Name: the mouse isolated perfused kidney technique
Uploaded: 2016-11-17T14:00:00.000+00:00
Duration: 8 min 19 s
Description: Watch this Scientific Journal Video about The Mouse Isolated Perfused Kidney Technique at JoVE.com

The authors would like to thank Hans-Joachim Schurek for invaluable scientific advice. The authors would like to thank Monique Carrel and Mich&#232;le Heidemeyer for excellent technical assistance, David Penton Ribas and Nourdine Faresse for a critical reading of the manuscript and Carsten Wagner and J&#252;rg Biber for the NaPi-2a antibody. This work was supported by the Swiss National Centre for Competence in Research "Kidney.CH" and by a project grant (310030_143929/1) from the Swiss National Science Foundation.

Tutte le procedure che coinvolgono gli animali descritti in questo manoscritto sono stati condotti secondo il diritto svizzero e approvati dall&#39;amministrazione veterinario del Cantone di Zurigo, Svizzera. 1. Preparazione Buffer <ol><li> Preparare soluzioni 1 - 4 e la soluzione ormone antidiuretico (ADH) (Tabella 1). </li><li> Preparare il tampone di dialisi (Tabella 1). NOTA: Questo è il buffer utilizzato come tampone di dialisi durante la perfusione. Successivamente, gli eritrociti vengono diluiti in questo tampone per formare il perfusato finale. </li><li> preparazione degli eritrociti. <ol><li> Diluire 250 ml di concentrato di eritrociti umani (materiale testato ottenuto dalla banca locale del sangue) a 500 ml con tampone di dialisi. Centrifugare a 2.000 g per 8 minuti. Rimuovere il tampone, facendo attenzione a non rimuovere eventuali eritrociti. Ripetere 3x. </li></ol></li><li> Preparare la (albumina sierica bovina; BSA) l&#39;albumina del buffer. <ol><li> In 200 ml di dialysibuffer di s, sciogliere 44 g di BSA con un ancoretta. Filtrare la soluzione con carta da filtro. </li></ol></li><li> Preparare il perfusato. <ol><li> Filtrare le eritrociti dal punto 1.3.1 attraverso carta da filtro nel buffer BSA. Riempire fino ad un volume totale di 800 ml con tampone di dialisi. NOTA: Questo è il perfusato finale. L&#39;ematocrito dovrebbe essere compresa tra 8 e 12%. Il perfusato può essere conservato per un massimo di 12 ore a 4 ° C. </li></ol></li></ol> 2. Avvio Dialisi e l&#39;ossigenazione <ol><li> Accendere bagnomaria circonda il serbatoio più grande del buffer, più piccolo serbatoio tampone, e la camera umida (una piccola camera doppia parete portata a 37 ° C e 100% di umidità per mantenere entro il rene) a 37 ° C (figura 2) . </li><li> Riempire il serbatoio buffer maggiore con il tampone di dialisi e il serbatoio più piccolo con perfusato. </li><li> Accendere il 5% di CO 2/95% O 2 afflusso di gas al tampone di dialisi. </li><li> Accendere dialisi continua del perfusato contro il tampone di dialisi. Fate attenzione ad usare a basso flusso tubo di dialisi. Passare al punto 3. </li></ol><img alt="figura 2" src="/files/ftp_upload/54712/54712fig2.jpg" /> Figura 2: Schema del circuito di perfusione. Schema mostra i principali componenti del circuito di perfusione e la direzione del flusso buffer. Tutti i componenti circondato da blu scuro vengono mantenute a 37 ° C con un bagno di acqua / termostato. 1: Dialisi tampone di almeno 3 volte il volume del buffer di perfusione è continuamente gorgogliare con 95% O 2/5% di CO 2. 2: Dialisi buffer e buffer di perfusione sono continuamente dializzati contro l&#39;altro in un tubo di dialisi da una pompa a rulli. 3: througho costante Per questo la dialisi, il buffer di perfusione è arricchito con 9% O 2/5% di CO 2 ed elettroliti livelli sono mantenutiut perfusione. 4: Una pompa rullo spinge il buffer di perfusione verso il rene. 5: Un windkessel rimuove onde peristaltiche e agisce come una trappola bolla. 6: trasduttore di pressione (collegato a 4. (pompa roller) per mantenere una pressione continua pur consentendo flusso liberamente alternata). 7: Durante la perfusione, il rene rimane in una camera umida per 100% di umidità e temperatura di 37 ° C renale. <a href="http://ecsource.jove.com/files/ftp_upload/54712/54712fig2large.jpg" target="_blank">Clicca qui per vedere una versione più grande di questa figura.</a> 3. Procedura chirurgica Part 1 (per un diagramma di tutte le legature, vedi Figura 3) Nota: Eseguire tutte le legature utilizzando 5-0 filo chirurgico. <ol><li> Anestetizzare un topo mediante iniezione intraperitoneale (10 ml / g di peso corporeo, 20 mg / ml di ketamina e 1 mg / ml xilazina disciolto in 0,9% NaCl). Confermare la sufficiente profondità di AnesthESIA testando per l&#39;assenza di riflessi retro-piede. </li><li> Fissare il mouse in posizione supina nella camera umida. Proteggere gli occhi con vet unguento. Inserire una siringa da 1 ml di sotto della colonna vertebrale per elevare i vasi lombari. </li><li> Eseguire una laparotomia mediana dalla cresta pubica per l&#39;apertura dello sterno prima la pelle, poi i muscoli addominali, con le forbici. </li><li> Rimuovere l&#39;intestino e posizionarlo sul lato sinistro del laterali del mouse dall&#39;addome. </li><li> Liberare la vescica da tessuto connettivo e di esplorare entrambi gli ureteri e l&#39;uretra. </li><li> Mettere una legatura intorno dell&#39;uretere sinistro (I legature). Chiudilo. </li><li> Inserire una legatura intorno all&#39;uretra (legature II). Chiudilo. </li><li> Posizionare un &quot;laccio&quot; legatura in giro per tutta la vescica (legature III). </li><li> Incidere la vescica 1 mm. </li><li> Incannulare l&#39;apertura cm 2 PE 50 tubi. </li><li> Chiudi legatura III attorno al tubo. </li><li> Tagliare l&#39;uretere sinistro e dell&#39;uretra distale dalle legature. il bladder è ora attaccato al dell&#39;uretere solo a destra e liberi di muoversi. </li><li> Cancellare l&#39;aorta addominale del tessuto connettivo e il grasso. </li><li> Inserire un addominale legatura metà dell&#39;aorta (legature IV). </li><li> Inserire una legatura intorno all&#39;aorta sotto il diaframma tra l&#39;arteria mesenterica superiore e il tronco celiaco (legature V). </li><li> Mettere una legatura intorno all&#39;arteria mesenterica superiore (legature VI). </li><li> Inserire un legatura aortica direttamente sotto destra e sopra l&#39;arteria renale sinistra (VII legatura). </li><li> Mettere una legatura attorno al pacchetto caudale vena (cava) (VIII legature). Passare al punto 4. </li></ol><img alt="Figura 3" src="/files/ftp_upload/54712/54712fig3.jpg" /> Figura 3: Schema del Legature poste durante l&#39;intervento chirurgico. Vista dell&#39;addome aperto dopo la laparotomia. L&#39;intestino viene spostato verso sinistra. L e R indicano il rene sinistro e destro. Illinee nere mostrano l&#39;area della rispettiva legatura. Legature prima immissione e poi chiusi, nella sequenza indicata nel testo. X indica la posizione di incisione per dell&#39;aorta cannulazione. <a href="http://ecsource.jove.com/files/ftp_upload/54712/54712fig3large.jpg" target="_blank">Clicca qui per vedere una versione più grande di questa figura.</a> 4. Adescamento del circuito di perfusione <ol><li> Avviare la pompa rotativa e riempire il tubo con perfusato. Fare attenzione a svuotare tutte le bolle d&#39;aria da esso. </li><li> Riempire il dispositivo windkessel a circa la metà del livello con perfusato. </li><li> Calibrare il trasduttore di pressione a 0 mm Hg quando tutti i tubi è riempita e il flusso è 0. Tenere l&#39;ago perfusione a livello renale durante questo periodo. </li><li> Mantenere il flusso ad un livello minimo costante (0,6 ml / min) e passare al punto 5. </li></ol> 5. Procedura chirurgica Parte 2 <ol><li> Posizionare un morsetto tra la legatura IV e la ramificazione della rena sinistral arteria. </li><li> Fai una piccola incisione nella caudale dell&#39;aorta di legatura IV, avendo cura di non tagliare la parete dorsale. </li><li> Dilata l&#39;apertura in aorta con un dilatatore vascolare. </li><li> Incannulare l&#39;aorta con un ago (2 cm di lunghezza, tirato PE 50), spingendo la punta solo al morsetto. </li><li> Aprire il morsetto. </li><li> Inserire la punta di un ago cranialmente fino a raggiungere il bivio dell&#39;arteria rene destro e l&#39;aorta. </li><li> Chiudere la legatura VII. </li><li> Chiudere la legatura IV. </li><li> Aprite la cassa con le forbici dalla dissezione del diaframma. Con un unico taglio, separare l&#39;aorta, vena cava, cuore e nervi vegetativi. Con questo passo, l&#39;animale viene sacrificato via rapida dissanguamento in anestesia profonda continua. </li><li> Avviare il controllo della pressione della pompa di perfusione. Mantenere la pressione media tra 80 e 100 mmHg. </li><li> Chiudi V. legatura </li><li> Chiudere la legatura VI. </li><li> Chiudere la legatura VIII. </li><li> Libero il diritto rene da tissu connettivoe e la sua incorporazione nella capsula adiposa con le forbici. </li><li> Tagliare l&#39;aorta prossimale di legatura V. </li><li> Tagliare l&#39;arteria mesenterica superiore distale di legatura VI. </li><li> Tagliare il fascio nave rene-out di supporto, avendo cura di non tagliare in vasi stessi. </li><li> Tagliare il fegato al collegamento al rene. Fare attenzione per liberare il rene, ma lasciare una piccola parte del aderenti fegato ad esso, in modo che la vena cava viene mantenuto aperto da esso. </li><li> Prendere il fascio di rene di topo. Rimuovere il mouse dalla camera umida. </li><li> Posizionare un &quot;laccio&quot; legatura attorno al collegamento di fegato e rene (legature IX). </li><li> Incannulare la vena cava con una linea venosa (2 centimetri PE 50). </li><li> Chiudere la legatura IX. deflusso venoso attraverso la linea venosa dovrebbe iniziare immediatamente. </li><li> Chiudere la camera umida. </li></ol> 6. Le analisi a valle <ol><li> Durante l&#39;ora successiva, monitorare continuamente il flusso di sangue e intravasculpressione ar 15. Raccogliere deflusso venoso, che può essere utilizzato per l&#39;analisi, ad esempio, renale rilascio di renina 7. Raccogliere le urine per l&#39;analisi della concentrazione di elettroliti e velocità di filtrazione glomerulare 14. Dopo 1 ora di perfusione, i reni possono essere snap-congelati per blotting occidentale o essere fissati per l&#39;imaging approcci 16. </li></ol>

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The authors have no competing financial interests and nothing else to disclose.

Il mouse isolato rene perfuso è uno strumento per lo studio della funzione renale in un ambiente controllato ex-vivo per 1 ora, colmando il divario tra esperimenti in vivo su animali intatti, che può essere viziata da l&#39;impatto di numerosi fattori sistemici, e in esperimenti in vitro in nefrone isolati o cellule in coltura, che necessariamente trascurare l&#39;impatto della struttura dell&#39;organo intatta sulla funzione. C&#39;è, alla conoscenza degli autori, non tecnica alte...

La perfusione isolata di organi è stato oggetto di uno sforzo continuo tra fisiologi per molti decenni 1. La tecnica consente la funzione dell&#39;organo, senza influenze sistemiche come la pressione sanguigna, ormoni, o nervi, da studiare. Carl Eduard Loebell è considerato il primo ad aver descritto la perfusione successo di un rene isolato, nel 1849 2. Da allora, l&#39;apparato perfusione ha subito notevole raffinatezza. Frey e Gruber ha introdotto un polmone artificiale per le pompe di ossigenazione e pulsatile per continua perfusione 2. Mentre i primi ricercatori hanno studiato principalmente i reni di grandi mammiferi, cioè, maiali e cani 2 3 -la prima relazione l&#39;uso di reni di ratto, di Weiss et al. , È stata una pietra miliare nello studio della perfusione piccolo-mammifero-organo 4. Schurek et al. riferito la necessità di aggiungere eritrociti di mammifero al perfusato se tubulare renale sufficienteossigenazione doveva essere raggiunto 5. Critico per esperimenti di lunga durata è stata l&#39;introduzione della dialisi continua del buffer dallo stesso gruppo di ricerca 6. Nel 2003, Schweda et al. sono stati i primi a segnalare un rene del mouse funzionale isolato perfuso (MIPK) 7, in seguito perfezionato da Rahgozar et al. 18 e Lindell et al. 14. Mentre tecnicamente più impegnativo rispetto al ratto isolato rene perfuso, l&#39;uso del MIPK porta il vantaggio di permettere l&#39;uso di una vasta gamma di topi geneticamente modificati. Questo documento presenta i dettagli del metodo degli autori per la perfusione isolata reni del mouse per 1 ora. Il metodo consente la valutazione continua della velocità di flusso renale, resistenza vascolare, rilascio di ormone, emogasanalisi, analisi delle urine, e l&#39;applicazione di farmaci. Seguendo la procedura, reni potrebbero essere trattati per analisi molecolari e biochimiche, fissati per la microscopia, otrapiantato in un topo ricevente (Figura 1). <img alt="Figura 1" src="/files/ftp_upload/54712/54712fig1.jpg" /> Figura 1: panoramica dei possibili Input / Output al rene isolato e perfuso. BGA: analisi dei gas del sangue. <a href="http://ecsource.jove.com/files/ftp_upload/54712/54712fig1large.jpg" target="_blank">Clicca qui per vedere una versione più grande di questa figura.</a> Questa tecnica probabile che riceverà maggiore attenzione nei prossimi anni, come molte applicazioni innovative sono in discussione con l&#39;alba di una prolungata perfusione del rene normotermico prima del trapianto (con o senza l&#39;applicazione di anti-rigetto o droghe genoma-editing) 8, 9, 10 , 11, la bioingegneria di interi reni da impalcature decellularized 12, e l&#39;applicazione di elevate dosi di coloranti fluorescenti per l&#39;imaging multiphoton 13 </sup&gt;. E &#39;anche un modello ideale con cui studiare il ruolo di specifici geni durante il danno renale acuto 14. Un protocollo step-by-step è dato per consentire ad altri laboratori di eseguire isolato mouse perfusione del rene con successo. Innanzitutto, è specificato la composizione e preparazione del buffer. Quindi, l&#39;intervento è descritto in dettaglio e sono presenti i punti critici. In terzo luogo, i dati vengono presentati che sono rappresentativi di una preparazione di successo: il flusso ematico renale, la resistenza vascolare, velocità di filtrazione glomerulare, e degli elettroliti frazionaria misurazioni funzionali di vitalità e di elettroni trasmissione micrografie della morfologia dei diversi segmenti nefrone di reni perfusi tutti escrezione- fisso dopo 1 ora di perfusione.

<table><tbody><tr><td>&lt;strong&gt;Perfusion Circuit:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Moist chamber 834/8</td><td>Harvard Apparatus/Hugo Sachs Elektronik GmbH</td><td>73-2901</td><td></td></tr><tr><td>Cannular with basket and side port</td><td>Harvard Apparatus/Hugo Sachs Elektronik GmbH</td><td>73-2947</td><td></td></tr><tr><td>Thermostat TC120-ST5&amp;nbsp;</td><td>Harvard Apparatus/Hugo Sachs Elektronik GmbH</td><td>73-4544</td><td></td></tr><tr><td>ISM 827/230V Roller Pump Reglo Analogue</td><td>Harvard Apparatus/Hugo Sachs Elektronik GmbH</td><td>73-0114</td><td></td></tr><tr><td>Reservoir jacketed for buffer solution 1 L</td><td>Harvard Apparatus/Hugo Sachs Elektronik GmbH</td><td>73-3438</td><td></td></tr><tr><td>Reservoir jacketed for buffer solution 0.5 L</td><td>Harvard Apparatus/Hugo Sachs Elektronik GmbH</td><td>73-3436</td><td></td></tr><tr><td>Pressure Transducer APT300&amp;nbsp;</td><td>Harvard Apparatus/Hugo Sachs Elektronik GmbH</td><td>73-3862</td><td></td></tr><tr><td>TAM-D Plugsys Transducer</td><td>Harvard Apparatus/Hugo Sachs Elektronik GmbH</td><td>73-1793</td><td></td></tr><tr><td>SCP Plugsys servo controller</td><td>Harvard Apparatus/Hugo Sachs Elektronik GmbH</td><td>73-2806</td><td></td></tr><tr><td>Windkessel&amp;nbsp;</td><td>Harvard Apparatus/Hugo Sachs Elektronik GmbH</td><td>73-3717</td><td></td></tr><tr><td>HSE-USB data acquisition</td><td>Harvard Apparatus/Hugo Sachs Elektronik GmbH</td><td>73-3330</td><td></td></tr><tr><td>Low-Flux Dialysator Diacap Polysulfone</td><td>B.Braun</td><td>7203525</td><td></td></tr><tr><td>PE-Tubing for aorta cannulation 1.19 mm I.D. x 1.70 mm O.D.</td><td>Scientific Commodities Inc.</td><td>BB31695-PE/8</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Buffer reagents:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Aminoplasmal 10%</td><td>B.Braun</td><td>134518064</td><td></td></tr><tr><td>Sodium pyruvate</td><td>Sigma-Aldrich</td><td>P2256-25G</td><td></td></tr><tr><td>L-Glutamic acid monosodium salt hydrate</td><td>Sigma-Aldrich</td><td>G1626-100G</td><td></td></tr><tr><td>L-(-)-Malic acid sodium salt</td><td>Sigma-Aldrich</td><td>M1125-25G</td><td></td></tr><tr><td>Sodium-L-Lactate</td><td>Sigma-Aldrich</td><td>L7022-10G</td><td></td></tr><tr><td>alpha-Ketoglutaric acid sodium salt</td><td>Sigma-Aldrich</td><td>K1875-25G</td><td></td></tr><tr><td>NaCl</td><td>Sigma-Aldrich</td><td>31434-1KG-R</td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>S5761-5KG</td><td></td></tr><tr><td>KCl</td><td>Sigma-Aldrich</td><td>60130-1KG</td><td></td></tr><tr><td>Urea</td><td>Sigma-Aldrich</td><td>U5378-500G</td><td></td></tr><tr><td>Creatinine</td><td>Sigma-Aldrich</td><td>C4255-10G</td><td></td></tr><tr><td>Ampicillin</td><td>Roche</td><td>10835242001</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt; * 6H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma-Aldrich</td><td>M2393-500G</td><td></td></tr><tr><td>D-Glucose</td><td>Sigma-Aldrich</td><td>G8270-1KG</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt; * 6H&lt;sub&gt;2&lt;/sub&gt;O&amp;nbsp;</td><td>Riedel-de-Ha&amp;euml;n</td><td>12074</td><td></td></tr><tr><td>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>S9638-500G</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>S0876-500G</td><td></td></tr><tr><td>Antidiuretic Hormone dDAVP</td><td>Sigma-Aldrich</td><td>V2013-1MG</td><td></td></tr><tr><td>FITC-Inulin</td><td>Sigma-Aldrich</td><td></td><td></td></tr><tr><td>Filter used for erythrocyte filtration</td><td>Macherey-Nagel</td><td>MN 615</td><td></td></tr><tr><td>BGA Analysis:</td><td></td><td></td><td></td></tr><tr><td>ABL 80 flex</td><td>Radiometer Medical ApS</td><td></td><td></td></tr><tr><td>Electron Microscope:</td><td></td><td></td><td></td></tr><tr><td>Philips CM100 TEM</td><td>FEI</td><td></td><td></td></tr></tbody></table>

the mouse isolated perfused kidney technique

L&#39;isolato rene perfuso mouse (MIPK) è una tecnica per mantenere un rene mouse sotto ex vivo condizioni perfusi e funzionali per 1 ora. Questo è un prerequisito per lo studio della fisiologia dell&#39;organo isolata e per molte applicazioni innovative che possono essere possibile in futuro, tra perfusione decellularization per la bioingegneria reni o la somministrazione di anti-rigetto o droghe genoma-editing in dosi elevate per innescare il rene per il trapianto. Durante il tempo della perfusione, il rene può essere manipolato, funzione renale può essere valutata, e vari farmaci somministrati. Dopo la procedura, il rene può essere trapiantato o trasformati per la biologia molecolare, l&#39;analisi biochimica, o al microscopio. Questo articolo descrive il perfusato e la tecnica chirurgica necessaria per la perfusione ex vivo di reni mouse. Dettagli dell&#39;apparato perfusione sono date e dati sono presentati mostra la vESPONSABILITÀ di preparazione del rene: il flusso ematico renale, la resistenza vascolare, e delle urine dati come, microscopio elettronico di trasmissione funzionali dei diversi segmenti del nefrone come letture morfologiche, e le macchie occidentali di proteine ​​di trasporto dei diversi segmenti del nefrone come lettura molecolare.

Con il metodo descritto, isolati reni mouse possono rimanere vitali per almeno 1 ora. Abbiamo testato la vitalità del tessuto dopo 1 ora di perfusione continua con funzionale (flusso ematico renale e la resistenza vascolare, analisi dei gas del sangue del deflusso venoso, velocità di filtrazione glomerulare, urinario frazionata Na + e K + escrezione, e nelle urine osmolalità) e morfologica (elettronico a trasmissione microscopia, TEM) metodi in quattro reni di ti...

Watch this Scientific Journal Video about The Mouse Isolated Perfused Kidney Technique at JoVE.com

The Mouse Isolated Perfused Kidney Technique

L&#39;isolato rene perfuso mouse (MIPK) è una tecnica per mantenere un rene mouse sotto ex vivo condizioni perfusi e funzionali per 1 ora. I buffer e tecnica chirurgica sono descritte in dettaglio.

La tecnica del rene perfusi mouse isolato

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 ...

the-mouse-isolated-perfused-kidney-technique

Research

JoVE Journal

Biology

21.6K Views.  University of Zürich.  L'isolato rene perfuso mouse (MIPK) è una tecnica per mantenere un rene mouse sotto ex vivo condizioni perfusi e funzionali per 1 ora. I buffer e tecnica chirurgica sono descritte in dettaglio. 

Video: The Mouse Isolated Perfused Kidney Technique

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.

L&#39;uso di un Hanging-peso di sistema per isolati di occlusione dell&#39;arteria renale

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 ...

Ricerca

Medicina

Isolation of Glomeruli and In Vivo Labeling of Glomerular Cell Surface Proteins

Proteinuria results from the disruption of the glomerular filter that is composed of the fenestrated endothelium, glomerular basement membrane, and podocytes with their slit diaphragms. The delicate structure of the glomerular filter, especially the slit diaphragm, relies on the interplay of diverse cell surface proteins. Studying these cell surface proteins has so far been limited to in vitro studies or histologic analysis. Here, we present a murine in vivo biotinylation labeling method, which enables the study of glomerular cell surface proteins under physiologic and pathophysiologic conditions. This protocol contains information on how to perfuse mouse kidneys, isolate glomeruli, and perform endogenous immunoprecipitation of a protein of interest. Semi-quantitation of glomerular cell surface abundance is readily available with this novel method, and all proteins accessible to biotin perfusion and immunoprecipitation can be studied. In addition, isolation of glomeruli with or without biotinylation enables further analysis of glomerular RNA and protein as well as primary glomerular cell culture (i.e., primary podocyte cell culture).

Isolation of Glomeruli and In Vivo Labeling of Glomerular Cell Surface Proteins

Here we present a protocol for murine in vivo labeling of glomerular cell surface proteins with biotin. This protocol contains information on how to perfuse mouse kidneys, isolate glomeruli, and perform endogenous immunoprecipitation of the protein of interest.

Isolamento dei Glomeruli e In Vivo di etichettatura delle proteine di superficie delle cellule glomerulari

Proteinuria results from the disruption of the glomerular filter that is composed of the fenestrated endothelium, glomerular basement membrane, and ...

Biochimica

Mouse Kidney Transplantation: Models of Allograft Rejection

Rejection of the transplanted kidney in humans is still a major cause of morbidity and mortality. The mouse model of renal transplantation closely replicates both the technical and pathological processes that occur in human renal transplantation. Although mouse models of allogeneic rejection in organs other than the kidney exist, and are more technically feasible, there is evidence that different organs elicit disparate rejection modes and dynamics, for instance the time course of rejection in cardiac and renal allograft differs significantly in certain strain combinations. This model is an attractive tool for many reasons despite its technical challenges. As inbred mouse strain haplotypes are well characterized it is possible to choose donor and recipient combinations to model acute allograft rejection by transplanting across MHC class I and II loci. Conversely by transplanting between strains with similar haplotypes a chronic process can be elicited were the allograft kidney develops interstitial fibrosis and tubular atrophy. We have modified the surgical technique to reduce operating time and improve ease of surgery, however a learning curve still needs to be overcome in order to faithfully replicate the model. This study will provide key points in the surgical procedure and aid the process of establishing this technique.

Here, we present a protocol to study the immunology of rejection. The surgical model presented reports a short operating time and a concise technique. Depending on the donor-recipient strain combination, the transplanted kidney may develop acute cellular rejection or chronic allograft damage, defined by interstitial fibrosis and tubular atrophy.

Mouse trapianto renale: Modelli di Allograft Rifiuto

Rejection of the transplanted kidney in humans is still a major cause of morbidity and mortality. The mouse model of renal transplantation closely ...

Reliable and High Efficiency Extraction of Kidney Immune Cells

Immune system activation occurs in multiple kidney diseases and pathophysiological processes. The immune system consists of both adaptive and innate components and multiple cell types. Sometimes, the cell type of interest is present in very low numbers among the large numbers of total cells isolated from the kidney. Hence, reliable and efficient isolation of kidney mononuclear cell populations is important in order to study the immunological problems associated with kidney diseases. Traditionally, tissue isolation of kidney mononuclear cells have been performed via enzymatic digestions using different varieties and strengths of collagenases/DNAses yielding varying numbers of viable immune cells. Recently, with the development of the mechanical tissue disruptors for single cell isolation, the collagenase digestion step is avoided and replaced by a simple mechanical disruption of the kidneys after extraction from the mouse. Herein, we demonstrate a simple yet efficient method for the isolation of kidney mononuclear cells for every day immune cell extractions. We further demonstrate an example of subset analysis of immune cells in the kidney. Importantly, this technique can be adapted to other soft and non-fibrous tissues such as the liver and brain.

Techniques that are reliable and efficient for the isolation of kidney immune cells are needed for downstream applications. This requires surface antibody labeling of a small number of kidney immune cells. Herein, we describe a concise method for isolation of kidney immune cells that seemingly achieves this goal.

Affidabile e di alta efficienza di estrazione del rene cellule immunitarie

Immune system activation occurs in multiple kidney diseases and pathophysiological processes.  The immune system consists of both adaptive and innate ...

Immunologia e infezioni

Comparative Proteomic Analysis of Whole Kidney, Medulla, and Cortical Tubules in Diabetic Pathogenesis of Kidney Injury in Mice

Defining the sequence of events in renal disease is the cornerstone of clinical practice in the nephrologist toolkit. Tissue proteomic analyses are a significant approach to understanding the fundamental physiological and molecular processes of renal pathophysiology. The methods and protocols we present here will allow for the molecular dissection of the kidney in each specific region of interest related to disease sequelae. To determine the effects of disease on specific kidney regions and structures with unique functions, the goals of this protocol are to demonstrate simplified mouse kidney compartmentalization and renal cortical tubule isolation techniques in tandem with streamlined label-free quantitative proteomic workflows. Combining these methods will assist in the identification of perturbed molecular patterns in the whole kidney, medullary compartments, and cortical tubule structures of kidneys, with the ultimate and eventual goal of single-cell proteomics in pathological contexts. Applying these methods in virtually any disease model will be helpful in delineating mechanisms of pathology related to kidney dysfunction.

Here, we present a detailed protocol for proteomic analysis of the whole kidney, isolated cortical tubule, and medullary proteomes. The study also compares regional proteomes in a diabetic mouse model and non-diabetic mice.

Analisi proteomica comparativa di interi reni, midollo e tubuli corticali nella patogenesi diabetica del danno renale nei topi

Defining the sequence of events in renal disease is the cornerstone of clinical practice in the nephrologist toolkit. Tissue proteomic analyses are a ...

Isolation of Mouse Kidney-Resident CD8+ T cells for Flow Cytometry Analysis

Tissue-resident memory T cell (TRM) is a rapidly expanding field of immunology research. Isolating T cells from various non-lymphoid tissues is one of the key steps to investigate TRMs. There are slight variations in lymphocyte isolation protocols for different organs. Kidney is an essential non-lymphoid organ with numerous immune cell infiltration especially after pathogen exposure or autoimmune activation. In recent years, multiple labs including our own have started characterizing kidney resident CD8+ T cells in various physiological and pathological settings in both mouse and human. Due to the abundance of T lymphocytes, kidney represents an attractive model organ to study TRMs in non-mucosal or non-barrier tissues. Here, we will describe a protocol commonly used in TRM-focused labs to isolate CD8+ T cells from mouse kidneys following systemic viral infection. Briefly, using an acute lymphocytic choriomeningitis virus (LCMV) infection model in C57BL/6 mice, we demonstrate intravascular CD8+ T cell labeling, enzymatic digestion, and density gradient centrifugation to isolate and enrich lymphocytes from mouse kidneys to make samples ready for the subsequent flow cytometry analysis.

Isolation of Mouse Kidney-Resident CD8+ T cells for Flow Cytometry Analysis

Following viral infection, kidney harbors a relatively large number of CD8+ T cells and offers an opportunity to study non-mucosal TRM cells. Here, we describe a protocol to isolate mouse kidney lymphocytes for flow cytometry analysis.

Isolamento delle cellule CD8 + T residenti neireni del topo per l'analisi della citometria a flusso

Tissue-resident memory T cell (TRM) is a rapidly expanding field of immunology research. Isolating T cells from various non-lymphoid tissues is one of the ...

Optical Clearing and Imaging of Immunolabeled Kidney Tissue

Optical clearing techniques render tissue transparent by equilibrating the refractive index throughout a sample for subsequent three-dimensional (3-D) imaging. They have received great attention in all research areas for the potential to analyze microscopic multicellular structures that extend over macroscopic distances. Given that kidney tubules, vasculature, nerves, and glomeruli extend in many directions, which have been only partially captured by traditional two-dimensional techniques so far, tissue clearing also opened up many new areas of kidney research. The list of optical clearing methods is rapidly growing, but it remains difficult for beginners in this field to choose the best method for a given research question. Provided here is a simple method that combines antibody labeling of thick mouse kidney slices; optical clearing with cheap, non-toxic and ready-to-use chemical ethyl cinnamate; and confocal imaging. This protocol describes how to perfuse kidneys and use an antigen-retrieval step to increase antibody- binding without requiring specialized equipment. Its application is presented in imaging different multicellular structures within the kidney, and how to troubleshoot poor antibody penetration into tissue is addressed. We also discuss the potential difficulties of imaging endogenous fluorophores and acquiring very large samples and how to overcome them. This simple protocol provides an easy-to-setup and comprehensive tool to study tissue in three dimensions.

The combination of antibody labeling, optical clearing, and advanced light microscopy allows three-dimensional analysis of complete structures or organs. Described here is a simple method to combine immunolabeling of thick kidney slices, optical clearing with ethyl cinnamate, and confocal imaging that enables visualization and quantification of three-dimensional kidney structures.

Cancellazione ottica e imaging del tessuto renale immunoetichettato

Optical clearing techniques render tissue transparent by equilibrating the refractive index throughout a sample for subsequent three-dimensional (3-D) ...

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 modello murino efficace di danno unilaterale da ischemia-riperfusione renale

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

Isolation, Characterization, And High Throughput Extracellular Flux Analysis of Mouse Primary Renal Tubular Epithelial Cells

Mitochondrial dysfunction in the renal tubular epithelial cells (TECs) can lead to renal fibrosis, a major cause of&#160;chronic kidney disease (CKD). Therefore, assessing mitochondrial function in primary TECs may provide valuable insight into the bioenergetic status of the cells, providing insight into the pathophysiology of CKD. While there are a number of complex protocols available for the isolation and purification of proximal tubules in different species, the field lacks a cost-effective method optimized for&#160;tubular cell isolation without the need for purification. Here, we provide an isolation protocol that allows for studies focusing on both primary mouse proximal and distal renal TECs. In addition to cost-effective reagents and minimal animal procedures required in this protocol, the isolated cells maintain high energy levels after isolation and can be sub-cultured up to four&#160;passages, allowing for continuous studies. Furthermore, using a high throughput extracellular flux analyzer, we assess the mitochondrial respiration directly in the isolated TECs in a 96-well plate for which we provide recommendations for the optimization of cell density and compound concentration. These observations suggest that this protocol can be used for renal tubular ex vivo studies with a consistent, well-standardized production of renal TECs. This protocol may have broader future applications to study mitochondrial dysfunction associated with renal disorders for drug discovery or drug characterization purposes.

This protocol provides a cost-effective approach to isolate and characterize mouse primary renal tubular cells that can subsequently be sub-cultured to assess renal biological functions ex vivo, including mitochondrial bioenergetics.

Isolamento, caratterizzazione e Throughput elevato flusso extracellulare analisi di cellule epiteliali tubolari renali primari del Mouse

Mitochondrial dysfunction in the renal tubular epithelial cells (TECs) can lead to renal fibrosis, a major cause of&#160;chronic kidney disease (CKD). ...

Biologia

Glomeruli Isolation: A Technique to Obtain Intact Glomeruli from Murine Kidney

Source: Eymael, J. et al. <a href="https://www.jove.com/video/60324" target="_blank">Glomerular Outgrowth as an Ex Vivo Assay to Analyze Pathways Involved in Parietal Epithelial Cell Activation.</a> J. Vis. Exp. (2020)
This video describes the method to isolate intact glomeruli from mouse kidneys. The encapsulated and decapsulated glomeruli thus obtained can further be used for culturing parietal epithelial cell outgrowths from the murine kidney.

Isolamento dei glomeruli: una tecnica per ottenere glomeruli intatti dal rene murino

Source: Eymael, J. et al. Glomerular Outgrowth as an Ex Vivo Assay to Analyze Pathways Involved in Parietal Epithelial Cell Activation. J. Vis. Exp. ...

La tecnica del rene perfusi mouse isolato

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L'uso di un Hanging-peso di sistema per isolati di occlusione dell'arteria renale