La ricerca è stata sostenuta dal NHLBI Research Scientist Development Grant (1K01HL135461-01) a JAG. Grazie a David Carmona-Berrio e Isabel Adarve-Rengifo per la loro assistenza tecnica.

I topi sono stati ospitati e curati presso la Vanderbilt University Medical Center (VUMC) Division of Animal Care seguendo le linee guida del National Institutes of Health (NIH) e la Guida per la cura e l'uso degli animali da laboratorio, Dipartimento della salute e dei servizi umani degli Stati Uniti. Tutte le procedure sugli animali sono state approvate dal VUMC Institutional Animal Care and Use Committee prima di iniziare gli esperimenti.
1. Preparazione e dissezione degli animali
<ol><li>Accendere il germinatore e la pompa dell'acqua della piastra riscaldante circa 30 minuti prima di iniziare l'intervento chirurgico.</li>
	<li>Tagliare tubi in poliuretano lunghi 0,5 mm con un bisturi affilato. Rimuovere 0,2 mm della circonferenza facendo un taglio longitudinale per fare un polsino. NOTA: Questa è una parte critica della procedura di stenosi dell'arteria renale che richiede estrema precisione, attenzione ai dettagli e pazienza. Prova a tagliare diversi pezzi di tubi di poliuretano alla volta. Eseguire tutta questa procedura al microscopio.</li>
	<li>Prima di procedere oltre, indossare guanti chirurgici sterili e una maschera chirurgica.</li>
	<li>Utilizzare topi C57BL / 6 wild-type (WT) di 6-8 settimane. Utilizzare un numero uguale di topi maschi e femmine per evitare qualsiasi pregiudizio sessuale.</li>
	<li>Registra il peso dei topi prima di eseguire un intervento chirurgico. Il peso ideale per eseguire la stenosi dell'arteria renale con tubo in poliuretano è 18-22 g. Maneggiare i topi delicatamente e non agitare durante l'iniezione dell'anestesia. Somministrare l'analgesia preoperatoria (Ketofgen, 5 mg/kg di larghezza corporea). NOTA: Durante il trasferimento dei topi dalla loro stanza di alloggio alla sala operatoria, portarli con grande delicatezza e cura per evitare agitazione. Si consiglia vivamente di portare gabbie per topi in mano invece di un carrello.</li>
	<li>Anestetizzare i topi con una miscela di ketamina (100 mg/kg di peso corporeo) e xilazina (10 mg/kg di peso corporeo) tramite iniezioni intraperitoneali (I.P.).</li>
	<li>Riposizionare il mouse all'interno della gabbia fino a completa anestetizzazione. Ci vogliono circa 4-5 minuti prima che il mouse sia completamente incosciente. Pizzicare la coda o la punta del piede con una pinza per verificare se il topo è completamente incosciente e pronto per l'intervento chirurgico.</li>
	<li>Appoggia il mouse sulla schiena su un tovagliolo di carta. Rimuovere i peli dell'addome laterale utilizzando un tagliacapelli elettrico seguendo la direzione opposta della crescita dei capelli. Dopo aver rasato il sito chirurgico, pulire l'area con uno scrub chirurgico contenente iodio (o clorexidina) seguito da un risciacquo con etanolo al 70% (o soluzione salina sterile).  Ripeti 3 volte. NOTA: La procedura di depilazione deve essere eseguita a una certa distanza o preferibilmente su un banco diverso rispetto al banco della procedura chirurgica per evitare interferenze e contaminazione dei capelli durante la procedura chirurgica.</li>
	<li>Coprire la piastra riscaldante con un foglio sterile. Portare il topo al banco chirurgico e posizionarlo sul foglio sterile, rivolto verso il lato laterale destro del topo verso il microscopio. Mantenere una temperatura costante del tampone di 37 °C con acqua circolante.</li>
	<li>Aprire la sacca sterilizzata contenente tutte le attrezzature chirurgiche. Utilizzando un microscopio da dissezione e forbici sterili taglienti, fare una piccola incisione sul fianco (vicino alla 13acostola toracica, l'ultima costola nel topo) e a circa 0,5 cm di distanza dalle vertebre. Procedere lungo le vertebre lombari e fare un'incisione di 1 pollice.</li>
	<li>Tirare indietro la pelle e il muscolo per esporre il rene.</li>
	<li>Pulire e rimuovere il grasso circostante utilizzando tamponi di cotone sterili per isolare l'arteria renale. Isolare il nervo renale dall'arteria renale usando una pinza curva.</li>
	<li>Quando si esegue l'intervento chirurgico fittizio, applicare punti di sutura per chiudere la pelle e applicare antibiotici alla ferita chiusa, quindi procedere alla cura post-operatoria. In caso contrario, procedere con la sezione seguente per stenosare l'arteria. NOTA: Ogni esperimento dovrebbe avere animali fittizi come controlli della procedura chirurgica. Gli animali fittizi sono costituiti da topi che hanno attraversato la procedura chirurgica di esporre l'arteria renale senza posizionare un bracciale su di essa.</li>
</ol>2. Stenosi dell'arteria renale destra
<ol><li>Posizionare due suture di nylon sotto l'arteria renale destra, sciogliere i nodi e quindi posizionare il bracciale attorno all'arteria renale principale approssimativamente equidistante tra il rene e la biforcazione dell'aorta</li>
	<li>Chiudere il bracciale usando le suture di nylon. Fai quattro nodi per ogni sutura per evitare la probabilità di perdere le suture dopo l'intervento.</li>
	<li>Chiudere l'incisione nel muscolo applicando una semplice sutura continua.</li>
	<li>Fai semplici punti di sutura interrotti per chiudere la pelle.</li>
	<li>Somministrare analgesici approvati dalla IACUC. NOTA: Strumenti chirurgici in autoclave prima di ogni utilizzo. Se viene eseguito più di un intervento chirurgico contemporaneamente, pulire tutti gli strumenti usati con un indicatore alcolico sterile e metterli in un germinatore caldo per 15-30 secondi dopo ogni intervento chirurgico. Cambiare guanti sterili anche per ogni topo.</li>
</ol>3. Assistenza post-operatoria
<ol><li>Riportare i topi nella loro gabbia e lasciare la gabbia metà accesa, metà spenta, una piastra riscaldante ad acqua circolante per 2 - 3 ore. Aggiungi cibo per il recupero della dieta gel all'interno della gabbia.</li>
	<li>Somministrare antidolorifico (ketoprofene) per via intraperitoneale (dose: 5 mg/kg p.c.) il giorno successivo.</li>
	<li>Pesare i topi per i prossimi due giorni; se alcuni topi perdono più del 20% del loro peso, consultarsi con il veterinario e decidere se l'animale deve essere eutanasia seguendo l'appropriata procedura autorizzata IACUC.</li>
	<li>Monitorare i topi ogni giorno per valutare arrossamento, gonfiore, dolore o infezione.</li>
	<li>Cercare un consulto veterinario per le complicanze postoperatorie in conformità con le politiche istituzionali IACUC.</li>
</ol>4. Raccolta dei tessuti
<ol><li>Raccogliere il tessuto 3 settimane dopo la procedura chirurgica. Registra il peso di ciascun mouse.</li>
	<li>Eutanasia del mouse con una procedura approvata IACUC.</li>
	<li>Posizionare il mouse su una piattaforma sterile in posizione supina per sezionare.</li>
	<li>Fissare ed estendere gli arti per limitare i movimenti.</li>
	<li>Spruzzare accuratamente i topi con etanolo al 70%.</li>
	<li>Fai un'incisione della linea mediana per aprire l'addome e la zona del torace usando forbici affilate.</li>
	<li>Tirare indietro la pelle e la parete peritoneale.</li>
	<li>Esporre attentamente il cuore e perforare il ventricolo destro ed essanguare il topo.</li>
	<li>Rimuovere entrambi i reni usando una pinza. I reni si trovano sul retro dei topi. NOTA: Non mescolare entrambi i reni. Essere consapevoli dei reni stenosi, finti e controlaterali.</li>
	<li>Rimuovere la capsula renale, pulirli da qualsiasi grasso e registrare il peso di ciascun rene separatamente.</li>
	<li>Tagliare una sezione longitudinale di entrambi i reni e fissarla in PFA al 4% durante la notte a 4 °C per essere successivamente elaborata per l'incorporazione di paraffina, per eseguire l'ibridazione in situ (ISH) e l'immunoistochimica (IHC). Seguire i protocolli ISH e IHC come riportato23,24.</li>
	<li>Isolare la corteccia del rene rimanente e congelare l'azoto liquido per eseguire il western blot. Conservare i campioni a -80 °C fino all'analisi.</li>
	<li>Quantificare le espressioni di renina e N-GAL con Western blot come descritto in letteratura23,24.</li>
</ol>5. Statistiche
<ol><li>Utilizzare ANOVA unidirezionale o bidirezionale per esperimenti con tre o più condizioni seguiti da test post-hoc Bonferroni per confronti tra singoli gruppi. Si consideri un valore p uguale o minore di 0,05 significativo. Utilizzare un software (ad esempio, GraphPad Prism 8.2) per eseguire tutte le analisi statistiche.</li>
</ol>

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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pressor+responses+to+vasopressin+in+rabbits+with+3-day+renal+artery+stenosis.">Pressor responses to vasopressin in rabbits with 3-day renal artery stenosis.</a> American Journal of Physiology. 240 (6), 862-867 (1981).</li><li>Eirin, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+glomerular+filtration+rate+after+renal+revascularization+correlate+with+microvascular+hemodynamics+and+inflammation+in+Swine+renal+artery+stenosis.">Changes in glomerular filtration rate after renal revascularization correlate with microvascular hemodynamics and inflammation in Swine renal artery stenosis.</a> Circulation: Cardiovascular Interventions. 5 (5), 720-728 (2012).</li><li>Ma, Z., Jin, X., He, L., Wang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CXCL16+regulates+renal+injury+and+fibrosis+in+experimental+renal+artery+stenosis.">CXCL16 regulates renal injury and fibrosis in experimental renal artery stenosis.</a> American Journal of Physiology-Heart and Circulatory. 311 (3), 815-821 (2016).</li><li>Cheng, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+analysis+of+signaling+pathways+activated+in+a+murine+model+of+two-kidney,+one-clip+hypertension.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-kidney,+one+clip+and+one-kidney,+one+clip+hypertension+in+mice.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Models+of+experimental+hypertension+in+mice.">Models of experimental hypertension in mice.</a> Hypertension. 28 (6), 1064-1069 (1996).</li></ol>

Nessun conflitto di interessi, finanziario o di altro tipo, è dichiarato dagli autori.

La stenosi dell'arteria renale è una causa importante di ipertensione secondaria o resistente e danno renale 1,29. Il modello Goldblatt a due rene una clip (2K1C) è stato impiegato per studiare l'ipertensione renovascolare indotta da RAStenosi 1,17,18,19. Numerosi studi precedenti che utilizzano vari modelli animali hanno dimostrato c...

La stenosi dell'arteria renale (RAStenosis) è un problema intrattabile che colpisce circa il 6% delle persone sopra i 65 anni e fino al 40% delle persone con malattia coronarica o vascolare periferica 1,2. I trattamenti attuali per la malattia sono limitati; pertanto, vi è una necessità critica di sviluppare nuove terapie per trattare l'ipertensione renovascolare o l'ipertensione resistente indotta da RAStenosis. Il sistema dell'angiotensina aldosterone renina (RAAS) è la via chiave coinvolta nella patogenesi dell'ipertensione indotta da RAStenosi o dell'ipertensione renovascolare 3,4. Le terapie note che prendono di mira la RAAS, come gli ACE-inibitori o i bloccanti del recettore dell'angiotensina, alleviano l'ipertensione, ma richiedono un attento esame per insufficienza renale e iperkaliemia 5,6,7. La renina catalizza la fase limitante del tasso in RAAS; converte l'angiotensinogeno in angiotensina I. Nell'aterosclerosi, la formazione di placche provoca il restringimento dell'arteria renale che guida la secrezione di renina, con conseguente ipertensione renovascolare e danno renale8. Numerosi studi hanno riportato un aumento dei livelli di stress ossidativo durante l'ipertensione renovascolare nell'uomo, che sono stati corroborati con il modello di topi a due rene una clip (2K1C) e altri modelli animali ipertesi 2,9,10,11,12,13,14,15,16 . Il meccanismo molecolare del controllo dell'espressione della renina durante l'ipertensione renovascolare indotta da RAStenosi non è ben compreso e giustifica ulteriori indagini.
Modelli animali sperimentali che ricapitolano RAStenosi in modo affidabile e riproducibile sono importanti per chiarire i meccanismi cellulari e molecolari del controllo dell'espressione della renina per lo sviluppo di nuove terapie. Il modello murino 2K1C è un modello sperimentale consolidato per studiare la patogenesi dell'ipertensione renovascolare17,18,19,20. Questo modello è generato dalla costrizione dell'arteria renale utilizzando una clip 17,20,21, producendo quindi occlusione dell'arteria renale che si traduce in un aumento dell'espressione della renina e ipertensione17,19,20,21. Tuttavia, non ci sono relazioni tecniche disponibili, che descrivono una procedura passo passo per generare stenosi dell'arteria renale in modelli animali.
Clip d'argento convenzionali a forma di U, tubi in poliuretano e altre clip sono stati utilizzati per restringere l'arteria renale per indurre la stenosi dell'arteria renale. Alcuni studi hanno dimostrato che il design e il materiale della clip sono fondamentali per ottenere dati affidabili e riproducibili con il modello animale 2K1C. Secondo Lorenz et al., l'uso di clip d'argento convenzionali progettate a U induce un basso tasso di successo di ipertensione (40-60%)21. A causa del design della clip, l'arteria renale viene premuta lateralmente, innescando alcune costrizioni e una maggiore probabilità di essere spostata dall'arteria renale. La malleabilità e la duttilità dell'argento possono consentire variazioni nella larghezza delle clip; pertanto, causando diversi livelli di ipertensione tra i topi. I biossidi d'argento sulla clip possono causare infiammazione perivascolare, proliferazione intimale e granulazione dei tessuti, alterando il diametro dell'arteria renale22. A causa della variabilità dei livelli di ipertensione ottenuti con la clip d'argento convenzionale U-design, Warner et al. e Lorenz et al. hanno utilizzato con successo un tubo in poliuretano dal design più rotondo per iniziare la stenosi dell'arteria renale nei topi, generando un'induzione più affidabile e coerente del modello animale a due rene una clip20,21.
In questo rapporto, descriviamo un protocollo chirurgico per generare RAStenosi sperimentale nei topi, utilizzando il tubo di poliuretano per restringere l'arteria renale. Il polsino rotondo in poliuretano è una clip più riproducibile, affidabile e a basso costo per generare stenosi nel mouse. L'obiettivo di questo modello sperimentale è quello di studiare e definire il meccanismo molecolare e cellulare del controllo dell'espressione della renina durante la stenosi dell'arteria renale. Abbiamo confermato il successo del modello di topi RAStenosis misurando l'espressione della renina e il marcatore di danno renale lipocalina associata alla gelatinasi neutrofila (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 stenosi dell'arteria renale è una condizione comune nei pazienti con malattia coronarica o vascolare periferica in cui il sistema renina-angiotensina aldosterone (RAAS) è iperattivato. In questo contesto, c'è un restringimento delle arterie renali che stimola un aumento dell'espressione e del rilascio di renina, la proteasi limitante nella RAAS. Il conseguente aumento dell'espressione della renina è un noto driver dell'ipertensione renovascolare, frequentemente associata a danno renale e danno agli organi terminali. Pertanto, vi è un grande interesse nello sviluppo di nuovi trattamenti per questa condizione. Il meccanismo molecolare e cellulare del controllo della renina nella stenosi dell'arteria renale non è completamente compreso e merita ulteriori indagini. Per indurre la stenosi dell'arteria renale nei topi, è stato sviluppato un modello murino Goldblatt modificato 2 rene 1 clip (2K1C). Il rene destro è stato stenoso in topi wild type e topi operati fittizi sono stati utilizzati come controllo. Dopo la stenosi dell'arteria renale, abbiamo determinato l'espressione della renina e il danno renale. I reni sono stati raccolti e sono state utilizzate cortecce fresche per determinare l'espressione di proteine e mRNA della renina. Questo modello animale è riproducibile e può essere utilizzato per studiare le risposte fisiopatologiche, le vie molecolari e cellulari coinvolte nell'ipertensione renovascolare e nel danno renale.

La costrizione dell'arteria renale aumenta l'espressione della renina nel rene stenoso mentre reprime l'espressione nel rene controlaterale. Il modello di stenosi a due rene una clip (2K1C) o Goldblatt induce un aumento dell'espressione di renina e danno renale. Questo è riconosciuto come il miglior modello rappresentativo di stenosi unilaterale dell'arteria renale nell'uomo.
L'espressione di renina e prorenina (precursore della renina) è stata misurata utilizzando immunoblotting. I dati mo...

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

Un modello murino Goldblatt modificato a 2 rene 1 clip (2K1C) è stato sviluppato utilizzando tubi in poliuretano per avviare la stenosi dell'arteria renale, inducendo un aumento dell'espressione della renina e un danno renale. Qui, descriviamo una procedura dettagliata di preparazione e posizionamento del bracciale sull'arteria renale per generare un modello murino 2K1C riproducibile e coerente.

Un modello di topo modificato a due rene una clip della regolazione della renina nella stenosi dell'arteria renale

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

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Research

JoVE Journal

Medicine

4.6K Views.  Vanderbilt University Medical Center. Un modello murino Goldblatt modificato a 2 rene 1 clip (2K1C) è stato sviluppato utilizzando tubi in poliuretano per avviare la stenosi dell'arteria renale, inducendo un aumento dell'espressione della renina e un danno renale. Qui, descriviamo una procedura dettagliata di preparazione e posizionamento del bracciale sull'arteria renale per generare un modello murino 2K1C riproducibile e coerente.

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.

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

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.

Renale ischemia riperfusione: un modello murino di infortuni e rigenerazione

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.

La valutazione transcutanea della funzione renale nei roditori Conscious

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

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 ...

Improved Renal Denervation Mitigated Hypertension Induced by Angiotensin II Infusion

The benefits of renal sympathetic denervation (RDN) on blood pressure have been proved in a large number of clinical trials in recent years. However, the regulatory mechanism of RDN on hypertension remains elusive. Thus, it&#39;s essential to establish a simpler RDN model in mice. In this study, osmotic mini pumps filled with Angiotensin II were implanted in 14-week-old C57BL/6 mice. One week after the implantation of the mini-osmotic pump, a modified RDN procedure was performed on bilateral renal arteries of the mice using phenol. Age-sex-matched mice were given saline and served as sham group. Blood pressure was measured at baseline and every week subsequently for 21 days. Then, renal artery, abdominal aorta and heart were collected for histological examination using H&#38;E and Masson staining. In this study, we present a simple, practical, repeatable, and standardized RDN model, which can control hypertension and alleviate cardiac hypertrophy. The technique can denervate peripheral renal sympathetic nerves without renal artery damage. Compared to previous models, the modified RDN facilitates the study of the pathobiology and pathophysiology of hypertension.

Here, we present a protocol for renal sympathetic denervation (RDN) in mice with hypertension induced by Angiotensin II infusion. The procedure is repeatable, convenient and allows to study the regulatory mechanisms of RDN on hypertension and cardiac hypertrophy.

Miglioramento della denervazione renale ha attenuato l'ipertensione indotta dall'infusione di angiotensina II

The benefits of renal sympathetic denervation (RDN) on blood pressure have been proved in a large number of clinical trials in recent years. However, the ...

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 modello murino di disfunzione della mano correlata all'accesso all'emodialisi

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 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 ...

Biologia

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 Mouse 5/6th Nephrectomy modello che induce cardiomiopatia uremica sperimentale

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>Nefrectomia standardizzata 5/6 nei ratti per insufficienza renale e malperfusione

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>Nefrectomia 5/6 con bipolectomia acuta tramite laparotomia della linea mediana nei ratti

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.

Nefrectomia 5/6 con bipolectomia acuta tramite laparotomia della linea mediana nei ratti

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

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Nefrectomia 5/6 con bipolectomia acuta tramite laparotomia della linea mediana nei ratti

Nefrectomia 5/6 con bipolectomia acuta tramite laparotomia della linea mediana nei ratti