A pesquisa foi apoiada pela NHLBI Research Scientist Development Grant (1K01HL135461-01) para a JAG. Obrigado a David Carmona-Berrio e Isabel Adarve-Rengifo por sua assistência técnica.

Os camundongos foram alojados e atendidos na Divisão de Cuidados com Animais do Centro Médico da Universidade Vanderbilt (VUMC) seguindo as diretrizes do National Institutes of Health (NIH) e o Guia para o Cuidado e Uso de Animais de Laboratório, Departamento de Saúde e Serviços Humanos dos EUA. Todos os procedimentos em animais foram aprovados pelo Comitê Institucional de Cuidados e Uso de Animais da VUMC antes do início dos experimentos.
1. Preparação e dissecção animal
<ol><li>Ligue o germinador e a bomba de água da almofada de aquecimento cerca de 30 minutos antes de iniciar a cirurgia.</li>
	<li>Corte tubo de poliuretano de 0,5 mm de comprimento com um bisturi afiado. Remova 0,2 mm da circunferência fazendo um corte longitudinal para fazer uma braçadeira. NOTA: Esta é uma parte crítica do procedimento de estenose da artéria renal que requer extrema precisão, atenção aos detalhes e paciência. Tente cortar vários pedaços de tubos de poliuretano de cada vez. Realize todo este procedimento sob microscópio.</li>
	<li>Antes de prosseguir, coloque luvas cirúrgicas estéreis e uma máscara cirúrgica.</li>
	<li>Use ratos C57BL/6 (WT) de 6-8 semanas. Use um número igual de ratos machos e fêmeas para evitar qualquer viés sexual.</li>
	<li>Grave o peso dos camundongos antes de realizar a cirurgia. O peso ideal para realizar estenose da artéria renal com tubo de poliuretano é de 18 a 22 g. Manuseie os ratos suavemente e não se agite ao injetar a anestesia. Administrar analgesia pré-operatória (Ketofgen, 5 mg/kg de musculação corporal). NOTA: Ao transferir os ratos de sua sala de alojamento para a sala de cirurgia, leve-os com grande gentileza e cuidado para evitar agitação. Carregar gaiolas de ratos nas mãos em vez de um carrinho é altamente recomendado.</li>
	<li>Anestesiar os camundongos com uma mistura de cetamina (100 mg/kg BW) e xilazina (10 mg/kg BW) através de injeções intraperitoneais (I.P.).</li>
	<li>Coloque o mouse de volta dentro da gaiola até anestesiar completamente. Leva cerca de 4-5 minutos antes que o rato esteja totalmente inconsciente. Aperte a cauda ou o dedo do pé com fórceps para verificar se o rato está totalmente inconsciente e pronto para a cirurgia.</li>
	<li>Coloque o rato de costas em uma toalha de papel. Remova o cabelo do abdômen lateral usando um cortador de cabelo elétrico seguindo a direção oposta do crescimento capilar. Após raspar o local cirúrgico, limpe a área com um esfoliante cirúrgico contendo iodo (ou clorexidina), seguido de uma lavagem com 70% de etanol (ou soro fisiológico estéril).  Repita 3 vezes. NOTA: O procedimento de depilação deve ser realizado a alguma distância ou preferencialmente em um banco diferente do banco do procedimento cirúrgico para evitar qualquer interferência capilar e contaminação capilar durante o procedimento cirúrgico.</li>
	<li>Cubra a almofada de aquecimento com uma folha estéril. Leve o mouse para o banco cirúrgico e coloque na folha estéril, de frente para o lado lateral direito do mouse em direção ao microscópio. Mantenha uma temperatura constante da almofada de 37 °C com água circulante.</li>
	<li>Abra o saco esterilizado contendo todo o equipamento cirúrgico. Usando um microscópio dissecando e uma tesoura afiada estéril, faça uma pequena incisão flanco (perto da costela torácica de13º , a última costela no rato) e cerca de 0,5 cm de distância das vértebras. Prossiga ao longo das vértebras lombares e faça uma incisão de 1 polegada.</li>
	<li>Puxe a pele e o músculo para expor o rim.</li>
	<li>Limpe e remova a gordura circundante usando cotonetes estéreis para isolar a artéria renal. Isole o nervo renal da artéria renal usando fórceps curvos.</li>
	<li>Ao realizar a cirurgia falsa, aplique suturas para fechar a pele e aplicar antibiótico na ferida fechada, em seguida, prossiga para cuidados pós-operatórios. Se não, prossiga com a seguinte seção para stenose a artéria. NOTA: Todo experimento deve ter animais falsos como controles do procedimento cirúrgico. Animais falsos consistem em camundongos que passaram pelo procedimento cirúrgico de expor a artéria renal sem colocar uma braçadeira sobre ela.</li>
</ol>2. Estenose da artéria renal direita
<ol><li>Coloque duas suturas de nylon sob a artéria renal direita, faça nós soltos e, em seguida, coloque o manguito ao redor da artéria renal principal aproximadamente equidistante entre o rim e a bifurcação da aorta</li>
	<li>Feche a braçadeira usando as suturas de nylon. Faça quatro nós para cada sutura para evitar a probabilidade de perder as suturas após a cirurgia.</li>
	<li>Feche a incisão no músculo aplicando uma sutura contínua simples.</li>
	<li>Faça suturas simples interrompidas para fechar a pele.</li>
	<li>Administrar analgésicos aprovados pela IACUC. NOTA: Ferramentas cirúrgicas autoclave antes de cada uso. Se mais de uma cirurgia estiver sendo realizada de uma só vez, limpe todas as ferramentas usadas com um medidor de álcool estéril e coloque-as em germinadores quentes para 15-30 s após cada cirurgia. Troque luvas estéreis também para cada rato.</li>
</ol>3. Cuidados pós-operatórios
<ol><li>Voltem os ratos para sua gaiola e deixem a gaiola meio em frente, meio fora, uma almofada de aquecimento de água circulante por 2 - 3 horas. Adicione alimentos de recuperação da dieta gel dentro da gaiola.</li>
	<li>Administrá-lo com analgésico (cetoprofeno) intraperitoneal (dose: 5 mg/kg BW) no dia seguinte.</li>
	<li>Pesar os ratos pelos próximos dois dias; se alguns camundongos perderem mais de 20% de seu peso consultar o veterinário e decidir se o animal precisa ser eutanizado após o procedimento autorizado pela IACUC.</li>
	<li>Monitore os camundongos diariamente para avaliar vermelhidão, inchaço, dor ou infecção.</li>
	<li>Procure consulta veterinária para complicações pós-operatórias de acordo com as políticas institucionais da IACUC..</li>
</ol>4. Colheita de tecidos
<ol><li>Colher tecido 3 semanas após o procedimento cirúrgico. Grave o peso de cada rato.</li>
	<li>Eutanize o mouse com um procedimento aprovado pela IACUC.</li>
	<li>Coloque o mouse em uma plataforma estéril em posição supina para dissecar.</li>
	<li>Segure e estenda os membros para limitar o movimento.</li>
	<li>Pulverize minuciosamente os ratos com 70% de etanol.</li>
	<li>Faça uma incisão midline para abrir o abdômen e a área do peito usando uma tesoura afiada.</li>
	<li>Puxe a pele e a parede peritoneal.</li>
	<li>Exponha cuidadosamente o coração e perfure o ventrículo direito e exsanguine o rato.</li>
	<li>Remova ambos os rins usando fórceps. Os rins estão localizados na parte de trás dos ratos. NOTA: Não misture os dois rins. Esteja ciente dos rins estenosed, falso e contralateral.</li>
	<li>Remova a cápsula renal, limpe-as de qualquer gordura e grave o peso de cada rim separadamente.</li>
	<li>Corte uma seção longitudinal de ambos os rins e fixada em 4% pfa durante a noite a 4 °C para ser posteriormente processada para incorporação de parafina, para realizar em situ hibridização (ISH) e imunohistoquímica (IHC). Siga os protocolos ISH e IHC conforme relatado23,24.</li>
	<li>Isole o córtex do rim restante e o flash congele em nitrogênio líquido para realizar a mancha ocidental. Armazene amostras a -80 °C até a análise.</li>
	<li>Quantifique expressões de renin, e N-GAL com mancha ocidental como descrito na literatura23,24.</li>
</ol>5. Estatísticas
<ol><li>Use ANOVA unidirecional ou bidirecional para experimentos com três ou mais condições seguidas de testes pós-hoc bonferroni para comparações entre grupos individuais. Considere um valor p igual ou inferior a 0,05 significativo. Use software (por exemplo, GraphPad Prism 8.2) para realizar todas as análises estatísticas.</li>
</ol>

<ol>
	<li>Kashyap, S., 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=Blockade+of+CCR2+reduces+macrophage+influx+and+development+of+chronic+renal+damage+in+murine+renovascular+hypertension.">Blockade of CCR2 reduces macrophage influx and development of chronic renal damage in murine renovascular hypertension.</a> American Journal of Physiology-Renal Physiology. 310 (5), 372-384 (2016).</li><li>Wang, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+inflammatory+biomarkers+after+renal+revascularization+in+atherosclerotic+renal+artery+stenosis.">Changes in inflammatory biomarkers after renal revascularization in atherosclerotic renal artery stenosis.</a> Nephrology Dialysis Transplantation. 31 (9), 1437-1443 (2016).</li><li>Yerram, P., Karuparthi, P. R., Chaudhary, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenesis+and+management+of+renovascular+hypertension+and+ischemic+nephropathy.">Pathogenesis and management of renovascular hypertension and ischemic nephropathy.</a> Minerva Urologica e Nefrologica. 64 (1), 63-72 (2012).</li><li>Covic, A., Gusbeth-Tatomir, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+the+renin-angiotensin-aldosterone+system+in+renal+artery+stenosis,+renovascular+hypertension,+and+ischemic+nephropathy:+diagnostic+implications.">The role of the renin-angiotensin-aldosterone system in renal artery stenosis, renovascular hypertension, and ischemic nephropathy: diagnostic implications.</a> Progress in Cardiovascular Diseases. 52 (3), 204-208 (2009).</li><li>Barreras, A., Gurk-Turner, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiotensin+II+receptor+blockers.">Angiotensin II receptor blockers.</a> Proceedings. 16 (1), 123-126 (2003).</li><li>Sica, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiotensin-converting+enzyme+inhibitors+side+effects--physiologic+and+non-physiologic+considerations.">Angiotensin-converting enzyme inhibitors side effects--physiologic and non-physiologic considerations.</a> Journal of Clinical Hypertension. 6 (7), 410-416 (2004).</li><li>Hill, R. D., Vaidya, P. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiotensin+II+Receptor+Blockers+(ARB,+ARb).">Angiotensin II Receptor Blockers (ARB, ARb).</a> StatPearls. , (2019).</li><li>Durante, 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=Role+of+the+renin-angiotensin-aldosterone+system+in+the+pathogenesis+of+atherosclerosis.">Role of the renin-angiotensin-aldosterone system in the pathogenesis of atherosclerosis.</a> Current Pharmaceutical Design. 18 (7), 981-1004 (2012).</li><li>Chen, 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=Plasma+reactive+carbonyl+species:+Potential+risk+factor+for+hypertension.">Plasma reactive carbonyl species: Potential risk factor for hypertension.</a> Free Radical Research. 45 (5), 568-574 (2011).</li><li>Zhang, X., 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=Angiotensin+receptor+blockade+has+protective+effects+on+the+poststenotic+porcine+kidney.">Angiotensin receptor blockade has protective effects on the poststenotic porcine kidney.</a> Kidney International. 84 (4), 767-775 (2013).</li><li>Zou, X., 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=Renal+scattered+tubular-like+cells+confer+protective+effects+in+the+stenotic+murine+kidney+mediated+by+release+of+extracellular+vesicles.">Renal scattered tubular-like cells confer protective effects in the stenotic murine kidney mediated by release of extracellular vesicles.</a> Scientific Reports. 8 (1), 1263 (2018).</li><li>Kinra, M., Mudgal, J., Arora, D., Nampoothiri, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+insight+into+the+role+of+cyclooxygenase+and+lipooxygenase+pathway+in+renal+ischemia.">An insight into the role of cyclooxygenase and lipooxygenase pathway in renal ischemia.</a> European Review for Medical and Pharmacological Sciences. 21 (21), 5017-5020 (2017).</li><li>Cavalcanti, C. 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=Inhibition+of+PDE5+Restores+Depressed+Baroreflex+Sensitivity+in+Renovascular+Hypertensive+Rats.">Inhibition of PDE5 Restores Depressed Baroreflex Sensitivity in Renovascular Hypertensive Rats.</a> Frontiers in Physiology. 7, 15 (2016).</li><li>Dias, A. T., 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=Sildenafil+ameliorates+oxidative+stress+and+DNA+damage+in+the+stenotic+kidneys+in+mice+with+renovascular+hypertension.">Sildenafil ameliorates oxidative stress and DNA damage in the stenotic kidneys in mice with renovascular hypertension.</a> Journal of Translational Medicine. 12, 35 (2014).</li><li>Lerman, L. O., Chade, A. R., Sica, V., Napoli, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+hypertension:+an+overview.">Animal models of hypertension: an overview.</a> Journal of Laboratory and Clinical Medicine. 146 (3), 160-173 (2005).</li><li>Reckelhoff, J. F., Romero, D. G., Yanes Cardozo, L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sex,+Oxidative+Stress,+and+Hypertension:+Insights+From+Animal+Models.">Sex, Oxidative Stress, and Hypertension: Insights From Animal Models.</a> Physiology (Bethesda). 34 (3), 178-188 (2019).</li><li>Goldblatt, H., Lynch, J., Hanzal, R. F., Summerville, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+Experimental+Hypertension+:+I.+The+Production+of+Persistent+Elevation+of+Systolic+Blood+Pressure+by+Means+of+Renal+Ischemia.">Studies on Experimental Hypertension : I. The Production of Persistent Elevation of Systolic Blood Pressure by Means of Renal Ischemia.</a> Journal of Experimental Medicine. 59 (3), 347-379 (1934).</li><li>Gollan, F., Richardson, E., Goldblatt, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypertension+in+the+systemic+blood+of+animals+with+experimental+renal+hypertension.">Hypertension in the systemic blood of animals with experimental renal hypertension.</a> Journal of Experimental Medicine. 88 (4), 389-400 (1948).</li><li>Lewis, H. A., Goldblatt, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+Experimental+Hypertension:+XVIII.+Experimental+Observations+on+the+Humoral+Mechanism+of+Hypertension.">Studies on Experimental Hypertension: XVIII. Experimental Observations on the Humoral Mechanism of Hypertension.</a> Bulletin of the New York Academy of Medicine. 18 (7), 459-487 (1942).</li><li>Warner, G. M., 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=Genetic+deficiency+of+Smad3+protects+the+kidneys+from+atrophy+and+interstitial+fibrosis+in+2K1C+hypertension.">Genetic deficiency of Smad3 protects the kidneys from atrophy and interstitial fibrosis in 2K1C hypertension.</a> American Journal of Physiology-Renal Physiology. 302 (11), 1455-1464 (2012).</li><li>Lorenz, J. N., 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=Renovascular+hypertension+using+a+modified+two-kidney,+one-clip+approach+in+mice+is+not+dependent+on+the+alpha1+or+alpha2+Na-K-ATPase+ouabain-binding+site.">Renovascular hypertension using a modified two-kidney, one-clip approach in mice is not dependent on the alpha1 or alpha2 Na-K-ATPase ouabain-binding site.</a> American Journal of Physiology-Renal Physiology. 301 (3), 615-621 (2011).</li><li>Ebina, K., Iwabuchi, T., Suzuki, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histological+change+in+permanently+clipped+or+ligated+cerebral+arterial+wall.+Part+II:+Autopsy+cases+of+aneurysmal+neck+clipping.">Histological change in permanently clipped or ligated cerebral arterial wall. Part II: Autopsy cases of aneurysmal neck clipping.</a> Acta Neurochirurgica. 66 (1-2), 23-42 (1982).</li><li>Saleem, M., 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=Sox6:+A+new+modulator+of+renin+expression+during+physiological+conditions.">Sox6: A new modulator of renin expression during physiological conditions.</a> bioRxiv. , (2019).</li><li>Saleem, M., 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=Sox6+as+a+new+modulator+of+renin+expression+in+the+kidney.">Sox6 as a new modulator of renin expression in the kidney.</a> American Journal of Physiology-Renal Physiology. , (2019).</li><li>Chade, A. R., Williams, M. L., Engel, J., Guise, E., Harvey, 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=A+translational+model+of+chronic+kidney+disease+in+swine.">A translational model of chronic kidney disease in swine.</a> American Journal of Physiology-Renal Physiology. 315 (2), 364-373 (2018).</li><li>Xue, Y., Xu, Z., Chen, H., Gan, W., Chong, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low-energy+shock+wave+preconditioning+reduces+renal+ischemic+reperfusion+injury+caused+by+renal+artery+occlusion.">Low-energy shock wave preconditioning reduces renal ischemic reperfusion injury caused by renal artery occlusion.</a> Acta Cir&#250;rgica Brasileira. 32 (7), 550-558 (2017).</li><li>Lalanne, A., Beaudeux, J. L., Bernard, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NGAL:+a+biomarker+of+acute+and+chronic+renal+dysfunction.">NGAL: a biomarker of acute and chronic renal dysfunction.</a> Annales de Biologie Clinique. 69 (6), 629-636 (2011).</li><li>Bolignano, 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=Neutrophil+gelatinase-associated+lipocalin+(NGAL)+as+a+marker+of+kidney+damage.">Neutrophil gelatinase-associated lipocalin (NGAL) as a marker of kidney damage.</a> American Journal of Kidney Diseases. 52 (3), 595-605 (2008).</li><li>Kashyap, S., 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=Development+of+renal+atrophy+in+murine+2+kidney+1+clip+hypertension+is+strain+independent.">Development of renal atrophy in murine 2 kidney 1 clip hypertension is strain independent.</a> Research in Veterinary Science. 107, 171-177 (2016).</li><li>Anderson, W. P., Woods, R. L., Kline, R. L., Korner, P. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+haemodynamic+responses+to+unilateral+renal+artery+stenosis+in+conscious+dogs.">Acute haemodynamic responses to unilateral renal artery stenosis in conscious dogs.</a> Clinical and Experimental Pharmacology and Physiology. 12 (3), 305-309 (1985).</li><li>Imanishi, M., 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=Critical+degree+of+renal+arterial+stenosis+that+causes+hypertension+in+dogs.">Critical degree of renal arterial stenosis that causes hypertension in dogs.</a> Angiology. 43 (10), 833-842 (1992).</li><li>Ziecina, R., Abramczyk, P., Lisiecka, A., Papierski, K., Przybylski, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adrenal-renal+portal+circulation+contributes+to+decrease+in+renal+blood+flow+after+renal+artery+stenosis+in+rats.">Adrenal-renal portal circulation contributes to decrease in renal blood flow after renal artery stenosis in rats.</a> Journal of Physiology and Pharmacology. 49 (4), 553-560 (1998).</li><li>Johnson, J. A., Ichikawa, S., Kurz, K. D., Fowler, W. L., Payne, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pressor+responses+to+vasopressin+in+rabbits+with+3-day+renal+artery+stenosis.">Pressor responses to vasopressin in rabbits with 3-day renal artery stenosis.</a> American Journal of Physiology. 240 (6), 862-867 (1981).</li><li>Eirin, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+glomerular+filtration+rate+after+renal+revascularization+correlate+with+microvascular+hemodynamics+and+inflammation+in+Swine+renal+artery+stenosis.">Changes in glomerular filtration rate after renal revascularization correlate with microvascular hemodynamics and inflammation in Swine renal artery stenosis.</a> Circulation: Cardiovascular Interventions. 5 (5), 720-728 (2012).</li><li>Ma, Z., Jin, X., He, L., Wang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CXCL16+regulates+renal+injury+and+fibrosis+in+experimental+renal+artery+stenosis.">CXCL16 regulates renal injury and fibrosis in experimental renal artery stenosis.</a> American Journal of Physiology-Heart and Circulatory. 311 (3), 815-821 (2016).</li><li>Cheng, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+analysis+of+signaling+pathways+activated+in+a+murine+model+of+two-kidney,+one-clip+hypertension.">Temporal analysis of signaling pathways activated in a murine model of two-kidney, one-clip hypertension.</a> American Journal of Physiology-Renal Physiology. 297 (4), 1055-1068 (2009).</li><li>Wiesel, P., Mazzolai, L., Nussberger, J., Pedrazzini, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-kidney,+one+clip+and+one-kidney,+one+clip+hypertension+in+mice.">Two-kidney, one clip and one-kidney, one clip hypertension in mice.</a> Hypertension. 29 (4), 1025-1030 (1997).</li><li>Johns, C., Gavras, I., Handy, D. E., Salomao, A., Gavras, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Models+of+experimental+hypertension+in+mice.">Models of experimental hypertension in mice.</a> Hypertension. 28 (6), 1064-1069 (1996).</li></ol>

Nenhum conflito de interesses, financeiro ou não, é declarado pelos autores.

A estenose da artéria renal é uma importante causa de hipertensão secundária ou resistente, e lesão renal 1,29. O modelo goldblatt de dois rins (2K1C) foi empregado para estudar a hipertensão renovascular induzida por RAStenosis 1,17,18,19. Vários estudos anteriores utilizando vários modelos de animais mostraram que a estenose n...

A estenose da artéria renal (RAStenosis) é um problema intratável que afeta cerca de 6% das pessoas com mais de 65 anos e em até 40% das pessoas com doença vascular coronariana ou periférica 1,2. Os tratamentos atuais para a doença são limitados; portanto, há uma necessidade crítica de desenvolver novas terapias para tratar hipertensão renovascular ou hipertensão resistente induzida por RAStenosis. O sistema de aldosterona de angiotensina renin (RAAS) é o caminho-chave envolvido na patogênese da hipertensão induzida por RAStenose ou hipertensão renovascular 3,4. Terapias conhecidas voltadas para o RAAS, como inibidores de ACE ou bloqueadores de receptores de angiotensina, aliviam a hipertensão, mas precisam de um exame próximo para insuficiência renal e hipercalemia 5,6,7. Renin catalisa o passo limitante de taxas no RAAS; converte angiotensinogênio em angiotensina I. Na aterosclerose, a formação da placa provoca o estreitamento da artéria renal que impulsiona a secreção de renin, resultando em hipertensão renovascular e danos nos rins8. Vários estudos relataram aumento dos níveis de estresse oxidativo durante a hipertensão renovascular em humanos, que foram corroborados com o modelo de dois camundongos de um rim (2K1C), bem como outros modelos animais hipertensos 2,9,10,11,12,13,14,15,16 . O mecanismo molecular de controle de expressão de renin durante a hipertensão renovascular induzida por RAStenosis não é bem compreendido e merece uma investigação mais aprofundada.
Modelos animais experimentais que recapitulam RAStenosis de forma confiável e reprodutiva são importantes na elucidação dos mecanismos celulares e moleculares do controle de expressão de renin para o desenvolvimento de novas terapias. O modelo de camundongo 2K1C é um modelo experimental bem estabelecido para estudar a patogênese da hipertensão renovascular 17,18,19,20. Este modelo é gerado pela constrição da artéria renal utilizando um clipe 17,20,21, produzindo assim oclusão da artéria renal que resulta em um aumento na expressão de renin e hipertensão 17,19,20,21. No entanto, não há relatórios técnicos disponíveis, que descrevam um procedimento passo a passo para gerar estenose da artéria renal em modelos animais.
Clipes de prata convencionais em forma de U, tubos de poliuretano e outros clipes têm sido usados para restringir a artéria renal para induzir estenose da artéria renal. Alguns estudos têm demonstrado que o design e o material do clipe são fundamentais para obter dados confiáveis e reprodutíveis com o modelo animal 2K1C. De acordo com Lorenz et al., o uso de clipes de prata convencionais projetados por U induz uma baixa taxa de sucesso de hipertensão (40-60%)21. Devido ao desenho do clipe, a artéria renal é pressionada lateralmente, desencadeando algumas constrições e maior probabilidade de ser desalojada da artéria renal. Maleabilidade prateada e ductilidade podem permitir alterações nas larguras do clipe; portanto, causando diferentes níveis de hipertensão entre camundongos. Os dióxidos de prata no clipe podem causar inflamação perivascular, proliferação intimal e granulação tecidual, alterando o diâmetro da artéria renal22. Devido à variabilidade nos níveis de hipertensão obtida com o clipe de prata u-design convencional, Warner et al. e Lorenz et al. usaram com sucesso um tubo de poliuretano de design arredondador para iniciar a estenose da artéria renal em camundongos, gerando uma indução mais confiável e consistente dos dois rins um clipe modelo animal20,21.
Neste relatório, descrevemos um protocolo cirúrgico para gerar RAStenosis experimental em camundongos, usando a tubulação de poliuretano para restringir a artéria renal. O manguito de design redondo de poliuretano é um clipe mais reprodutível, confiável e de baixo custo para gerar estenose no mouse. O objetivo deste modelo experimental é estudar e definir o mecanismo molecular e celular do controle da expressão de renin durante a estenose da artéria renal. Confirmamos o sucesso do modelo de camundongos RAStenosis medindo a expressão de renin e o marcador de lesão renal neutrófilo gelatinase-associado lipocalina (N-GAL).

<table>
	<tbody>
		<tr>
			<td>Diet Gel</td>
			<td>Clear H2O</td>
			<td>Diet-Gel 76A</td>
			<td>Surgery recovery diet</td>
		</tr>
		<tr>
			<td>EMC Heated Hard pad</td>
			<td>Hallowell</td>
			<td>000A2788B</td>
			<td>Heating pads were used to keep mice warm</td>
		</tr>
		<tr>
			<td>Ethilon Nylon Suture</td>
			<td>Ethicon</td>
			<td>662G</td>
			<td>4-0 (1.5 metric), This suture was used to close the peritoneum, and skin</td>
		</tr>
		<tr>
			<td>Ethilon Nylon Suture</td>
			<td>Ethicon</td>
			<td>2815 G</td>
			<td>8-0 (0.4 metric), This suture was used to close cuff to tie and constrict the artery</td>
		</tr>
		<tr>
			<td>Germinator 500</td>
			<td>Braintree Scientific Inc.</td>
			<td>GER 5287</td>
			<td>Sterilize surgical tools between surgeries</td>
		</tr>
		<tr>
			<td>Ketoprofen</td>
			<td>Zoetis</td>
			<td>Ketofen</td>
			<td>Painkiller</td>
		</tr>
		<tr>
			<td>Polyurethane</td>
			<td>Braintree Scientific Inc.</td>
			<td>MRE-025</td>
			<td>This tube was used to initiate stenosis</td>
		</tr>
		<tr>
			<td>Povidone-iodine antiseptic swabsticks</td>
			<td>Medline</td>
			<td>MDS093901</td>
			<td>It was applied after hair removal and surgery on the skin</td>
		</tr>
		<tr>
			<td>Reflex 7 Clip Applier</td>
			<td>Roboz Surgical Instrument Co</td>
			<td>204-1000</td>
			<td>This clip applier was used to apply clip in case one or more sutures went off</td>
		</tr>
		<tr>
			<td>Sterile towel drapes</td>
			<td>Dynarex</td>
			<td>4410</td>
			<td>It was used as a bedsheet for mice during surgery</td>
		</tr>
		<tr>
			<td>Triple antibiotic ointment</td>
			<td>Medi-First</td>
			<td>22312</td>
			<td></td>
		</tr>
		<tr>
			<td>Water pump</td>
			<td>Stryker</td>
			<td>T/pump Professionals</td>
			<td>Used to warm and circulate water in the heating hard pad to keep mice warm during and post-surgery</td>
		</tr>
	</tbody>
</table>

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

A estenose da artéria renal é uma condição comum em pacientes com doença vascular coronariana ou periférica onde o sistema de aldosterona renin angiotensin (RAAS) é superativado. Nesse contexto, há um estreitamento das artérias renais que estimulam o aumento da expressão e liberação de renin, a protease que limita a taxa no RAAS. O aumento resultante da expressão de renin é um conhecido condutor da hipertensão renovascular, frequentemente associada a lesões renais e danos nos órgãos finais. Assim, há um grande interesse em desenvolver novos tratamentos para essa condição. O mecanismo molecular e celular do controle de renin na estenose da artéria renal não é totalmente compreendido e merece uma investigação mais aprofundada. Para induzir estenose da artéria renal em camundongos, foi desenvolvido um modelo de rato Goldblatt modificado de 2 rins 1 (2K1C). O rim direito foi estenoso em camundongos do tipo selvagem e camundongos operados por vergonha foram usados como controle. Após estenose da artéria renal, determinamos expressão de renin e lesão renal. Os rins foram colhidos, e cortices frescos foram usados para determinar a expressão proteica e mRNA de renin. Este modelo animal é reprodutível e pode ser usado para estudar respostas fiofoficológicas, vias moleculares e celulares envolvidas na hipertensão renovascular e lesão renal.

A constrição da artéria renal aumenta a expressão de renin no rim estenoso enquanto reprime a expressão no rim contralateral. O dois rim um clipe (2K1C) ou modelo Goldblatt de estenose induz aumento da expressão renin e lesão renal. Este é reconhecido como o melhor modelo representativo de estenose unilateral da artéria renal em humanos.
A expressão de renin e prorenina (precursora da renin) foram medidas utilizando-se imunoblotting. Os dados mostram que a expressão de renin e pror...

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

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

Um modelo modificado de 2 rim 1 (2K1C) O modelo de rato Goldblatt foi desenvolvido usando tubos de poliuretano para iniciar a estenose da artéria renal, induzindo um aumento na expressão de renin e lesão renal. Aqui, descrevemos um procedimento detalhado de preparação e colocação da braçadeira na artéria renal para gerar um modelo de mouse 2K1C reprodutível e consistente.

Um modelo modificado de rato de dois rins um clipe de regulação de renin em estenose da artéria renal

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

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Research

JoVE Journal

Medicine

4.5K visualizações.  Vanderbilt University Medical Center. Um modelo modificado de 2 rim 1 (2K1C) O modelo de rato Goldblatt foi desenvolvido usando tubos de poliuretano para iniciar a estenose da artéria renal, induzindo um aumento na expressão de renin e lesão renal. Aqui, descrevemos um procedimento detalhado de preparação e colocação da braçadeira na artéria renal para gerar um modelo de mouse 2K1C reprodutível e consistente.

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

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

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

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

Uso de um sistema de suspensão de peso para oclusão da artéria renal isolada

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

Medicina

Mouse Model of Middle Cerebral Artery Occlusion

Stroke is the most common fatal neurological disease in the United States 1. The majority of strokes (88%) result from blockage of blood vessels in the brain (ischemic stroke) 2. Since most ischemic strokes (~80%) occur in the territory of middle cerebral artery (MCA) 3, many animal stroke models that have been developed have focused on this artery. The intraluminal monofilament model of middle cerebral artery occlusion (MCAO) involves the insertion of a surgical filament into the external carotid artery and threading it forward into the internal carotid artery (ICA) until the tip occludes the origin of the MCA, resulting in a cessation of blood flow and subsequent brain infarction in the MCA territory 4. The technique can be used to model permanent or transient occlusion 5. If the suture is removed after a certain interval (30 min, 1 h, or 2 h), reperfusion is achieved (transient MCAO); if the filament is left in place (24 h) the procedure is suitable as a model of permanent MCAO. This technique does not require craniectomy, a neurosurgical procedure to remove a portion of skull, which may affect intracranial pressure and temperature 6. It has become the most frequently used method to mimic permanent and transient focal cerebral ischemia in rats and mice 7,8. To evaluate the extent of cerebral infarction, we stain brain slices with 2,3,5-triphenyltetrazolium chloride (TTC) to identify ischemic brain tissue 9. In this video, we demonstrate the MCAO method and the determination of infarct size by TTC staining.

We demonstrate in the video a method for producing a middle cerebral artery occlusion in adult mice using an intraluminal monofilament. We also show how to evaluate the extent of cerebral infarction by 2,3,5-triphenyltetrazolium chloride (TTC) staining.

Modelo do rato de Oclusão da Artéria Cerebral Média

Stroke is the most common fatal neurological disease in the United States 1. The majority of strokes (88%) result from blockage of blood vessels in 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.

Avaliação da função vascular em pacientes com doença renal crônica

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

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

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

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

Renal Isquemia Reperfusão: um modelo do rato de lesão e regeneração

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

Murine Kidney Transplant Technique

The first mouse kidney transplant technique was published in 19731 by the Russell laboratory. Although it took some years for other labs to become proficient in and utilize this technique, it is now widely used by many laboratories around the world. A significant refinement to the original technique using the donor aorta to form the arterial anastomosis instead of the renal artery was developed and reported in 1993 by Kalina and Mottram 2 with a further advancement coming from the same laboratory in 1999 3. While one can become proficient in this model, a search of the literature reveals that many labs still experience a high proportion of graft loss due to arterial thrombosis. We describe here a technique that was devised in our laboratory that vastly reduces the arterial thrombus reported by others 4,5. This is achieved by forming a heel-and-toe cuff of the donor infra-renal aorta that facilitates a larger anastomosis and straighter blood flow into the kidney.

The goal of this manuscript is to describe the steps required to perform a kidney transplant in a mouse, paying particular attention to the details of the arterial anastomosis.

Rim murino técnica de transplante

The first mouse kidney transplant technique was published in 19731 by the Russell laboratory. Although it took some years for other labs to become ...

Mouse Model for Pancreas Transplantation Using a Modified Cuff Technique

Mouse models have several advantages in transplantation research, including easy handling, a variety of genetically well-defined strains, and the availability of the widest range of molecular probes and reagents to perform in vivo as well as in vitro studies. Based on our experience with various murine transplantation models, we developed a heterotopic pancreas transplantation model in mice with the intent to analyze mechanisms underlying severe ischemia reperfusion injury-associated early graft damage. In contrast to previously described techniques using suture techniques, herein we describe a new procedure using a non-suture cuff technique. 
In recent years, we have performed more than 300 pancreas transplantations in mice with an overall success rate of &gt;90%, a success rate never described before in mouse pancreas transplantation. The backbone of this non-suture cuff technique for graft revascularization consists of two major steps: (I) pulling the recipient vessel over a polyethylene/polyamide cuff and fixing it with a circumferential ligature, and (II) placing the donor vessel over the everted recipient vessel and fixing it with a second circumferential ligature. The resultant continuity of the endothelial layer results in less thrombogenic lesions with high patency rates and, finally, high success rates.
In this model, arterial anastomosis is achieved by pulling the abdominal aorta of the donor graft over the everted common carotid artery of the recipient animal. Venous drainage of the graft is achieved by pulling the portal vein of the graft over the everted external jugular vein of the recipient. This manuscript provides details and crucial steps of the organ recovery and organ implantation procedures, which will allow researchers with microsurgical skills to perform the transplantation successfully in their laboratories.

Among abdominal solid organ transplantation, pancreatic grafts are prone to develop severe ischemia reperfusion injury-associated graft damage, leading eventually to early graft loss. This protocol describes a model of murine pancreas transplantation using a non-suture cuff technique, ideally suited for analyzing these early, deleterious damages.

Modelo do rato para transplante de pâncreas, usando uma técnica modificada do manguito

Mouse models have several advantages in transplantation research, including easy handling, a variety of genetically well-defined strains, and the ...

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.

Avaliação da função renal em modelos do rato da doença Glomerular

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

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

Medição de contratilidade isométrica da artéria mesentérica Mouse usando fio miografia

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

An Efficient Sieving Method to Isolate Intact Glomeruli from Adult Rat Kidney

Preservation of glomerular structure and function is pivotal in the prevention of glomerulonephritis, a category of kidney disease characterized by proteinuria which can eventually lead to chronic and end-stage renal disease. The glomerulus is a complex apparatus responsible for the filtration of plasma from the body. In disease, structural integrity is lost and allows for the abnormal leakage of plasma contents into the urine. A method to isolate and examine glomeruli in culture is critical for the study of these diseases. In this protocol, an efficient method of retrieving intact glomeruli from adult rat kidneys while conserving structural and morphological characteristics is described. This process is capable of generating high yields of glomeruli per kidney with minimal contamination from other nephron segments. With these glomeruli, injury conditions can be mimicked by incubating them with a variety of chemical toxins, including protamine sulfate, which causes foot process effacement and proteinuria in animal models. Degree of injury can be assessed using transmission electron microscopy, immunofluorescence staining, and western blotting. Nephrin and Wilms Tumor 1 (WT1) levels can also be assessed from these cultures. Due to the ease and flexibility of this protocol, the isolated glomeruli can be utilized as described or in a way that best suits the needs of the researcher to help better study glomerular health and structure in diseased states.

The main focus of this protocol is to efficiently isolate viable primary glomeruli cultures with minimal contaminants for use in a variety of downstream applications. The isolated glomeruli retain structural relationships between component cell types and can be cultured ex vivo for a short time.

Um método eficiente de crivagem para isolar intactos glomérulos do rim de rato adulto

Preservation of glomerular structure and function is pivotal in the prevention of glomerulonephritis, a category of kidney disease characterized by ...

Direct Drug Delivery to Kidney via the Renal Artery

There is a need for directed injections to enable increased and specific renal exposure for efficient evaluation of drug targets in the renal research field. Accumulation of drugs in certain organs may give rise to adverse and unwanted effects, depending on the nature of injectate. To minimize spillover and/or accumulation in other tissues, the herein described method directs the formulation into the renal artery bloodstream by inserting a catheter in the infra renal aorta, just below where it branches into the renal artery, resulting in the kidney as first reached organ and distributing of formulation throughout the kidney.
This manuscript provides a detailed description of the method, as well as its challenges and difficulties. It guides the experimenter to become skillful with this type of microsurgery that requires accuracy under sterile conditions. Speed is crucial for minimizing the ischemia and practicing the procedure will increase the chance of successful injections without adverse effects. By modulating the time between injection and reperfusion as well as the injected volume, the risk of spillover to other organs is mitigated. 
Note that this technique is suitable for single dosing strategies.

This manuscript describes a method for targeted delivery to a single kidney via a catheter placed in the infrarenal abdominal aorta in the mouse.

Entrega direta de medicamentos ao rim através da artéria renal

There is a need for directed injections to enable increased and specific renal exposure for efficient evaluation of drug targets in the renal research ...