Ringraziamo Krista den Ouden per l&#39;eccellente assistenza tecnica. Questa tecnica è stata sostenuta finanziariamente dalla Fondazione olandese del rene, concessione C06.2174. MCV è sostenuta dalla organizzazione olandese per la ricerca scientifica (NWO) Vidi-sovvenzione 016.096.359.

Tutti gli esperimenti sono eseguiti in conformità agli animali sperimentali linee guida etiche del comitato animale sperimentale Utrecht. Il protocollo è eseguita sotto la guida e l&#39;approvazione di cura degli animali dell&#39;istituzione dell&#39;autore e comitato utilizzo. CKD è indotta in ratti maschi inbred Lewis (Charles River, Sulzfeld, Germania) all&#39;età di 8 settimane. I ratti sono alloggiati in condizioni standard in un ambiente di luce, temperatura e umidità controllata. 1. Chirurgia Preparazione <ol><li> Sterilizzare strumenti chirurgici: <ul><li> 1 tessuto studente pinza 1-2 denti di 12 centimetri </li><li> 1 studente pinza stile internazionale </li><li> 2 Semken forcipe </li><li> 1 Mayo forbici </li><li> 1 studente iris forbici </li><li> 1 Olsen-Hegar porta-aghi con le forbici </li><li> Coperta </li></ul></li><li> Lista di controllo di inventario: <ul><li> Tavolo operatorio con pad riscaldamento e lampada </li><li> O <sub&gt; 2, isoflurano </li><li> Tessuti </li><li> Shaver </li><li> Bilance </li><li> Set operatoria sterile </li><li> 5x5 garza sterile e 10x10 </li><li> 70% di alcol </li><li> Soluzione di NaCl allo 0,9% </li><li> 1 ml siringa </li><li> Aghi (25G) </li><li> Gel cuscinetti in schiuma: Spongostan </li><li> Vicryl 4.0 e 5.0 </li><li> Buprenorfina 0,03 mg / kg (diluito 1:10 in sale fisiologico) </li><li> Gabbia pulita per i ratti dopo l&#39;intervento chirurgico </li></ul></li></ol> 2. Diritto Uninephrectomy Side <ol><li> Disinfettare il tavolo con il 70% di alcol. </li><li> Luogo topo nella scatola induzione e indurre l&#39;anestesia con il 4% isoflurano e 1 litro flusso di O 2. Nota: a seconda del ceppo di ratto usato, pre e perioperatoria analgesia sono prescritti, questo deve essere discusso con il veterinario locale. </li><li> Trasferimento ratto a tavola e luogo naso a cono. Ratto e cono sono posti su una piastra elettrica per mantenere la temperatura corporea del ratto. Il ratto is ventilato con una miscela di 2% isoflurano e 1 litro O 2. </li><li> Shave fianco del ratto e disinfettare con alcool. Lavare e disinfettare le mani e indossare guanti chirurgici sterili. Indossare una protezione della testa e una maschera. Fai per 1 - 1,5 centimetri incisione parallela alle costole con pinze anatomiche e forbici smussate. Esporre il rene per via smussa dei muscoli della schiena. </li><li> Il polo inferiore del rene è visibile, utilizzare con attenzione le piccole pinze smussate per afferrare il tessuto adiposo perirenale. Esternare il rene tirando delicatamente il grasso perirenale con una pinza. Fare attenzione a non disturbare la ghiandola surrenale durante questa procedura. La ghiandola surrenale può essere facilmente eliminati mettendo una pinza nella sede mediale nel tessuto grasso e delicatamente spostata verso l&#39;alto fra la ghiandola surrenale e il rene come illustrato nella Figura 2. </li><li> Posizionare rene delicatamente sulla garza e chiara circostante grasso e tessuto connettivo. Identificare l&#39;arteria e la vena renale e inserire una legatura (5,0Vicryl) con un singolo nodo intorno i vasi, ma non legare il nodo. </li><li> Spostare il nodo sciolto accuratamente lungo i vasi verso l&#39;aorta di circa 0,5 centimetri, per creare spazio tra il rene e il nodo. Questo viene fatto per evitare la legatura di scivolare dopo il taglio. </li><li> Legare nodo con due nodi doppi. Se perfusione viene interrotta, il colore del rene passerà immediatamente marrone. Non tagliare le estremità della legatura. </li><li> Tagliare i vasi renali vicino al rene e rimuovere il rene. Tirare delicatamente le lunghe estremità della legatura per controllare il sanguinamento dei vasi renali. La legatura deve rimanere sul posto a bordo della nave. Tagliare le lunghe estremità della legatura. Nota, i vasi renali remnant si ritrae immediatamente nella cavità addominale. </li><li> Asciugare rene asportato asciughi e si pesa. </li><li> Chiudere l&#39;incisione del muscolo scheletrico con l&#39;esecuzione di suture con Vicryl 5.0. Iniettare 0,10 ml (0,03 mg / kg) buprenorfina im in dell&#39;arto posteriore. Chiudere ilpelle con Vicryl 4.0 con suture intracutanee. Ciò impedisce che i ratti di aprire la ferita. Nota: a causa di depressione respiratoria in ratti Lewis, diamo solo l&#39;analgesia post-operatoria poco prima di spegnere l&#39;isoflurano. </li><li> La casa del topo in una gabbia pulita solitario, con facile accesso al cibo e all&#39;acqua. Posizionare la metà della gabbia sul riscaldamento pad O / N e controllare il topo con cura il giorno successivo. Quando la ferita è chiusa e il ratto è attiva, che è normalmente compresa tra 6 e 12 ore dopo l&#39;intervento chirurgico, il ratto può essere riposto nel contenitore gruppo. </li></ol> 3. Lato sinistro nefrectomia Subtotale <ol><li> Sette giorni dopo, gli stessi preparati sono fatte sul lato sinistro come per il lato destro come descritto 2,1-2,5. Prima dell&#39;intervento chirurgico, la quantità di tessuto renale che deve essere rimosso corrispondente a circa 2/3rds del peso del rene destro è calcolato. Preparare piccoli pezzi di schiuma gel (Spongostan, Johnson &amp; Johnson, New Jersey) prima di un intervento chirurgico. </li&gt; <li> Collocare forbici taglienti attorno al polo superiore del rene sinistro e resecare il polo superiore in un colpo (per l&#39;ubicazione forbice, vedi Figura 3). Coprire immediatamente con un pezzo di schiuma gel ed esercitare una leggera pressione con una garza sterile. </li><li> Ripetere con il polo inferiore del rene come in 3.3. </li><li> Sollevare rene per prevenire la coagulazione sulla pelle. Mantenere una pressione delicata su entrambe le imbottiture in gel con garza sterile fino a quando l&#39;emorragia si ferma. Quando il sanguinamento persiste, aggiungere un altro rilievo di gomma piuma. </li><li> Posizionare residuo rene con aderenti imbottiture in gel all&#39;interno dell&#39;addome. Chiudere il muscolo e pelle e seguire 2,9-2,12. </li></ol> 4. Sham Chirurgia <ol><li> Sham controlli azionati subiscono la stessa procedura, al fine di esporre i reni. Invece di estirpare il rene o il taglio dei pali una settimana più tardi, entrambi i reni sono decapsulated ad un intervallo di una settimana, facendo attenzione a non disturbare le ghiandole surrenali. Chiusura della ferita e di assistenza post-operatoria sono identical a 2.11 e 2.12. </li></ol>

<ol>
	<li>AS, G. o., Chertow, G. M., Fan, D., McCulloch, C. E., Hsu, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+kidney+disease+and+the+risks+of+death,+cardiovascular+events,+and+hospitalization.">Chronic kidney disease and the risks of death, cardiovascular events, and hospitalization.</a> N. Engl. J. Med. 351 (13), 1296-1305 (2004).</li><li>Fleck, C., Appenroth, D., 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=Suitability+of+5/6+nephrectomy+(5/6NX)+for+the+induction+of+interstitial+renal+fibrosis+in+rats--influence+of+sex,+strain,+and+surgical+procedure.">Suitability of 5/6 nephrectomy (5/6NX) for the induction of interstitial renal fibrosis in rats--influence of sex, strain, and surgical procedure.</a> Exp. Toxicol. Pathol. 57 (3), 195-205 (2006).</li><li>Griffin, K. A., Picken, M. M., Churchill, M., Churchill, P., Bidani, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+and+structural+correlates+of+glomerulosclerosis+after+renal+mass+reduction+in+the+rat.">Functional and structural correlates of glomerulosclerosis after renal mass reduction in the rat.</a> J. Am. Soc. Nephrol. 11 (3), 497-506 (2000).</li><li>Kang, D. H., Kanellis, 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=Role+of+the+microvascular+endothelium+in+progressive+renal+disease.">Role of the microvascular endothelium in progressive renal disease.</a> J. Am. Soc. 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. K., De N, G., Zatz, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+nitric+oxide+synthase+inhibition+aggravates+glomerular+injury+in+rats+with+subtotal+nephrectomy.">Chronic nitric oxide synthase inhibition aggravates glomerular injury in rats with subtotal nephrectomy.</a> J. Am. Soc. Nephrol. 5 (7), 1498-1507 (1995).</li><li>Fujihara, C. K., Sena, C. R., Malheiros, D. M., Mattar, A. L., Zatz, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short-term+nitric+oxide+inhibition+induces+progressive+nephropathy+after+regression+of+initial+renal+injury.">Short-term nitric oxide inhibition induces progressive nephropathy after regression of initial renal injury.</a> Am. J. Physiol. Renal Physiol. 290 (3), F632-F640 (2006).</li><li>Dikow, R., Kihm, L. P., 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=Increased+infarct+size+in+uremic+rats:+reduced+ischemia+tolerance?.">Increased infarct size in uremic rats: reduced ischemia tolerance?.</a> J. Am. Soc. Nephrol. 15 (6), 1530-1536 (2004).</li><li>Elrashidy, R. A., Asker, M. E., Mohamed, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pioglitazone+attenuates+cardiac+fibrosis+and+hypertrophy+in+a+rat+model+of+diabetic+nephropathy.">Pioglitazone attenuates cardiac fibrosis and hypertrophy in a rat model of diabetic nephropathy.</a> J. Cardiovasc. Pharmacol. 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. M. <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+two+protocols+of+uremic+rat+model:+partial+nephrectomy+and.">Evaluation of two protocols of uremic rat model: partial nephrectomy and.</a> 25 (6), 935-943 (2003).</li><li>Griffin, K. A., Picken, M., Bidani, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+of+renal+mass+reduction+is+a+critical+modulator+of+subsequent+hypertension+and+glomerular+injury.">Method of renal mass reduction is a critical modulator of subsequent hypertension and glomerular injury.</a> J. Am. Soc. Nephrol. 4 (12), 2023-2031 (1994).</li><li>Meyer, F., Ioshii, S. 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>

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La rimozione chirurgica di 5/6th di massa renale nel ratto Lewis, in combinazione con una dieta ad alto contenuto di sale e NOS temporanea inibizione porta ad un modello di CKD che assomiglia CKD umana e permette lo studio dei meccanismi causali e l&#39;efficacia degli interventi terapeutici in CKD. Il modello di nefrectomia 5/6th è un modello ben noto e ampiamente descritto per la CKD. Tuttavia, la semplice rimozione 5/6th di massa renale non porta a insufficienza renale immediato in tutti...

A causa della sua natura progressiva, conseguente fine malattia renale stadio, e associati morbilità e mortalità cardiovascolare, insufficienza renale cronica è un crescente problema di salute pubblica 1. Rallentare la progressione della malattia renale cronica è quindi una priorità di sanità. Dal CKD è caratterizzata da alterazioni complesse di omeostasi, modelli animali integrativi sono necessari per studiare lo sviluppo e la progressione della malattia renale cronica. Il rene costituito da una vasta gamma di tipi cellulari che interagiscono tra di loro. Questa complessità non può essere imitato in vitro. Per studiare nuovi interventi terapeutici in CKD, si usa il modello 5/6th nefrectomia ablazione, un modello sperimentale noto di malattia renale progressiva, simile ad alcuni aspetti della CKD umana 2,3. La riduzione lorda in massa renale provoca progressiva glomerulare e lesioni tubulo-interstiziale, perdita di nefroni residue e lo sviluppo di ipertensione sistemica e glomerulare. Si è associato con progressive perdita capillare intrarenale 4, l&#39;infiammazione e la glomerulosclerosi. Fattori di rischio per CKD invariabilmente impatto sulla funzione endoteliale 5. Abbiamo utilizzato un ceppo di ratto (Lewis) che è relativamente resistente a sviluppo di CKD e quindi abbiamo combinato rimozione di 5/6th di massa renale con l&#39;ossido nitrico (NO) esaurimento 6, 7, 8 e una dieta ricca di sale 9. Dopo l&#39;arrivo e acclimatazione, gli animali ricevono un inibitore della NO sintetasi (L-NNA) integrato per l&#39;acqua potabile (20 mg / L) per un periodo di 4 settimane, seguito da uninephrectomy lati destro (UNX) con la continuazione di L-NNA dopo due giorni . Una settimana più tardi, totale parziale nefrectomia (SNX) ossia la rimozione di 2/3rds di massa renale è eseguita sul lato sinistro. Dopo SNX, animali viene permesso di recuperare per 2 giorni, seguiti nuovamente da 20 mg / L LNNA in acqua potabile per un periodo di 4 settimane. Una dieta ricca di sale (6%), integrati in terra chow (vedi linea del tempo figura 1), è proseguito per tutto l&#39;esperimento. Tha motivo di eseguire la UNX sul lato destro e il SNX sul lato sinistro è che i vasi renali sono più lunghi sul lato sinistro, che rende più facile l&#39;accesso al rene senza allungare i vasi troppo quando il rene è esposto all&#39;esterno del corpo . In letteratura sono descritti i modelli in cui i poli del rene sinistro vengono rimossi prima, seguita dalla UNX del rene destro una settimana dopo 10,11,12. Nelle nostre mani questo modello ha mostrato uno sviluppo molto più rapido di insufficienza renale, ma anche una variazione molto più grande nella perdita della funzione renale. Progressione dell&#39;insufficienza renale è seguita nel tempo misurando plasma urea, la pressione sanguigna sistolica e proteinuria. Con sei settimane dopo SNX, insufficienza renale si è sviluppata, caratterizzata da marcata riduzione della velocità di filtrazione glomerulare (69%) e del flusso plasmatico renale (62%) 13 l&#39;ipertensione (pressione sistolica&gt; 150 mmHg), proteinuria (&gt; 50 mg/24 hr) e mite uremia (&gt; 10 mm). Caratteristiche istologiche comprendono Tubuldanni o-interstiziale riflessa da infiammazione, atrofia tubulare e fibrosi e glomerulosclerosi focale che porta alla massiccia riduzione dei glomeruli sani nella popolazione residuo (&lt;10%). Il follow-up fino a 12 settimane dopo SNX mostra ulteriore progressione della malattia renale cronica, fornendo una finestra di opportunità per la valutazione degli interventi terapeutici.

<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

Malattia renale cronica (CKD) è un problema globale. Rallentare la progressione della malattia renale cronica è una delle principali priorità di salute. Dal CKD è caratterizzata da alterazioni complesse di omeostasi, modelli animali integrativi sono necessari per studiare lo sviluppo e la progressione della malattia renale cronica. Per studiare lo sviluppo di interventi terapeutici CKD e romanzo in CKD, si usa il modello ablazione nefrectomia 5/6th, un modello sperimentale ben noto di malattia renale progressiva, simile a molti aspetti della CKD umana. La riduzione lorda in massa renale provoca progressiva glomerulare e lesioni tubulo-interstiziale, perdita di nefroni residue e lo sviluppo di ipertensione sistemica e glomerulare. Esso è anche associato con progressiva perdita intrarenale capillare, infiammazione e glomerulosclerosi. Fattori di rischio per CKD invariabilmente impatto sulla funzione endoteliale. Per simulare questo, uniamo la rimozione di 5/6th di massa renale con l&#39;ossido nitrico (NO), l&#39;esaurimento e una dieta ricca di sale. Dopo l&#39;arrivo e acclimatazione, animali riCeive un inibitore della NO sintasi (NG-nitro-L-arginina) (L-NNA) addizionato all&#39;acqua potabile (20 mg / L) per un periodo di 4 settimane, seguite da destra uninephrectomy lati. Una settimana più tardi, una nefrectomia totale parziale (SNX) viene eseguita sul lato sinistro. Dopo SNX, sono ammessi animali di recuperare per due giorni seguiti da LNNA in acqua potabile (20 mg / L) per un ulteriore periodo di 4 settimane. Una dieta ricca di sale (6%), integrati in terra chow (vedi linea del tempo figura 1), è proseguito per tutto l&#39;esperimento. Progressione dell&#39;insufficienza renale è seguita nel tempo misurando plasma urea, la pressione sanguigna sistolica e proteinuria. Con sei settimane dopo SNX, insufficienza renale si è sviluppata. La funzione renale è misurata con inulina &#39;gold standard&#39; e l&#39;acido ippurico para-amino (PAH), la tecnologia di liquidazione. Questo modello di CKD è caratterizzata da una riduzione della velocità di filtrazione glomerulare (GFR) e del flusso plasmatico renale (ERPF), ipertensione (pressione sistolica&gt; 150 mmHg), proteinuria (&gt; 50 mg/24 ore) E uremia lieve (&gt; 10 mm). Caratteristiche istologiche comprendono danno tubulo-interstiziale riflessa da infiammazione, atrofia tubulare e fibrosi e glomerulosclerosi focale che porta alla massiccia riduzione dei glomeruli sani nella popolazione residuo (&lt;10%). Il follow-up fino a 12 settimane dopo SNX mostra ulteriore progressione della malattia renale cronica.

Dopo nefrectomia subtotale, circa 1/6th della massa renale totale è a sinistra. Figura 4 mostra il peso della parte rimossa del rene destro con deviazione media e standard in due esperimenti precedenti. Si dovrebbe tenere presente che nella settimana dopo UNX, ipertrofia del rene sinistro si verifica, indicante che il peso che deve essere rimosso calcolato sulla base del peso del rene destro provoca sempre in meno di rimozione 5/6th. Tuttavia, poiché non è possibile determinare il peso del rene sinis...

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

Un metodo a due stadi per stabilire malattia renale cronica (CKD) nel ratto Lewis rimuovere chirurgicamente 5/6th di massa renale è descritta. Combinazione della procedura chirurgica, NOS-inibizione e di una dieta ad alto contenuto di sale porta a un modello simile CKD umano, permettendo lo studio di meccanismi e di sviluppo di nuovi interventi terapeutici causali.

5/6th nefrectomia in combinazione con dieta di sale e di ossido nitrico sintasi Inibizione di indurre la malattia renale cronica nel ratto 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 Views.  University Medical Center Utrecht. Un metodo a due stadi per stabilire malattia renale cronica (CKD) nel ratto Lewis rimuovere chirurgicamente 5/6th di massa renale è descritta. Combinazione della procedura chirurgica, NOS-inibizione e di una dieta ad alto contenuto di sale porta a un modello simile CKD umano, permettendo lo studio di meccanismi e di sviluppo di nuovi interventi terapeutici causali.

Video: 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.

Tecniche analitiche per il saggio Bioattività Ossido Nitrico

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 ...

Ricerca

Medicina

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.

Ablazione transcatetere in combinazione con i tasti chiusura appendice atriale per la fibrillazione atriale

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.

Cariotipo spettrale allo studio anomalie cromosomiche negli esseri umani e nei topi con malattia del rene policistico

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 modello del Chronic Infusion nutrienti nel ratto

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.

Valutazione della funzione vascolare nei pazienti con malattia renale cronica

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.

Lento rilascio Drug Delivery attraverso Elvax 40W al Rat Retina: Implicazioni per il trattamento di condizioni croniche

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 come mielosoppressiva agente per la generazione stabile ad alto livello di midollo osseo chimerismo nei topi

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.

Consegna intravascolare di Biologics al rene di ratto

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.

Valutazione della funzione del rene in modelli murini di malattia glomerulare

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.

Rilevazione basata su piastrine di ossido nitrico nel sangue misurando VASP fosforilazione

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