). NOTA: Al transferir los ratones de su habitación de alojamiento a la sala de cirugía, llévelos con gran delicadeza y cuidado para evitar la agitación. Llevar jaulas de ratones en las manos en lugar de un carrito es muy recomendable.</li>
	<li>Anestesiar a los ratones con una mezcla de ketamina (100 mg/kg de peso corporal) y xilazina (10 mg/kg de peso corporal) mediante inyecciones intraperitoneales (I.P.).</li>
	<li>Coloque el ratón de nuevo dentro de la jaula hasta que esté completamente anestesiado. Toma alrededor de 4-5 minutos antes de que el ratón esté completamente inconsciente. Pellizque la cola o el dedo del pie con fórceps para verificar si el ratón está completamente inconsciente y listo para la cirugía.</li>
	<li>Coloque el mouse boca arriba sobre una toalla de papel. Retire el vello del abdomen lateral con un cortapelos eléctrico siguiendo la dirección opuesta al crecimiento del vello. Después de afeitarse el sitio quirúrgico, limpie el área con un exfoliante quirúrgico que contenga yodo (o clorhexidina) seguido de un enjuague con etanol al 70% (o solución salina estéril).  Repita 3 veces. NOTA: El procedimiento de depilación debe realizarse a cierta distancia o preferiblemente en un banco diferente al del banco de procedimiento de cirugía para evitar cualquier interferencia de vello y contaminación del vello durante el procedimiento quirúrgico.</li>
	<li>Cubra la almohadilla térmica con una lámina estéril. Lleve el ratón al banco quirúrgico y colóquelo sobre la lámina estéril, mirando hacia el lado lateral derecho del ratón hacia el microscopio. Mantener una temperatura constante de la almohadilla de 37 °C con agua circulante.</li>
	<li>Abra la bolsa esterilizada que contiene todo el equipo quirúrgico. Usando un microscopio de disección y tijeras estériles para objetos punzantes, haga una pequeña incisión en el flanco (cerca de la13ª costilla torácica, la última costilla en el ratón) y a unos 0,5 cm de distancia de las vértebras. Proceda a lo largo de las vértebras lumbares y haga una incisión de 1 pulgada.</li>
	<li>Retire la piel y el músculo para exponer el riñón.</li>
	<li>Limpie y elimine la grasa circundante con hisopos de algodón estériles para aislar la arteria renal. Aislar el nervio renal de la arteria renal usando fórceps curvos.</li>
	<li>Al realizar la cirugía simulada, aplique suturas para cerrar la piel y aplique antibióticos a la herida cerrada, luego proceda a los cuidados postoperatorios. Si no es así, proceda con la siguiente sección para estenosis de la arteria. NOTA: Cada experimento debe tener animales simulados como controles del procedimiento quirúrgico. Los animales simulados consisten en ratones que han pasado por el procedimiento quirúrgico de exponer la arteria renal sin colocar un manguito sobre ella.</li>
</ol>2. Estenosis de la arteria renal derecha
<ol><li>Coloque dos suturas de nylon debajo de la arteria renal derecha, haga nudos sueltos y luego coloque el manguito alrededor de la arteria renal principal aproximadamente equidistante entre el riñón y la bifurcación de la aorta</li>
	<li>Cierre el manguito con las suturas de nylon. Haga cuatro nudos para cada sutura para evitar la probabilidad de perder las suturas después de la cirugía.</li>
	<li>Cierre la incisión en el músculo aplicando una sutura continua simple.</li>
	<li>Haga suturas simples interrumpidas para cerrar la piel.</li>
	<li>Administrar analgésicos aprobados por IACUC. NOTA: Herramientas quirúrgicas de autoclave antes de cada uso. Si se realiza más de una cirugía a la vez, limpie todas las herramientas usadas con un medidor de alcohol estéril y colóquelas en un germinador caliente durante 15-30 s después de cada cirugía. Cambie los guantes estériles también para cada ratón.</li>
</ol>3. Cuidados postoperatorios
<ol><li>Devuelva a los ratones a su jaula y deje la jaula medio encendida, mitad apagada, una almohadilla de calentamiento de agua circulante durante 2 a 3 horas. Agregue alimentos de recuperación de la dieta en gel dentro de la jaula.</li>
	<li>Administrar analgésico (ketoprofeno) por vía intraperitoneal (dosis: 5 mg/kg pc) al día siguiente.</li>
	<li>Pesar los ratones durante los próximos dos días; si algunos ratones pierden más del 20% de su peso consulte con el veterinario y decida si el animal necesita ser sacrificado siguiendo el procedimiento apropiado autorizado por IACUC.</li>
	<li>Controle a los ratones diariamente para evaluar si hay enrojecimiento, hinchazón, dolor o infección.</li>
	<li>Buscar consulta veterinaria por complicaciones postoperatorias de acuerdo con las políticas institucionales de IACUC.</li>
</ol>4. Recolección de tejidos
<ol><li>Cosechar tejido 3 semanas después del procedimiento quirúrgico. Registra el peso de cada ratón.</li>
	<li>Eutanasia del ratón con un procedimiento aprobado por IACUC.</li>
	<li>Coloque el ratón en una plataforma estéril en posición supina para diseccionar.</li>
	<li>Asegure y extienda las extremidades para limitar el movimiento.</li>
	<li>Rocíe bien los ratones con etanol al 70%.</li>
	<li>Haga una incisión en la línea media para abrir el abdomen y el área del pecho con tijeras afiladas.</li>
	<li>Retire la piel y la pared peritoneal.</li>
	<li>Exponga cuidadosamente el corazón y perfore el ventrículo derecho y exangre al ratón.</li>
	<li>Extirpar ambos riñones con fórceps. Los riñones se encuentran en la parte posterior de los ratones. NOTA: No mezcle ambos riñones. Tenga cuidado con los riñones estenosados, simulados y contralaterales.</li>
	<li>Retire la cápsula de riñón, límpielas de cualquier grasa y registre el peso de cada riñón por separado.</li>
	<li>Cortar una sección longitudinal de ambos riñones y fijar en PFA al 4% durante la noche a 4 °C para ser procesado posteriormente para la incrustación de parafina, para realizar hibridación in situ (ISH) e inmunohistoquímica (IHC). Siga los protocolos ISH e IHC como se informó23,24.</li>
	<li>Aísle la corteza del riñón restante y congele rápidamente en nitrógeno líquido para realizar Western blot. Almacenar las muestras a -80 °C hasta su análisis.</li>
	<li>Cuantificar las expresiones de renina y N-GAL con Western blot como se describe en la literatura23,24.</li>
</ol>5. Estadísticas
<ol><li>Utilice ANOVA unidireccional o bidireccional para experimentos con tres o más condiciones seguidas de pruebas post-hoc de Bonferroni para comparaciones entre grupos individuales. Considere un valor p igual o menor que 0.05 significativo. Utilice software (por ejemplo, GraphPad Prism 8.2) para realizar todos los análisis estadísticos.</li>
</ol>

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<li>Cheng, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Temporal+analysis+of+signaling+pathways+activated+in+a+murine+model+of+two-kidney%2C+one-clip+hypertension%5BTitle%5D)+AND+%22American+Journal+of+Physiology-Renal+Physiology%22%5BJournal%5D)">Temporal analysis of signaling pathways activated in a murine model of two-kidney, one-clip hypertension.</a> American Journal of Physiology-Renal Physiology. 297 (4), 1055-1068 (2009).</li>
<li>Wiesel, P., Mazzolai, L., Nussberger, J., Pedrazzini, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Two-kidney%2C+one+clip+and+one-kidney%2C+one+clip+hypertension+in+mice%5BTitle%5D)+AND+%22Hypertension%22%5BJournal%5D)">Two-kidney, one clip and one-kidney, one clip hypertension in mice.</a> Hypertension. 29 (4), 1025-1030 (1997).</li>
<li>Johns, C., Gavras, I., Handy, D. E., Salomao, A., Gavras, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Models+of+experimental+hypertension+in+mice%5BTitle%5D)+AND+%22Hypertension%22%5BJournal%5D)">Models of experimental hypertension in mice.</a> Hypertension. 28 (6), 1064-1069 (1996).</li>
</ol>

Los autores no declaran ningún conflicto de intereses, financieros o de otro tipo.

La estenosis de la arteria renal es una causa importante de hipertensión secundaria o resistente, y lesión renal 1,29. El modelo Goldblatt de dos riñones y un clip (2K1C) se ha empleado para estudiar la hipertensión renovascular inducida por RAStenosis 1,17,18,19. Varios estudios previos con varios modelos animales han demostrado qu...

La estenosis de la arteria renal (RAStenosis) es un problema intratable que afecta a alrededor del 6% de las personas mayores de 65 años y en hasta el 40% de las personas con enfermedad coronaria o vascular periférica 1,2. Los tratamientos actuales para la enfermedad son limitados; por lo tanto, existe una necesidad crítica de desarrollar nuevas terapias para tratar la hipertensión renovascular o la hipertensión resistente inducida por RAStenosis. El sistema renina angiotensina aldosterona (SRAA) es la vía clave implicada en la patogénesis de la hipertensión inducida por RAStenosis o hipertensión renovascular 3,4. Las terapias conocidas dirigidas al SRAA, como los inhibidores de la ECA o los bloqueadores de los receptores de angiotensina, alivian la hipertensión, pero necesitan un examen minucioso para detectar insuficiencia renal e hiperpotasemia 5,6,7. La renina cataliza el paso de limitación de velocidad en RAAS; convierte el angiotensinógeno en angiotensina I. En la aterosclerosis, la formación de placa causa el estrechamiento de la arteria renal que impulsa la secreción de renina, lo que resulta en hipertensión renovascular y daño renal8. Varios estudios han reportado un aumento de los niveles de estrés oxidativo durante la hipertensión renovascular en humanos, que fueron corroborados con el modelo de dos ratones de riñón un clip (2K1C), así como otros modelos animales hipertensos 2,9,10,11,12,13,14,15,16 . El mecanismo molecular del control de la expresión de renina durante la hipertensión renovascular inducida por RAStenosis no se comprende bien y justifica una mayor investigación.
Los modelos animales experimentales que recapitulan RAStenosis de manera confiable y reproducible son importantes para dilucidar los mecanismos celulares y moleculares del control de la expresión de renina para el desarrollo de nuevas terapias. El modelo de ratón 2K1C es un modelo experimental bien establecido para estudiar la patogénesis de la hipertensión renovascular17,18,19,20. Este modelo es generado por la constricción de la arteria renal utilizando un clip 17,20,21, produciendo así la oclusión de la arteria renal que resulta en un aumento de la expresión de renina e hipertensión17,19,20,21. Sin embargo, no hay informes técnicos disponibles, que describen un procedimiento paso a paso para generar estenosis de la arteria renal en modelos animales.
Se han utilizado clips de plata convencionales en forma de U, tubos de poliuretano y otros clips para constreñir la arteria renal para inducir la estenosis de la arteria renal. Algunos estudios han demostrado que el diseño y el material del clip son críticos para obtener datos confiables y reproducibles con el modelo animal 2K1C. Según Lorenz et al., el uso de clips de plata convencionales diseñados en U induce una baja tasa de éxito de la hipertensión arterial (40-60%)21. Debido al diseño del clip, la arteria renal se presiona lateralmente, lo que desencadena algunas constricciones y una mayor probabilidad de desprenderse de la arteria renal. La maleabilidad y ductilidad de la plata pueden permitir cambios en los anchos de los clips; por lo tanto, causando diferentes niveles de hipertensión entre los ratones. Los dióxidos de plata en el clip pueden causar inflamación perivascular, proliferación de la íntima y granulación del tejido, alterando el diámetro de la arteria renal22. Debido a la variabilidad en los niveles de hipertensión obtenidos con el clip de plata convencional U-design, Warner et al. y Lorenz et al. han utilizado con éxito un tubo de poliuretano de diseño más redondo para iniciar la estenosis de la arteria renal en ratones, generando una inducción más confiable y consistente de los dos riñones un clip animal modelo20,21.
En este informe, describimos un protocolo quirúrgico para generar RAStenosis experimental en ratones, utilizando el tubo de poliuretano para constreñir la arteria renal. El manguito de diseño redondo de poliuretano es un clip más reproducible, confiable y de bajo costo para generar estenosis en el ratón. El objetivo de este modelo experimental es estudiar y definir el mecanismo molecular y celular del control de la expresión de renina durante la estenosis de la arteria renal. Confirmamos el éxito del modelo de ratones RAStenosis midiendo la expresión de renina y el marcador de lesión renal de lipocalina asociada a gelatinasa de neutrófilos (N-GAL).

<table><tbody><tr><td>Diet Gel</td><td>Clear H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Diet-Gel 76A</td><td>Surgery recovery diet</td></tr><tr><td>EMC Heated Hard pad</td><td>Hallowell</td><td>000A2788B</td><td>Heating pads were used to keep mice warm</td></tr><tr><td>Ethilon Nylon Suture</td><td>Ethicon</td><td>662G</td><td>4-0 (1.5 metric), This suture was used to close the peritoneum, and skin</td></tr><tr><td>Ethilon Nylon Suture</td><td>Ethicon</td><td>2815 G</td><td>8-0 (0.4 metric), This suture was used to close cuff to tie and constrict the artery</td></tr><tr><td>Germinator 500</td><td>Braintree Scientific Inc.</td><td>GER 5287</td><td>Sterilize surgical tools between surgeries</td></tr><tr><td>Ketoprofen</td><td>Zoetis</td><td>Ketofen</td><td>Painkiller</td></tr><tr><td>Polyurethane</td><td>Braintree Scientific Inc.</td><td>MRE-025</td><td>This tube was used to initiate stenosis</td></tr><tr><td>Povidone-iodine antiseptic swabsticks</td><td>Medline</td><td>MDS093901</td><td>It was applied after hair removal and surgery on the skin</td></tr><tr><td>Reflex 7 Clip Applier</td><td>Roboz Surgical Instrument Co</td><td>204-1000</td><td>This clip applier was used to apply clip in case one or more sutures went off</td></tr><tr><td>Sterile towel drapes</td><td>Dynarex</td><td>4410</td><td>It was used as a bedsheet for mice during surgery</td></tr><tr><td>Triple antibiotic ointment</td><td>Medi-First</td><td>22312</td><td></td></tr><tr><td>Water pump</td><td>Stryker</td><td>T/pump Professionals</td><td>Used to warm and circulate water in the heating hard pad to keep mice warm during and post-surgery</td></tr></tbody></table>

a modified two kidney one clip mouse model of renin regulation in renal artery stenosis

La estenosis de la arteria renal es una afección común en pacientes con enfermedad coronaria o vascular periférica donde el sistema renina angiotensina aldosterona (SRAA) está sobreactivado. En este contexto, hay un estrechamiento de las arterias renales que estimulan un aumento en la expresión y liberación de renina, la proteasa limitante de la velocidad en RAAS. El aumento resultante en la expresión de renina es un impulsor conocido de la hipertensión renovascular, frecuentemente asociada con lesión renal y daño a los órganos terminales. Por lo tanto, existe un gran interés en desarrollar nuevos tratamientos para esta condición. El mecanismo molecular y celular del control de la renina en la estenosis de la arteria renal no se comprende completamente y justifica una mayor investigación. Para inducir estenosis de la arteria renal en ratones, se desarrolló un modelo modificado de ratón Goldblatt de 2 riñones 1 clip (2K1C). El riñón derecho se estenosó en ratones de tipo salvaje y se utilizaron ratones operados con simulacro como control. Después de la estenosis de la arteria renal, determinamos la expresión de renina y la lesión renal. Se recolectaron riñones y se utilizaron cortezas frescas para determinar la expresión de proteínas y ARNm de renina. Este modelo animal es reproducible y se puede utilizar para estudiar respuestas fisiopatológicas, vías moleculares y celulares involucradas en la hipertensión renovascular y la lesión renal.

La constricción de la arteria renal aumenta la expresión de renina en el riñón estenosado mientras reprime la expresión en el riñón contralateral. Los dos riñones de un clip (2K1C) o el modelo de Goldblatt de estenosis inducen un aumento de la expresión de renina y lesión renal. Esto es reconocido como el modelo mejor representativo de estenosis unilateral de la arteria renal en humanos.
La expresión de renina y prorenina (precursor de la renina) se midió mediante inmunotransferen...

Watch this Scientific Journal Video about A Modified Two Kidney One Clip Mouse Model of Renin Regulation in Renal Artery Stenosis at JoVE.com

A Modified Two Kidney One Clip Mouse Model of Renin Regulation in Renal Artery Stenosis

Se desarrolló un modelo modificado de ratón Goldblatt de 2 riñones 1 clip (2K1C) utilizando tubos de poliuretano para iniciar la estenosis de la arteria renal, induciendo un aumento en la expresión de renina y lesión renal. Aquí, describimos un procedimiento detallado de preparación y colocación del manguito en la arteria renal para generar un modelo de ratón 2K1C reproducible y consistente.

Un modelo modificado de dos riñones y un ratón clip de regulación de renina en la estenosis de la arteria renal

Renal artery stenosis is a common condition in patients with coronary or peripheral vascular disease where the renin angiotensin aldosterone system (RAAS) ...

a-modified-two-kidney-one-clip-mouse-model-renin-regulation-renal

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

4.7K vistas.  Vanderbilt University Medical Center. Se desarrolló un modelo modificado de ratón Goldblatt de 2 riñones 1 clip (2K1C) utilizando tubos de poliuretano para iniciar la estenosis de la arteria renal, induciendo un aumento en la expresión de renina y lesión renal. Aquí, describimos un procedimiento detallado de preparación y colocación del manguito en la arteria renal para generar un modelo de ratón 2K1C reproducible y consistente.

Video: A Modified Two Kidney One Clip Mouse Model of Renin Regulation in Renal Artery Stenosis

Use of a Hanging-weight System for Isolated Renal Artery Occlusion

In hospitalized patients, over 50% of cases of acute kidney injury (AKI) are caused by renal ischemia 1-3. A recent study of hospitalized patients revealed that only a mild increase in serum creatinine levels (0.3 to 0.4 mg/dl) is associated with a 70% greater risk of death than in persons without any increase 1. Along these lines, surgical procedures requiring cross-clamping of the aorta and renal vessels are associated with a renal failure rates of up to 30% 4. Similarly, AKI after cardiac surgery occurs in over 10% of patients under normal circumstances and is associated with dramatic increases in mortality. AKI are also common complications after liver transplantation. At least 8-17% of patients end up requiring renal replacement therapy 5. Moreover, delayed graft function due to tubule cell injury during kidney transplantation is frequently related to ischemia-associated AKI 6. Moreover, AKI occurs in approximately 20% of patients suffering from sepsis 6.The occurrence of AKI is associated with dramatic increases of morbidity and mortality 1. Therapeutic approaches are very limited and the majority of interventional trials in AKI have failed in humans. Therefore, additional therapeutic modalities to prevent renal injury from ischemia are urgently needed 3, 7-9. 
To elucidate mechanisms of renal injury due to ischemia and possible therapeutic strategies murine models are intensively required 7-13. Mouse models provide the possibility of utilizing different genetic models including gene-targeted mice and tissue specific gene-targeted mice (cre-flox system). However, murine renal ischemia is technically challenging and experimental details significantly influence results. We performed a systematic evaluation of a novel model for isolated renal artery occlusion in mice, which specifically avoids the use of clamping or suturing the renal pedicle 14. This model requires a nephrectomy of the right kidney since ischemia can be only performed in one kidney due to the experimental setting. In fact, by using a hanging-weight system, the renal artery is only instrumented once throughout the surgical procedure. In addition, no venous or urethral obstruction occurs with this technique. We could demonstrate time-dose-dependent and highly reproducible renal injury with ischemia by measuring serum creatinine. Moreover, when comparing this new model with conventional clamping of the whole pedicle, renal protection by ischemic preconditioning is more profound and more reliable. Therefore his new technique might be useful for other researchers who are working in the field of acute kidney injury.

A precise murine model for acute kidney injury (AKI) due to ischemia is an important tool to investigate acute kidney injury and possibly find therapeutic tools to treat renal injury. The hanging weight system offers a tool for immediate and reliable renal artery occlusion and reperfusion without causing renal congestion.

El uso de un sistema de suspensión de peso para la oclusión de la arteria renal aislada

In hospitalized patients, over 50% of cases of acute kidney injury (AKI) are caused by renal ischemia 1-3. A recent study of hospitalized patients ...

Investigación

Medicina

Renal Ischaemia Reperfusion Injury: A Mouse Model of Injury and Regeneration

Renal ischaemia reperfusion injury (IRI) is a common cause of acute kidney injury (AKI) in patients and occlusion of renal blood flow is unavoidable during renal transplantation. Experimental models that accurately and reproducibly recapitulate renal IRI are crucial in dissecting the pathophysiology of AKI and the development of novel therapeutic agents. Presented here is a mouse model of renal IRI that results in reproducible AKI. This is achieved by a midline laparotomy approach for the surgery with one incision allowing both a right nephrectomy that provides control tissue and clamping of the left renal pedicle to induce ischaemia of the left kidney. By careful monitoring of the clamp position and body temperature during the period of ischaemia this model achieves reproducible functional and structural injury. Mice sacrificed 24 hr&#160;following surgery demonstrate loss of renal function with elevation of the serum or plasma creatinine level as well as structural kidney damage with acute tubular necrosis evident. Renal function improves and the acute tissue injury resolves during the course of 7 days following renal IRI such that this model may be used to study renal regeneration. This model of renal IRI has been utilized to study the molecular and cellular pathophysiology of AKI as well as analysis of the subsequent renal regeneration.

The mouse model of renal ischaemia reperfusion injury described here comprises of a right nephrectomy that provides control tissue and clamping of the left renal pedicle to induce ischaemia that results in acute kidney injury. This model uses a midline laparotomy approach with all steps performed via one incision.

La isquemia reperfusión renal: un modelo de ratón de la lesión y regeneración

Renal ischaemia reperfusion injury (IRI) is a common cause of acute kidney injury (AKI) in patients and occlusion of renal blood flow is unavoidable ...

Transcutaneous Assessment of Renal Function in Conscious Rodents

Glomerular filtration rate (GFR) is the gold standard to assess overall kidney function. However, traditional methods to evaluate GFR are cumbersome and time-consuming. In addition, serial blood or urine samples are required, with the associated stress for the experimental animals. A recent technique significantly reduces the investment in time and resources, minimizing the invasiveness and the animal stress, but being equally valid as the traditional approaches. The method measures transcutaneously renal function. Using an optical device and the exogenous renal marker fluorescein isothiocyanate (FITC)-sinistrin, this technique is capable of measuring the elimination kinetics of the marker through the skin. With neither blood nor urine samples nor the associated laboratory assays needed, the results of the transcutaneous measurement are almost instantaneously available. The method has been already validated in different species and successfully applied in several models of renal pathology. Moreover, due to its minimally invasive characteristics, it is suitable for sequential measurements within the same animal. Here is provided a detailed protocol to carry out the transcutaneous assessment of renal function in rodents.

Determination of glomerular filtration rate (GFR) is the gold standard to assess overall kidney function. However, traditional procedures to measure this parameter are cumbersome and require a large investment of time. Here we describe a faster and minimally invasive method to determinate GFR transcutaneously.

Evaluación transcutánea de la función renal en roedores Conscious

Glomerular filtration rate (GFR) is the gold standard to assess overall kidney function. However, traditional methods to evaluate GFR are cumbersome and ...

Assessment of Kidney Function in Mouse Models of Glomerular Disease

The use of murine models to mimic human kidney disease is becoming increasingly common. Our research is focused on the assessment of glomerular function in diabetic nephropathy and podocyte-specific VEGF-A knock-out mice; therefore, this protocol describes the full kidney work-up used in our lab to assess these mouse models of glomerular disease, enabling a vast amount of information regarding kidney and glomerular function to be obtained from a single mouse. In comparison to alternative methods presented in the literature to assess glomerular function, the use of the method outlined in this paper enables the glomerular phenotype to be fully evaluated from multiple aspects. By using this method, the researcher can determine the kidney phenotype of the model and assess the mechanism as to why the phenotype develops. This vital information on the mechanism of disease is required when examining potential therapeutic avenues in these models. The methods allow for detailed functional assessment of the glomerular filtration barrier through measurement of the urinary albumin creatinine ratio and individual glomerular water permeability, as well as both structural and ultra-structural examination using the Periodic Acid Schiff stain and electron microscopy. Furthermore, analysis of the genes dysregulated at the mRNA and protein level enables mechanistic analysis of glomerular function. This protocol outlines the generic but adaptable methods that can be applied to all mouse models of glomerular disease.

This protocol describes a full kidney work-up that should be carried out in mouse models of glomerular disease. The methods allow for detailed functional, structural, and mechanistic analysis of glomerular function, which can be applied to all mouse models of glomerular disease.

Evaluación de la función renal en modelos de ratón de la enfermedad Glomerular

The use of murine models to mimic human kidney disease is becoming increasingly common. Our research is focused on the assessment of glomerular function ...

An Effective Mouse Model of Unilateral Renal Ischemia-Reperfusion Injury

Ischemia-reperfusion injury (IRI) is the leading cause of acute renal failure and is a significant contributor to delayed graft function. Animal models are the only available resources that mimic the complexities of the IRI-associated damage encountered in vivo. This paper describes an effective mouse model of unilateral renal IRI that delivers highly reproducible data. Ischemia is induced by occluding the right renal pedicle for 30 min followed by reperfusion. In addition to the surgical procedure, a sequential overview of the expected physiological and histopathological changes following renal IRI will be provided by comparing data from seven different reperfusion times (4 h, 8 h, 16 h, 1 day, 2 days, 4 days, and 7 days). Critical data for planning experiments ahead, such as mean surgical time, average anesthetic consumption, and body weight changes over time, will be shared. This work will help researchers implement a reliable renal IRI model and select the appropriate reperfusion time that aligns with their intended investigative goals.

Renal ischemia-reperfusion injury is associated with high morbidity and mortality in hospitalized patients. Here, we present a simple and effective mouse model of unilateral renal ischemia-reperfusion injury and provide a sequential overview of representative pathological changes observed in the kidney.

Un modelo de ratón eficaz de lesión unilateral por isquemia-reperfusión renal

Ischemia-reperfusion injury (IRI) is the leading cause of acute renal failure and is a significant contributor to delayed graft function. Animal models ...

A Murine Model of Hemodialysis Access-Related Hand Dysfunction

Chronic kidney disease is a major public health problem, and the prevalence of end-stage renal disease (ESRD) requiring chronic renal replacement therapies such as hemodialysis continues to increase. Autogenous arteriovenous fistula (AVF) placement remains a primary vascular access option for ESRD patients. Unfortunately, approximately half of the hemodialysis patients experience dialysis access-related hand dysfunction (ARHD), ranging from subtle paresthesia to digital gangrene. Notably, the underlying biologic drivers responsible for ARHD are poorly understood, and no adequate animal model exists to elucidate the mechanisms and/or develop novel therapeutics for the prevention/treatment of ARHD. Herein, we describe a new mouse model in which an AVF is created between the left common iliac artery and vein, thereby facilitating the assessment of limb pathophysiology. The microsurgery includes vessel isolation, longitudinal venotomy, creation of arteriovenous anastomosis, and venous reconstruction. Sham surgeries include all the critical steps except for AVF creation. Iliac AVF placement results in clinically relevant alterations in central hemodynamics, peripheral ischemia, and impairments in hindlimb neuromotor performance. This novel preclinical AVF model provides a useful platform that recapitulates common neuromotor perturbations reported by hemodialysis patients, allowing researchers to investigate the mechanisms of ARHD pathophysiology and test potential therapeutics.

This protocol details the surgical steps of murine common iliac arteriovenous fistula creation. We developed this model to study hemodialysis access-related limb pathophysiology.

Un modelo murino de disfunción de la mano relacionada con el acceso a la hemodiálisis

Chronic kidney disease is a major public health problem, and the prevalence of end-stage renal disease (ESRD) requiring chronic renal replacement ...

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 ...

Biología

A Mouse 5/6th Nephrectomy Model That Induces Experimental Uremic Cardiomyopathy

Chronic kidney disease (CKD) is a great risk factor for cardiovascular disease events and mortality, and progressively develops to the clinical phenotype called "uremic cardiomyopathy". We describe here an experimental CKD mouse model, named 5/6th partial nephrectomy (PNx) with pole ligation, which developed uremic cardiomyopathy at four weeks post-surgery. This PNx model was performed by a two-step surgery. In step-one surgery, both poles of the left kidney were ligated. In step-two surgery, which was performed 7 days after the step-one surgery, the right kidney was removed. For the sham surgery, the same surgery procedures were performed but without pole ligation of the left kidney or removal of the right kidney. The surgical procedures are easier and less time-consuming, compared to other methods. However, the remnant functional renal mass is not as easily controlled as the renal artery ligation. Four weeks after surgery, in comparison with the sham-operated mice, the PNx mice developed impaired renal function, anemia, cardiac hypertrophy, cardiac fibrosis, and decreased heart systolic and diastolic function.

A Mouse 5/6th Nephrectomy Model That Induces Experimental Uremic Cardiomyopathy

This manuscript provides a detailed two-step surgical procedure to perform mouse 5/6th partial nephrectomy (PNx) with pole ligation. Four weeks after surgery, in comparison with sham-operated mice, the PNx mice developed impaired renal function, anemia, cardiac hypertrophy, cardiac fibrosis, and decreased heart systolic and diastolic function.

Un modelo de nefrectomía de ratón 5/6th que induce miocardiopatía urémica Experimental

Chronic kidney disease (CKD) is a great risk factor for cardiovascular disease events and mortality, and progressively develops to the clinical phenotype ...

<meta charset="utf8"></head><body>Nefrectomía estandarizada 5/6 en ratas para insuficiencia renal y malperfusión

5/6 Nephrectomy Using Sharp Bipolectomy Via Midline Laparotomy in Rats

Chronic kidney disease (CKD) affects over 10% of the global population, amounting to more than 800 million individuals worldwide. The advances in the treatment of CKD have had a significant impact on patient outcomes. While in the past, CKD was often considered a death sentence, with many patients succumbing to the complications of end-stage renal disease, it is now increasingly being managed as a chronic condition with the availability of dialysis and kidney transplantation, as well as new pharmaceutical developments such as SGLT2 inhibitors or nonsteroidal mineralocorticoid receptor antagonists. 
Yet, there continues to be a growing demand for further exploration into the pathophysiological processes and potential therapeutic interventions. Reliable biological models play a crucial role in facilitating this research. Given the multifaceted nature of renal disease, which encompasses not only cell biology but also vascular microanatomy and endocrine signaling, an appropriate model must possess a level of biological complexity that only an animal model can offer, rendering rodents an obvious choice.
This manuscript, therefore, provides an intricate, systematic protocol for surgically reducing renal parenchyma through midline laparotomy and combined total and partial nephrectomy in rats for survival as well as non-survival applications. It emphasizes the critical role of precise surgical techniques in ensuring consistent and reliable outcomes. Prime examples of potential applications for this model include biomolecular and pharmaceutical studies as well as the development of innovative intraoperative imaging modalities, such as hyperspectral imaging, to objectively visualize and differentiate renal malperfusion.

<meta charset="utf8"></head><body>5/6 Nefrectomía con bipolectomía aguda mediante laparotomía de línea media en ratas

This manuscript introduces a standardized protocol for a 5/6 nephrectomy by sharp bipolectomy via midline laparotomy in a rat model, aiming to induce a state of renal insufficiency via renal parenchyma reduction with a great degree of methodical precision and low risk of technical error.

5/6 Nefrectomía con bipolectomía aguda mediante laparotomía de línea media en ratas

Chronic kidney disease (CKD) affects over 10% of the global population, amounting to more than 800 million individuals worldwide. The advances in the ...

Un modelo modificado de dos riñones y un ratón clip de regulación de renina en la estenosis de la arteria renal

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