Nous remercions Krista den Ouden pour son excellente assistance technique. Cette technique a été soutenu financièrement par la Fondation canadienne du rein néerlandais, subvention C06.2174. MCV est soutenue par l&#39;organisation néerlandaise pour la recherche scientifique (NWO) Vidi-subvention 016.096.359.

Toutes les expériences sont exécutées conformément aux lignes expérimentales de guide éthique animale du comité d&#39;animal expérimental Utrecht. Le protocole est réalisé sous la direction et l&#39;approbation du soin des animaux de l&#39;institution de l&#39;auteur et du comité de l&#39;emploi. CKD est induite chez le mâle consanguin rats Lewis (Charles River, Sulzfeld, Allemagne) à l&#39;âge de 8 semaines. Les rats sont logés dans des conditions standard dans un environnement, de la lumière à température et humidité contrôlées. 1. Préparation de chirurgie <ol><li> Stériliser les instruments chirurgicaux: <ul><li> 1 tissu étudiant forceps 1-2 dents 12 cm </li><li> 1 étudiant au forceps de modèle standard </li><li> 2 Semken forceps </li><li> 1 Ciseaux Mayo </li><li> 1 étudiant ciseaux à iris </li><li> 1 Olsen-Hegar porte-aiguille avec des ciseaux </li><li> Blanket </li></ul></li><li> Consultez la liste d&#39;inventaire: <ul><li> Table d&#39;opération avec coussin chauffant et lampe </li><li> O <sub&gt; 2, l&#39;isoflurane </li><li> Tissus </li><li> Shaver </li><li> Scales </li><li> Ensemble opératoire stérile </li><li> 5x5 de gaze stérile et 10x10 </li><li> 70% d&#39;alcool </li><li> Une solution à 0,9% de NaCl </li><li> Seringue de 1 ml </li><li> Aiguilles (25G) </li><li> Tapis de mousse: Spongostan </li><li> Vicryl 4.0 et 5.0 </li><li> La buprénorphine 0,03 mg / kg (1:10 dilué dans du sel physiologique) </li><li> Cage propre pour les rats après une chirurgie </li></ul></li></ol> 2. Right Side uninéphrectomie <ol><li> Désinfecter la table avec 70% d&#39;alcool. </li><li> Lieu rat dans une boîte à induction et induire une anesthésie avec 4% d&#39;isoflurane et 1 litre flux de O 2. Remarque: en fonction de la souche de rat utilisée, avant et analgésie périopératoire sont prescrits, cela doit être discuté avec le vétérinaire local. </li><li> Transfert rat à table et lieu nez dans le cône. Le rat et le cône de nez sont placés sur un bloc de chauffage pour maintenir la température du corps du rat. Le rat is aérée avec un mélange de 2% d&#39;isoflurane et 1 litre d&#39;O 2. </li><li> Raser le flanc du rat et désinfecter avec de l&#39;alcool. Laver et désinfecter les mains et porter des gants chirurgicaux stériles. Portez un chapeau à tête et un masque. Faire un 1 à 1,5 cm incision parallèle aux côtes à l&#39;aide de forceps anatomiques et des ciseaux émoussés. Exposer le rein par dissection des muscles du dos. </li><li> Le pôle inférieur du rein est visible, utiliser soigneusement les petits forceps émoussé pour saisir le tissu adipeux périrénal. Externaliser le rein en tirant doucement sur la graisse péri-rénale avec une pince. Prenez soin de ne pas perturber la glande surrénale au cours de cette procédure. La glande surrénale peut être facilement retiré en plaçant une pince sur le site médial dans le tissu adipeux et déplacée légèrement vers le haut entre les glandes surrénales et les reins comme le montre la figure 2. </li><li> Placez les reins doucement sur de la gaze et claire qui entoure le tissu adipeux et conjonctif. Identifier l&#39;artère et la veine rénales et placer une ligature (5.0vicryl) avec un seul noeud autour des vaisseaux, mais ne pas attacher le noeud. </li><li> Déplacer le noeud lâche soigneusement le long des vaisseaux vers l&#39;aorte par environ 0,5 cm, pour créer un espace entre le rein et le noeud. Ceci est fait pour empêcher la ligature de glisser après la coupe. </li><li> Noeud de cravate avec deux doubles noeuds. Si la perfusion est arrêtée, la couleur du rein change immédiatement au brun. Ne pas couper les extrémités de la ligature. </li><li> Couper les vaisseaux rénaux proches du rein et enlever le rein. Tirez doucement sur les longues extrémités de la ligature pour vérifier le saignement des vaisseaux rénaux. La ligature doit rester en place sur le navire. Couper les longues extrémités de la ligature. Remarque, les artères rénales restantes se rétractent dans la cavité abdominale immédiatement. </li><li> Épongez les reins excisée sécher et peser. </li><li> Fermer l&#39;incision du muscle squelettique à l&#39;exploitation sutures avec Vicryl 5.0. Injecter 0,10 ml (0,03 mg / kg) buprénorphine im dans le membre postérieur. Fermez lepeau avec Vicryl 4.0 avec sutures intracutanées. Cela empêche les rats de l&#39;ouverture de la plaie. Note: en raison d&#39;une dépression respiratoire chez le rat Lewis, nous ne donnons analgésie post-opératoire juste avant d&#39;éteindre le isoflurane. </li><li> Abriter le rat dans une cage solitaire propre avec un accès facile à la nourriture et à l&#39;eau. Placez la moitié de la cage sur le réchauffement pad O / N et vérifier le rat soigneusement le lendemain. Lorsque la plaie est fermée et le rat est actif, ce qui est normalement comprise entre 6 et 12 h après l&#39;opération, le rat peut être placé en arrière dans le logement de groupe. </li></ol> 3. Côté gauche de la néphrectomie total <ol><li> Sept jours plus tard, les mêmes préparations sont réalisées sur le côté gauche que pour la droite, comme décrit de 2,1 à 2,5. Avant l&#39;intervention, la quantité de tissu rénal qui doit être éliminée correspond à environ 2/3rds du poids du rein droit est calculé. Préparez de petits morceaux de mousse gel (Spongostan, Johnson &amp; Johnson, New Jersey) avant la chirurgie. </li&gt; <li> Placez ciseaux pointus autour du pôle supérieur du rein gauche et la résection du pôle supérieur d&#39;un seul coup (pour l&#39;emplacement des ciseaux, voir figure 3). Couvrir immédiatement avec un morceau de mousse de gel et d&#39;exercer une légère pression avec une gaze stérile. </li><li> Répéter avec le pôle inférieur du rein comme dans 3.3. </li><li> Soulevez rein pour empêcher la coagulation sur la peau. Maintenir une légère pression sur les deux coussinets de mousse de gel avec de la gaze stérile jusqu&#39;à ce que le saignement s&#39;arrête. Lorsque le saignement persiste, ajouter un tapis de mousse. </li><li> Placez reste rein avec coussinets en mousse de gel adhérentes à l&#39;intérieur de l&#39;abdomen. Fermez le muscle et la peau et suivez 02.09 à 02.12. </li></ol> 4. Sham chirurgie <ol><li> Contrôles opérés Sham subissent la même procédure afin d&#39;exposer les reins. Au lieu d&#39;extirper le rein ou de couper les poteaux une semaine plus tard, les deux reins sont décapsulés à un intervalle d&#39;une semaine, en prenant soin de ne pas perturber les glandes surrénales. Fermeture de la plaie et les soins post-opératoires sont identical à 2.11 et 2.12. </li></ol>

<ol>
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Nephrol. 13 (3), 806-816 (2002).</li><li>Baylis, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nitric+oxide+synthase+derangements+and+hypertension+in+kidney+disease.">Nitric oxide synthase derangements and hypertension in kidney disease.</a> Curr. Opin. Nephrol. Hypertens. 21 (1), 1-6 (2012).</li><li>Bongartz, L. G., Braam, B., 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=Transient+nitric+oxide+reduction+induces+permanent+cardiac+systolic+dysfunction+and+worsens+kidney+damage+in+rats+with+chronic+kidney+disease.">Transient nitric oxide reduction induces permanent cardiac systolic dysfunction and worsens kidney damage in rats with chronic kidney disease.</a> Am. J. Physiol. Regul. Integr. Comp. Physiol. 298 (3), 815-823 (2010).</li><li>Fujihara, C. 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Ther. 17 (3), 324-333 (2012).</li><li>Haylor, J., Schroeder, 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=Skin+gadolinium+following+use+of+MR+contrast+agents+in+a+rat+model+of+nephrogenic+systemic+fibrosis.">Skin gadolinium following use of MR contrast agents in a rat model of nephrogenic systemic fibrosis.</a> Radiology. 263 (1), 107-116 (2012).</li><li>Moriguchi, Y., Yogo, K., 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=Left+ventricular+hypertrophy+is+associated+with+inflammation+in+sodium+loaded+subtotal+nephrectomized+rats.">Left ventricular hypertrophy is associated with inflammation in sodium loaded subtotal nephrectomized rats.</a> Biomed. Res. 32 (2), 83-90 (2011).</li><li>van Koppen, A., Joles, J. 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=Healthy+bone+marrow+cells+reduce+progression+of+kidney+failure+better+than+CKD+bone+marrow+cells+in+rats+with+established+chronic+kidney+disease.">Healthy bone marrow cells reduce progression of kidney failure better than CKD bone marrow cells in rats with established chronic kidney disease.</a> Cell Transplant. , (2012).</li><li>Baylis, C., Corman, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+aging+kidney:+insights+from+experimental+studies.">The aging kidney: insights from experimental studies.</a> J. Am. Soc. Nephrol. 9 (4), 699-709 (1998).</li><li>Szabo, A. J., Muller, V., Chen, G. F., Samsell, L. J., Erdely, A., Baylis, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nephron+number+determines+susceptibility+to+renal+mass+reduction-induced+CKD+in+Lewis+and+Fisher+344+rats:+implications+for+development+of+experimentally+induced+chronic+allograft+nephropathy.">Nephron number determines susceptibility to renal mass reduction-induced CKD in Lewis and Fisher 344 rats: implications for development of experimentally induced chronic allograft nephropathy.</a> Nephrol. Dial Transplant. 23 (8), 2492-2495 (2008).</li><li>Darling, I. M., Morris, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+"true"+creatinine+clearance+in+rats+reveals+extensive+renal+secretion.">Evaluation of "true" creatinine clearance in rats reveals extensive renal secretion.</a> Pharm. Res. 8 (10), 1318-1322 (1991).</li><li>Levey, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+renal+function+in+chronic+renal+disease.">Measurement of renal function in chronic renal disease.</a> Kidney Int. 38 (1), 167-184 (1990).</li><li>Myers, G. L., Miller, W. G., 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=Recommendations+for+improving+serum+creatinine+measurement:+a+report+from+the+Laboratory+Working+Group+of+the+National+Kidney+Disease+Education+Program.">Recommendations for improving serum creatinine measurement: a report from the Laboratory Working Group of the National Kidney Disease Education Program.</a> Clin. Chem. 52 (1), 5-18 (2006).</li><li>Hostetter, T. H., Meyer, T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+of+clearance+methods+for+measurement+of+glomerular+filtration+and+tubular+reabsorption.">The development of clearance methods for measurement of glomerular filtration and tubular reabsorption.</a> Am. J. Physiol. Renal Physiol. 287 (5), F868-F870 (2004).</li><li>Koeners, M. P., Racasan, S., Koomans, H. A., Joles, J. A., Braam, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nitric+oxide,+superoxide+and+renal+blood+flow+autoregulation+in+SHR+after+perinatal+L-arginine+and+antioxidants.">Nitric oxide, superoxide and renal blood flow autoregulation in SHR after perinatal L-arginine and antioxidants.</a> Acta. Physiol. (Oxf). 190 (4), 329-338 (2007).</li><li>van den Brandt, J., Wang, D., Kwon, S. H., Heinkelein, M., Reichardt, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lentivirally+generated+eGFP-transgenic+rats+allow+efficient+cell+tracking+in+vivo.">Lentivirally generated eGFP-transgenic rats allow efficient cell tracking in vivo.</a> Genesis. 39 (2), 94-99 (2004).</li><li>Kreutz, R., Kovacevic, L., Schulz, A., Rothermund, L., Ketteler, M., Paul, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+high+NaCl+diet+on+spontaneous+hypertension+in+a+genetic+rat+model+with+reduced+nephron+number.">Effect of high NaCl diet on spontaneous hypertension in a genetic rat model with reduced nephron number.</a> J. Hypertens. 18 (6), 777-782 (2000).</li><li>Liu, Z. C., Chow, K. M., Chang, T. 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O., 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=Laparoscopic+partial+nephrectomy+in+rats.">Laparoscopic partial nephrectomy in rats.</a> Acta. Cir. Bras. 22 (2), 152-156 (2007).</li></ol>

Les auteurs n&#39;ont rien à révéler.

L&#39;ablation chirurgicale de 5/6e de la masse rénale chez le rat Lewis, combiné avec un régime riche en sel et l&#39;inhibition de la NOS temporaire conduit à un modèle de CKD qui ressemble CKD humaine et permet d&#39;étudier les mécanismes de causalité et l&#39;efficacité des interventions thérapeutiques en CKD. Le modèle de néphrectomie 5/6e est un modèle bien connu et largement décrit pour CKD. Toutefois, la simple élimination 5/6e de la masse rénale n&#39;entraîne pas ...

En raison de sa nature progressive, qui a suivi une maladie rénale en phase terminale, et la morbidité cardiovasculaire et la mortalité associée, CKD est un problème croissant de santé publique 1. Le ralentissement de la progression CKD est donc une priorité majeure de santé. Depuis CKD est caractérisée par des désordres complexes de l&#39;homéostasie, les modèles animaux d&#39;intégration sont nécessaires pour étudier le développement et la progression de l&#39;IRC. Le rein est constitué d&#39;un large éventail de différents types de cellules qui interagissent les uns avec les autres. Cette complexité ne peut être mimée in vitro. Pour étudier les interventions thérapeutiques en CKD, nous utilisons le modèle 5/6e de néphrectomie d&#39;ablation, un modèle expérimental bien connu de la maladie rénale progressive, ressemblant à plusieurs aspects de la MRC humaine 2,3. La réduction brute de la masse rénale glomérulaire progressive et provoque des blessures tubulo-interstitielle, la perte de néphrons résiduelles et le développement de l&#39;hypertension systémique et glomérulaire. Il est associé avec progressive intrarénal perte capillaire 4, l&#39;inflammation et la glomérulosclérose. Les facteurs de risque pour CKD invariablement impact sur ​​la fonction endothéliale 5. Nous avons utilisé une souche de rats (Lewis) qui est relativement résistant au développement de la MRC et donc nous avons combiné suppression des 5/6e de la masse rénale avec de l&#39;oxyde nitrique (NO) épuisement 6, 7, 8 et un régime élevé de sel 9. Après l&#39;arrivée et l&#39;acclimatation, les animaux reçoivent un inhibiteur de la NO synthase (L-NNA) additionné à l&#39;eau potable (20 mg / L) pour une période de 4 semaines, suivie par droit uninéphrectomie face (UNX) avec la poursuite de la L-NNA après deux jours . Une semaine plus tard, la néphrectomie subtotale (SNX), à savoir l&#39;élimination de 2/3rds de la masse rénale est effectuée sur le côté gauche. Après SNX, les animaux sont autorisés à récupérer pendant 2 jours, puis à nouveau de 20 mg / L dans l&#39;eau potable LNNA pour une période de 4 semaines. Un régime élevé en sel (6%), complété en rez-de-chow (voir la ligne du temps Figure 1), se poursuit tout au long de l&#39;expérience. Til raison pour effectuer la UNX sur le côté droit et le SNX sur le côté gauche, c&#39;est que les vaisseaux rénaux sont plus longs sur le côté gauche, ce qui facilite l&#39;accès du rein sans étirer les vaisseaux trop quand le rein est exposée à l&#39;extérieur du corps . Dans la littérature, les modèles sont décrits dans laquelle les pôles du rein gauche sont éliminés en premier, suivi par UNX du rein droit une semaine plus tard 10,11,12. Dans nos mains ce modèle a montré un développement beaucoup plus rapide de l&#39;insuffisance rénale, mais aussi une plus grande variation de la perte de la fonction rénale. Progression de l&#39;insuffisance rénale est suivie dans le temps par la mesure de l&#39;urée plasmatique, la pression artérielle systolique et de la protéinurie. Par six semaines après SNX, l&#39;insuffisance rénale a développé, caractérisé par une réduction marquée du taux de filtration glomérulaire (69%) et une circulation efficace de plasma rénal (62%) 13 sur l&#39;hypertension (pression artérielle systolique&gt; 150 mmHg), protéinurie (&gt; 50 mg/24 h) et doux urémie (&gt; 10 mm). Ses caractéristiques histologiques comprennent Tubuldommage o-interstitielle traduit par une inflammation, une atrophie tubulaire et une fibrose et une glomérulosclérose focale conduisant à une réduction massive des glomérules saines au sein de la population restante (&lt;10%). Suivi jusqu&#39;à 12 semaines après SNX montre la progression de l&#39;IRC, en fournissant une fenêtre d&#39;opportunité pour l&#39;évaluation des interventions thérapeutiques.

<table border="1">
	<tbody>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Reagent</td>
		</tr>
		<tr>
			<td>L-NNA</td>
			<td>Sigma-aldrich</td>
			<td>N5501</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Spongostan dental: gel foam pads 1x1x1 cm</td>
			<td>Johnson&amp;Johnson</td>
			<td>Ms0005</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Ethicon Vicryl FS-2S naald 4/0 V392H p/36</td>
			<td>Ethicon</td>
			<td>V303H</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Ethicon Vicryl RB-1+ naald 5/0 V303H p/36</td>
			<td>Ethicon</td>
			<td>V392H</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Buprenorphine (0.3 mg/ml)</td>
			<td>Via local pharmacist ordered by Reckitt Benckiser pharmaceuticals</td>
			<td>unknown</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Student Tissue Forceps - 1x2 Teeth 12 cm</td>
			<td>Fine Science Tools (FST)</td>
			<td>91121-12</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Student Standard Pattern Forceps</td>
			<td>FST</td>
			<td>91100-12</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Mayo Scissors</td>
			<td>FST</td>
			<td>14010-15</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>2X Semken Forceps</td>
			<td>FST</td>
			<td>11008-13</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Student Iris Scissors</td>
			<td>FST</td>
			<td>91460-11</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Olsen-Hegar Needle Holder</td>
			<td>FST</td>
			<td>12002-14</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

5/6th nephrectomy in combination with high salt diet and nitric oxide synthase inhibition to induce chronic kidney disease in the lewis rat

L&#39;insuffisance rénale chronique (IRC) est un problème mondial. Le ralentissement de la progression CKD est une priorité majeure de santé. Depuis CKD est caractérisée par des désordres complexes de l&#39;homéostasie, les modèles animaux d&#39;intégration sont nécessaires pour étudier le développement et la progression de l&#39;IRC. Pour étudier le développement d&#39;interventions thérapeutiques CKD et roman en CKD, nous utilisons le modèle d&#39;ablation par néphrectomie 5/6e, un modèle expérimental bien connu de la maladie rénale progressive, ressemblant à plusieurs aspects de la MRC de l&#39;homme. La réduction brute de la masse rénale glomérulaire progressive et provoque des blessures tubulo-interstitielle, la perte de néphrons résiduelles et le développement de l&#39;hypertension systémique et glomérulaire. Elle est également associée avec la perte progressive intrarénal capillaire, l&#39;inflammation et la glomérulosclérose. Les facteurs de risque pour CKD invariablement impact sur la fonction endothéliale. Pour imiter cela, nous combinons l&#39;enlèvement de 5/6e de la masse rénale avec de l&#39;oxyde nitrique (NO) de l&#39;épuisement et une alimentation riche en sel. Après l&#39;arrivée et l&#39;acclimatation, les animaux recevoir un inhibiteur de la NO-synthase (NG-nitro-L-arginine) (L-NNA) additionné à de l&#39;eau potable (20 mg / L) pendant une période de 4 semaines, suivie de droite uninéphrectomie verso. Une semaine plus tard, une néphrectomie subtotale (SNX) est réalisée sur le côté gauche. Après SNX, les animaux sont autorisés à récupérer pendant deux jours, puis LNNA dans l&#39;eau potable (20 mg / L) pour une nouvelle période de 4 semaines. Un régime élevé en sel (6%), complété en rez-de-chow (voir la ligne du temps Figure 1), se poursuit tout au long de l&#39;expérience. Progression de l&#39;insuffisance rénale est suivie dans le temps par la mesure de l&#39;urée plasmatique, la pression artérielle systolique et de la protéinurie. Par six semaines après SNX, l&#39;insuffisance rénale s&#39;est développée. La fonction rénale est mesurée en utilisant l&#39;inuline «gold standard» et acide para-amino hippurique (PAH) technologies de déminage. Ce modèle de CKD est caractérisée par une réduction du taux de filtration glomérulaire (DFG) et le flux plasmatique rénal efficace (ERPF), hypertension (pression artérielle systolique&gt; 150 mmHg), protéinurie (&gt; 50 mg/24 h) Et l&#39;urémie légère (&gt; 10 mm). Ses caractéristiques histologiques incluent des dommages tubulo-interstitielle réfléchie par une inflammation, une atrophie tubulaire et une fibrose et une glomérulosclérose focale conduisant à une réduction massive des glomérules saines au sein de la population restante (&lt;10%). Suivi jusqu&#39;à 12 semaines après SNX montre la progression de l&#39;IRC.

Après néphrectomie subtotale, environ 1/6e de la masse rénale totale est laissée. Figure 4 montre le poids de la partie enlevée du rein droit avec moyenne et l&#39;écart dans deux expériences précédentes. Il convient de garder à l&#39;esprit que dans la semaine après UNX, l&#39;hypertrophie du rein gauche se produit, indiquant que le poids qui doit être retiré calculé en fonction du poids du rein droit se traduit toujours en moins de 5/6e enlèvement. Cependant, comme il n&#39;est pas poss...

Watch this Scientific Journal Video about 5/6th Nephrectomy in Combination with High Salt Diet and Nitric Oxide Synthase Inhibition to Induce Chronic Kidney Disease in the Lewis Rat at JoVE.com

5/6th Nephrectomy in Combination with High Salt Diet and Nitric Oxide Synthase Inhibition to Induce Chronic Kidney Disease in the Lewis Rat

Une méthode en deux étapes pour établir l&#39;insuffisance rénale chronique (IRC) chez le rat Lewis par l&#39;ablation chirurgicale 5/6e de la masse rénale est décrite. Combinaison de la procédure chirurgicale, NOS-inhibition et une alimentation riche en sel conduit à un modèle ressemblant CKD humaine, permettant l&#39;étude des mécanismes et le développement de nouvelles interventions thérapeutiques causales.

5/6e néphrectomie en association avec High Salt Diet Nitric Oxide Synthase et inhibition pour provoquer la maladie rénale chronique chez le rat Lewis

Chronic kidney disease (CKD) is a global problem. Slowing CKD progression is a major health priority. Since CKD is characterized by complex derangements ...

56th-nephrectomy-combination-with-high-salt-diet-nitric-oxide

Research

JoVE Journal

Medicine

23.4K vues.  University Medical Center Utrecht. Une méthode en deux étapes pour établir l'insuffisance rénale chronique (IRC) chez le rat Lewis par l'ablation chirurgicale 5/6e de la masse rénale est décrite. Combinaison de la procédure chirurgicale, NOS-inhibition et une alimentation riche en sel conduit à un modèle ressemblant CKD humaine, permettant l'étude des mécanismes et le développement de nouvelles interventions thérapeutiques causales.

Vidéo: 5/6th Nephrectomy in Combination with High Salt Diet and Nitric Oxide Synthase Inhibition to Induce Chronic Kidney Disease in the Lewis Rat

Analytical Techniques for Assaying Nitric Oxide Bioactivity

Nitric oxide (NO) is a diatomic free radical that is extremely short lived in biological systems (less than 1 second in circulating blood)1. NO may be considered one of the most important signaling molecules produced in our body, regulating essential functions including but not limited to regulation of blood pressure, immune response and neural communication. Therefore its accurate detection and quantification in biological matrices is critical to understanding the role of NO in health and disease. With such a short physiological half life of NO, alternative strategies for the detection of reaction products of NO biochemistry have been developed. The quantification of relevant NO metabolites in multiple biological compartments provides valuable information with regards to in vivo NO production, bioavailability and metabolism. Simply sampling a single compartment such as blood or plasma may not always provide an accurate assessment of whole body NO status, particularly in tissues. The ability to compare blood with select tissues in experimental animals will help bridge the gap between basic science and clinical medicine as far as diagnostic and prognostic utility of NO biomarkers in health and disease. Therefore, extrapolation of plasma or blood NO status to specific tissues of interest is no longer a valid approach. As a result, methods continue to be developed and validated which allow the detection and quantification of NO and NO-related products/metabolites in multiple compartments of experimental animals in vivo. The established paradigm of NO biochemistry from production by NO synthases to activation of soluble guanylyl cyclase (sGC) to eventual oxidation to nitrite (NO2-) and nitrate (NO3-) may only represent part of NO's effects in vivo. The interaction of NO and NO-derived metabolites with protein thiols, secondary amines, and metals to form S-nitrosothiols (RSNOs), N-nitrosamines (RNNOs), and nitrosyl-heme respectively represent cGMP-independent effects of NO and are likely just as important physiologically as activation of sGC by NO. A true understanding of NO in physiology is derived from in vivo experiments sampling multiple compartments simultaneously. Nitric oxide (NO) methodology is a complex and often confusing science and the focus of many debates and discussion concerning NO biochemistry. The elucidation of new mechanisms and signaling pathways involving NO hinges on our ability to specifically, selectively and sensitively detect and quantify NO and all relevant NO products and metabolites in complex biological matrices. Here, we present a method for the rapid and sensitive analysis of nitrite and nitrate by HPLC as well as detection of free NO in biological samples using in vitro ozone based chemiluminescence with chemical derivitazation to determine molecular source of NO as well as ex vivo with organ bath myography.

The endogenous production of nitric oxide (NO) regulates a wide variety of biological functions. It is becoming increasingly clear that disruption or dysregulation of NO based signaling is involved in many human diseases. Methods to quantify relevant NO metabolites may provide novel diagnostic or prognostic biomarkers for human disease.

Techniques d&#39;analyse permettant de doser la bioactivité d&#39;oxyde nitrique

Nitric oxide (NO) is a diatomic free radical that is extremely short lived in biological systems (less than 1 second in circulating blood)1. NO may be ...

Recherche

Médecine

Catheter Ablation in Combination With Left Atrial Appendage Closure for Atrial Fibrillation

Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia, affecting millions of individuals worldwide 1-3. The rapid, irregular, and disordered electrical activity in the atria gives rise to palpitations, fatigue, dyspnea, chest pain and dizziness with or without syncope 4, 5. Patients with AF have a five-fold higher risk of stroke 6.
Oral anticoagulation (OAC) with warfarin is commonly used for stroke prevention in patients with AF and has been shown to reduce the risk of stroke by 64% 7. Warfarin therapy has several major disadvantages, however, including bleeding, non-tolerance, interactions with other medications and foods, non-compliance and a narrow therapeutic range 8-11. These issues, together with poor appreciation of the risk-benefit ratio, unawareness of guidelines, or absence of an OAC monitoring outpatient clinic may explain why only 30-60% of patients with AF are prescribed this drug 8.
The problems associated with warfarin, combined with the limited efficacy and/or serious side effects associated with other medications used for AF 12,13, highlight the need for effective non-pharmacological approaches to treatment. One such approach is catheter ablation (CA), a procedure in which a radiofrequency electrical current is applied to regions of the heart to create small ablation lesions that electrically isolate potential AF triggers 4. CA is a well-established treatment for AF symptoms 14, 15, that may also decrease the risk of stroke. Recent data showed a significant decrease in the relative risk of stroke and transient ischemic attack events among patients who underwent ablation compared with those undergoing antiarrhythmic drug therapy 16.
Since the left atrial appendage (LAA) is the source of thrombi in more than 90% of patients with non-valvular atrial fibrillation 17, another approach to stroke prevention is to physically block clots from exiting the LAA. One method for occluding the LAA is via percutaneous placement of the WATCHMAN LAA closure device. The WATCHMAN device resembles a small parachute. It consists of a nitinol frame covered by fabric polyethyl terephthalate that prevents emboli, but not blood, from exiting during the healing process. Fixation anchors around the perimeter secure the device in the LAA (Figure 1). To date, the WATCHMAN is the only implanted percutaneous device for which a randomized clinical trial has been reported. In this study, implantation of the WATCHMAN was found to be at least as effective as warfarin in preventing stroke (all-causes) and death (all-causes) 18. This device received the Conformit&eacute; Europ&eacute;enne (CE) mark for use in the European Union for warfarin eligible patients and in those who have a contraindication to anticoagulation therapy 19.
Given the proven effectiveness of CA to alleviate AF symptoms and the promising data with regard to reduction of thromboembolic events with both CA and WATCHMAN implantation, combining the two procedures is hoped to further reduce the incidence of stroke in high-risk patients while simultaneously relieving symptoms. The combined procedure may eventually enable patients to undergo implantation of the WATCHMAN device without subsequent warfarin treatment, since the CA procedure itself reduces thromboembolic events. This would present an avenue of treatment previously unavailable to patients ineligible for warfarin treatment because of recurrent bleeding 20 or other warfarin-associated problems.
The combined procedure is performed under general anesthesia with biplane fluoroscopy and TEE guidance. Catheter ablation is followed by implantation of the WATCHMAN LAA closure device. Data from a non-randomized trial with 10 patients demonstrates that this procedure can be safely performed in patients with a CHADS2 score of greater than 1 21. Further studies to examine the effectiveness of the combined procedure in reducing symptoms from AF and associated stroke are therefore warranted.

Catheter ablation is combined with placement of the WATCHMAN Left Atrial Appendage Closure Device to prevent ischemic stroke in a patient with non-valvular atrial fibrillation.

Ablation par cathéter en association avec fermeture appendice auriculaire gauche pour la fibrillation auriculaire

Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia, affecting millions of individuals worldwide 1-3. The rapid, irregular, and ...

Spectral Karyotyping to Study Chromosome Abnormalities in Humans and Mice with Polycystic Kidney Disease

Conventional method to identify and classify individual chromosomes depends on the unique banding pattern of each chromosome in a specific species being analyzed 1, 2. This classical banding technique, however, is not reliable in identifying complex chromosomal aberrations such as those associated with cancer. To overcome the limitations of the banding technique, Spectral Karyotyping (SKY) is introduced to provide much reliable information on chromosome abnormalities.
SKY is a multicolor fluorescence in-situ hybridization (FISH) technique to detect metaphase chromosomes with spectral microscope 3, 4. SKY has been proven to be a valuable tool for the cytogenetic analysis of a broad range of chromosome abnormalities associated with a large number of genetic diseases and malignancies 5, 6. SKY involves the use of multicolor fluorescently-labelled DNA probes prepared from the degenerate oligonucleotide primers by PCR. Thus, every chromosome has a unique spectral color after in-situ hybridization with probes, which are differentially labelled with a mixture of fluorescent dyes (Rhodamine, Texas Red, Cy5, FITC and Cy5.5). The probes used for SKY consist of up to 55 chromosome specific probes 7-10.
The procedure for SKY involves several steps (Figure 1). SKY requires the availability of cells with high mitotic index from normal or diseased tissue or blood. The chromosomes of a single cell from either a freshly isolated primary cell or a cell line are spread on a glass slide. This chromosome spread is labeled with a different combination of fluorescent dyes specific for each chromosome. For probe detection and image acquisition,the spectral imaging system consists of sagnac interferometer and a CCD camera. This allows measurement of the visible light spectrum emitted from the sample and to acquire a spectral image from individual chromosomes. HiSKY, the software used to analyze the results of the captured images, provides an easy identification of chromosome anomalies. The end result is a metaphase and a karyotype classification image, in which each pair of chromosomes has a distinct color (Figure 2). This allows easy identification of chromosome identities and translocations. For more details, please visit Applied Spectral Imaging website (<a href="http://www.spectral-imaging.com/" target="_blank">http://www.spectral-imaging.com/</a>).
SKY was recently used for an identification of chromosome segregation defects and chromosome abnormalities in humans and mice with Autosomal Dominant Polycystic Kidney Disease (ADPKD), a genetic disease characterized by dysfunction in primary cilia 11-13. Using this technique, we demonstrated the presence of abnormal chromosome segregation and chromosomal defects in ADPKD patients and mouse models 14. Further analyses using SKY not only allowed us to identify chromosomal number and identity, but also to accurately detect very complex chromosomal aberrations such as chromosome deletions and translocations (Figure 2).

Spectral Karyotyping (SKY) is an advanced cytogenetics technique to identify genomic and chromosomal aberrations. This technique takes advantage of chromosome painting probes, which allow classification of all chromosomes. SKY can also identify complex chromosome aberrations and segregation defects in mice and humans with various diseases, including polycystic kidney disease.

Caryotype spectral pour l&#39;étude d&#39;anomalies chromosomiques dans les humains et les souris de la maladie polykystique des reins

Conventional method to identify and classify individual chromosomes depends on the unique banding pattern of each chromosome in a specific species being ...

A Model of Chronic Nutrient Infusion in the Rat

Chronic exposure to excessive levels of nutrients is postulated to affect the function of several organs and tissues and to contribute to the development of the many complications associated with obesity and the metabolic syndrome, including type 2 diabetes. To study the mechanisms by which excessive levels of glucose and fatty acids affect the pancreatic beta-cell and the secretion of insulin, we have established a chronic nutrient infusion model in the rat. The procedure consists of catheterizing the right jugular vein and left carotid artery under general anesthesia; allowing a 7-day recuperation period; connecting the catheters to the pumps using a swivel and counterweight system that enables the animal to move freely in the cage; and infusing glucose and/or Intralipid (a soybean oil emulsion which generates a mixture of approximately 80% unsaturated/20% saturated fatty acids when infused with heparin) for 72 hr. This model offers several advantages, including the possibility to finely modulate the target levels of circulating glucose and fatty acids; the option to co-infuse pharmacological compounds; and the relatively short time frame as opposed to dietary models. It can be used to examine the mechanisms of nutrient-induced dysfunction in a variety of organs and to test the effectiveness of drugs in this context.

A protocol for chronic infusions of glucose and Intralipid in rats is described. This model can be used to study the impact of nutrient excess on organ function and physiological parameters.

Un modèle d&#39;infusion de nutriments chronique chez le rat

Chronic exposure to excessive levels of nutrients is postulated to  affect the function of several organs and tissues and to contribute to the  ...

Assessment of Vascular Function in Patients With Chronic Kidney Disease

Patients with chronic kidney disease (CKD) have significantly increased risk of cardiovascular disease (CVD) compared to the general population, and this is only partially explained by traditional CVD risk factors. Vascular dysfunction is an important non-traditional risk factor, characterized by vascular endothelial dysfunction (most commonly assessed as impaired endothelium-dependent dilation [EDD]) and stiffening of the large elastic arteries. While various techniques exist to assess EDD and large elastic artery stiffness, the most commonly used are brachial artery flow-mediated dilation (FMDBA) and aortic pulse-wave velocity (aPWV), respectively. Both of these noninvasive measures of vascular dysfunction are independent predictors of future cardiovascular events in patients with and without kidney disease. Patients with CKD demonstrate both impaired FMDBA, and increased aPWV. While the exact mechanisms by which vascular dysfunction develops in CKD are incompletely understood, increased oxidative stress and a subsequent reduction in nitric oxide (NO) bioavailability are important contributors. Cellular changes in oxidative stress can be assessed by collecting vascular endothelial cells from the antecubital vein and measuring protein expression of markers of oxidative stress using immunofluorescence. We provide here a discussion of these methods to measure FMDBA, aPWV, and vascular endothelial cell protein expression.

The degree of vascular dysfunction and contributing physiological mechanisms can be assessed in patients with chronic kidney disease by measuring brachial artery flow-mediated dilation, aortic pulse-wave velocity, and vascular endothelial cell protein expression.

L&#39;évaluation de la fonction vasculaire chez les patients atteints de maladie rénale chronique

Patients with chronic kidney disease (CKD) have significantly increased risk of cardiovascular disease (CVD) compared to the general population, and this ...

Slow-release Drug Delivery through Elvax 40W to the Rat Retina: Implications for the Treatment of Chronic Conditions

Diseases of the retina are difficult to treat as the retina lies deep within the eye. Invasive methods of drug delivery are often needed to treat these diseases. Chronic retinal diseases such as retinal oedema or neovascularization usually require multiple intraocular injections to effectively treat the condition. However, the risks associated with these injections increase with repeated delivery of the drug. Therefore, alternative delivery methods need to be established in order to minimize the risks of reinjection. Several other investigations have developed methods to deliver drugs over extended time, through materials capable of releasing chemicals slowly into the eye. In this investigation, we outline the use of Elvax 40W, a copolymer resin, to act as a vehicle for drug delivery to the adult rat retina. The resin is made and loaded with the drug. The drug-resin complex is then implanted into the vitreous cavity, where it will slowly release the drug over time. This method was tested using 2-amino-4-phosphonobutyrate (APB), a glutamate analogue that blocks the light response of the retina. It was demonstrated that the APB was slowly released from the resin, and was able to block the retinal response by 7 days after implantation. This indicates that slow-release drug delivery using this copolymer resin is effective for treating the retina, and could be used therapeutically with further testing.

This paper details how Elvax 40W can be used as a slow-release method for drug delivery to the adult rat retina. The protocol for preparing, loading, and delivering the drug-resin complex to the eye is described.

À libération lente Drug Delivery par Elvax 40W pour le Retina Rat: Implications pour le traitement des maladies chroniques

Diseases of the retina are difficult to treat as the retina lies deep within the eye. Invasive methods of drug delivery are often needed to treat these ...

Busulfan as a Myelosuppressive Agent for Generating Stable High-level Bone Marrow Chimerism in Mice

Bone marrow transplantation (BMT) is often used to replace the bone marrow (BM) compartment of recipient mice with BM cells expressing a distinct biomarker isolated from donor mice. This technique allows for identification of donor-derived hematopoietic cells within the recipient mice, and can be used to isolate and characterize donor cells using various biochemical techniques. BMT typically relies on myeloablative conditioning with total body irradiation to generate niche space within the BM compartment of recipient mice for donor cell engraftment. The protocol we describe here uses myelosuppressive conditioning with the chemotherapeutic agent busulfan. Unlike irradiation, which requires the use of specialized facilities, busulfan conditioning is performed using intraperitoneal injections of 20 mg/kg busulfan until a total dose of 60-100 mg/kg has been administered. Moreover, myeloablative irradiation can have toxic side effects and requires successful engraftment of donor cells for survival of recipient mice. In contrast, busulfan conditioning using these doses is generally well tolerated and mice survive without donor cell support. Donor BM cells are isolated from the femurs and tibiae of mice ubiquitously expressing green fluorescent protein (GFP), and injected into the lateral tail vein of conditioned recipient mice. BM chimerism is estimated by quantifying the number of GFP+ cells within the peripheral blood following BMT. Levels of chimerism&nbsp;>80% are typically observed in the peripheral blood 3-4 weeks post-transplant and remain established for at least 1 year. As with irradiation, conditioning with busulfan and BMT allows for the accumulation of donor BM-derived cells within the central nervous system (CNS), particularly in mouse models of neurodegeneration. This busulfan-mediated CNS accumulation may be more physiological than total body irradiation, as the busulfan treatment is less toxic and CNS inflammation appears to be less extensive. We hypothesize that these cells can be genetically engineered to deliver therapeutics to the CNS.

We describe a protocol whereby busulfan conditioning permits the bone marrow of a recipient mouse to be replaced with bone marrow cells from donor mice ubiquitously expressing green fluorescent protein, in the absence of irradiation. This technique is useful to study bone marrow cell accumulation in the central nervous system.

Busulfan comme agent myélosuppressive pour générer de haut niveau stable de moelle osseuse chez des souris chimérisme

Bone marrow transplantation (BMT) is often used to replace the bone marrow (BM) compartment of recipient mice with BM cells expressing a distinct ...

Intravascular Delivery of Biologics to the Rat Kidney

The renal microvascular compartment plays an important role in the progression of kidney disease and hypertension, leading to the development of End Stage Renal Disease with high risk of death for cardiovascular events. Moreover, recent clinical studies have shown that renovascular structure and function may have a great impact on functional renal recovery after surgery. Here, we describe a protocol for the delivery of drugs into the renal artery of rats. This procedure offers significant advantages over the frequently used systemic administration as it may allow a more localized therapeutic effect. In addition, the use of rodents in pharmacodynamic analysis of preclinical studies may be cost effective, paving the way for the design of translational experiments in larger animal models. Using this technique, infusion of rat recombinant Vascular Endothelial Growth Factor (VEGF) protein in rats has induced activation of VEGF signaling as shown by increased expression of FLK1, pAKT/AKT, pERK/ERK. In summary, we established a protocol for the intrarenal delivery of drugs in rats, which is simple and highly reproducible.

The administration of drugs for recovery of kidney function requires control of the localization and distribution of the therapeutic compound. Here, we describe in detail a simple technique for intrarenal delivery of drugs in rats. This procedure may be easily performed with no mortality and high reproducibility.

Livraison intravasculaire de produits biologiques du rein de rat

The renal microvascular compartment plays an important role in the progression of kidney disease and hypertension, leading to the development of End Stage ...

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.

Évaluation de la fonction rénale chez les modèles murins de la maladie glomérulaire

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

Platelet-based Detection of Nitric Oxide in Blood by Measuring VASP Phosphorylation

Platelets are the blood components responsible for proper blood clotting. Their function is highly regulated by various pathways. One of the most potent vasoactive agents, nitric oxide (NO), can also act as a powerful inhibitor of platelet aggregation. Direct NO detection in blood is very challenging due to its high reactivity with cell-free hemoglobin that limits NO half-life to the millisecond range. Currently, NO changes after interventions are only estimated based on measured changes of nitrite and nitrate (members of the nitrate-nitrite-NO metabolic pathway). However precise, these measurements are rather difficult to interpret vis a vis actual NO changes, due to the naturally high baseline nitrite and nitrate levels that are several orders of magnitude higher than expected changes of NO itself. Therefore, the development of direct and simple methods that would allow one to detect NO directly is long overdue. This protocol addresses a potential use of platelets as a highly sensitive NO sensor in blood. It describes initial platelet rich plasma (PRP) and washed platelet preparations and the use of nitrite and deoxygenated red blood cells as NO generators. Phosphorylation of VASP at serine 239 (P-VASPSer239) is used to detect the presence of NO. The fact that VASP protein is highly expressed in platelets and that it is rapidly phosphorylated when NO is present leads to a unique opportunity to use this pathway to directly detect NO presence in blood.

Here, we present a protocol to address the potential use of platelets as a highly sensitive nitric oxide sensor in blood. It describes initial platelet preparation and the use of nitrite and deoxygenated red blood cells as nitric oxide generators.

Détection du monoxyde d’azote dans le sang en mesurant Phosphorylation VASP plaquettaire

Platelets are the blood components responsible for proper blood clotting. Their function is highly regulated by various pathways. One of the most potent ...