研究得到了NHLBI研究科学家发展补助金（1K01HL135461-01）对JAG的支持。感谢大卫·卡莫纳-贝里奥和伊莎贝尔·阿达尔维-伦吉福的技术援助。

根据美国国立卫生研究院（NIH）指南和美国卫生与公众服务部的范德比尔特大学医学中心（VUMC）动物护理部对小鼠进行饲养和护理。在开始实验之前，所有动物程序都得到了VUMC机构动物护理和使用委员会的批准。
1. 动物准备和解剖
<ol><li>在开始手术前约30分钟打开加热垫的发芽器和水泵。</li>
	<li>用锋利的手术刀切割0.5毫米长的聚氨酯管。取下 0.2 毫米的周长，纵向切开，做成袖口。 注意：这是肾动脉狭窄手术的关键部分，需要极高的精确度，注重细节和耐心。尝试一次切割几块聚氨酯管。在显微镜下执行所有这些程序。</li>
	<li>在继续之前，戴上外科无菌手套和外科口罩。</li>
	<li>使用6-8周的C57BL / 6野生型（WT）小鼠。使用相同数量的雄性和雌性小鼠以避免任何性别偏见。</li>
	<li>在进行手术之前记录小鼠的体重。用聚氨酯管进行肾动脉狭窄的理想重量为18-22克。轻轻地处理小鼠，注射麻醉剂时不要搅拌。给予术前镇痛（酮促，5毫克/ kg身体重量）。 注意：在将小鼠从其住房室转移到手术室时，请非常温和和小心地携带它们，以避免激动。强烈建议将小鼠笼子放在手中而不是手推车中。</li>
	<li>通过腹膜内（I.P.）注射用氯胺酮（100mg / kg BW）和甲苯噻嗪（10mg / kg BW）的混合物麻醉小鼠。</li>
	<li>将鼠标放回笼内，直到完全麻醉。鼠标完全失去知觉大约需要4-5分钟。用镊子捏住尾巴或脚趾，检查鼠标是否完全失去知觉并准备好进行手术。</li>
	<li>将鼠标仰面放在纸巾上。沿着毛发生长的相反方向，使用电动理发器去除外腹部的毛发。剃除手术部位后，用含有碘（或氯己定的）的手术磨砂膏清洁该区域，然后用70%乙醇（或无菌盐水）冲洗。 重复3次。 注意：脱毛手术必须在一定距离上进行，或者最好在与手术手术台不同的工作台上进行，以避免手术过程中的任何头发干扰和头发污染。</li>
	<li>用无菌片盖住加热垫。将鼠标带到手术台上，放在无菌片上，面向小鼠的右侧侧向显微镜。用循环水保持37°C的恒定焊盘温度。</li>
	<li>打开装有所有手术设备的灭菌袋。使用解剖显微镜和无菌利器剪刀，做一个小的侧腹切口（靠近第 13胸肋骨，小鼠的最后一根肋骨），距离椎骨约0.5厘米。沿着腰椎继续，做一个1英寸的切口。</li>
	<li>拉回皮肤和肌肉以暴露肾脏。</li>
	<li>使用无菌棉签清洁并去除周围的脂肪，以隔离肾动脉。使用弯曲的镊子将肾神经与肾动脉隔离开来。</li>
	<li>进行假手术时，应用缝合线以闭合皮肤并将抗生素应用于闭合的伤口，然后进行 术后护理。 如果没有，请继续执行以下部分以切断动脉狭窄。 注意：每个实验都应该有假动物作为外科手术的对照。假动物由经过暴露肾动脉而不在其上放置袖带的外科手术的小鼠组成。</li>
</ol>2. 右肾动脉狭窄
<ol><li>在右肾动脉下方放置两条尼龙缝合线，打出松散的结，然后将袖带放在肾脏和主动脉分叉之间大致等距的肾主动脉周围</li>
	<li>使用尼龙缝合线关闭袖口。每个缝合线打四个结，以避免手术后失去缝合线的可能性。</li>
	<li>通过应用简单的连续缝合线来关闭肌肉中的切口。</li>
	<li>进行简单的断续缝合以闭合皮肤。</li>
	<li>使用IACUC批准的镇痛药。 注意：每次使用前高压灭菌手术工具。如果一次进行多次手术，请用无菌酒精计擦拭所有使用过的工具，并在每次手术后将其放入热发芽器中15-30秒。也为每只老鼠更换无菌手套。</li>
</ol>3. 术后护理
<ol><li>将小鼠放回笼子里，让笼子一半打开，一半关闭，循环水加热垫2-3小时。在笼子内添加凝胶日粮恢复食品。</li>
	<li>第二天腹膜内注射止痛药（酮洛芬）（剂量：5毫克/千克体重）。</li>
	<li>称量接下来两天的小鼠;如果一些小鼠体重减轻超过20%，请咨询兽医，并决定动物是否需要按照适当的IACUC授权程序进行安乐死。</li>
	<li>每天监测小鼠以评估发红，肿胀，疼痛或感染。</li>
	<li>根据机构IACUC政策寻求兽医咨询术后并发症。</li>
</ol>4. 组织采集
<ol><li>手术后3周收获组织。记录每只鼠标的重量。</li>
	<li>用IACUC批准的程序对小鼠实施安乐死。</li>
	<li>将鼠标放在无菌平台上，以仰卧位解剖。</li>
	<li>固定并伸展四肢以限制运动。</li>
	<li>用70%乙醇彻底喷洒小鼠。</li>
	<li>做一个中线切口，用锋利的剪刀打开腹部和胸部区域。</li>
	<li>拉回皮肤和腹膜壁。</li>
	<li>小心地暴露心脏并穿刺右心室并释放小鼠。</li>
	<li>使用镊子取出两个肾脏。肾脏位于小鼠的背部。 注意：不要混合两个肾脏。注意狭窄的肾脏、假肾和对侧肾脏。</li>
	<li>取出肾囊，从任何脂肪中清除它们，并分别记录每个肾脏的重量。</li>
	<li>切开两个肾脏的纵向切片，并在4°C下固定在4%PFA中过夜，以便随后处理石蜡包埋，以进行原位杂交（ISH）和免疫组织化学（IHC）。遵循23，24 报告的 ISH 和 IHC 协议。</li>
	<li>分离剩余肾脏的皮质，并在液氮中快速冷冻以进行蛋白质印迹。将样品储存在-80°C直至分析。</li>
	<li>用蛋白质印迹量化肾素和N-GAL表达，如文献23，24中所述。</li>
</ol>5. 统计
<ol><li>使用单向或双向方差分析进行具有三个或更多条件的实验，然后使用 Bonferroni 事后检验来比较各个组之间的比较。考虑 p 值等于或小于 0.05 的显著性。使用软件（例如，图形键盘棱镜 8.2）执行所有统计分析。</li>
</ol>

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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pressor+responses+to+vasopressin+in+rabbits+with+3-day+renal+artery+stenosis.">Pressor responses to vasopressin in rabbits with 3-day renal artery stenosis.</a> American Journal of Physiology. 240 (6), 862-867 (1981).</li><li>Eirin, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+glomerular+filtration+rate+after+renal+revascularization+correlate+with+microvascular+hemodynamics+and+inflammation+in+Swine+renal+artery+stenosis.">Changes in glomerular filtration rate after renal revascularization correlate with microvascular hemodynamics and inflammation in Swine renal artery stenosis.</a> Circulation: Cardiovascular Interventions. 5 (5), 720-728 (2012).</li><li>Ma, Z., Jin, X., He, L., Wang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CXCL16+regulates+renal+injury+and+fibrosis+in+experimental+renal+artery+stenosis.">CXCL16 regulates renal injury and fibrosis in experimental renal artery stenosis.</a> American Journal of Physiology-Heart and Circulatory. 311 (3), 815-821 (2016).</li><li>Cheng, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+analysis+of+signaling+pathways+activated+in+a+murine+model+of+two-kidney,+one-clip+hypertension.">Temporal analysis of signaling pathways activated in a murine model of two-kidney, one-clip hypertension.</a> American Journal of Physiology-Renal Physiology. 297 (4), 1055-1068 (2009).</li><li>Wiesel, P., Mazzolai, L., Nussberger, J., Pedrazzini, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-kidney,+one+clip+and+one-kidney,+one+clip+hypertension+in+mice.">Two-kidney, one clip and one-kidney, one clip hypertension in mice.</a> Hypertension. 29 (4), 1025-1030 (1997).</li><li>Johns, C., Gavras, I., Handy, D. E., Salomao, A., Gavras, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Models+of+experimental+hypertension+in+mice.">Models of experimental hypertension in mice.</a> Hypertension. 28 (6), 1064-1069 (1996).</li></ol>

作者不申报任何利益冲突，包括财务或其他方面。

肾动脉狭窄是继发性或难治性高血压和肾损伤的重要病因1，29.两个肾脏一夹（2K1C）Goldblatt模型已被用于研究RAS狭窄诱导的肾血管高血压1，17，18，19。以前使用各种动物模型的一些研究表明，肾动脉狭窄是肾素过表达和释放的强大刺激因子，肾损伤<sup class=...

肾动脉狭窄（RAStenosis）是一种棘手的问题，影响约6%的65岁以上人群和高达40%的冠状动脉或外周血管疾病患者1，2。目前对这种疾病的治疗是有限的;因此，迫切需要开发新的疗法来治疗肾血管性高血压或由RAStenosis诱导的抵抗性高血压。肾素血管紧张素醛固酮系统（RAAS）是参与RAS狭窄诱导的高血压或肾血管性高血压发病机制的关键途径3，4。针对 RAAS 的已知疗法（如血管紧张素转换酶抑制剂或血管紧张素受体阻滞剂）可缓解高血压，但需要仔细检查肾衰竭和高钾血症5，6，7。肾素催化RAAS中的速率限制步骤;它将血管紧张素原转化为血管紧张素I。在动脉粥样硬化中，斑块形成导致肾动脉变窄，从而驱动肾素分泌，导致肾血管高血压和肾脏损伤8。许多研究报告了人类肾血管性高血压期间氧化应激水平升高，这与两个肾脏一夹（2K1C）小鼠模型以及其他高血压动物模型2，9，10，11，12，13，14，15，16证实。.RAS狭窄诱发的肾血管性高血压期间肾素表达控制的分子机制尚不清楚，需要进一步研究。
可靠且可重复地概括RAStenosis的实验动物模型对于阐明肾素表达控制的细胞和分子机制以开发新疗法非常重要。2K1C小鼠模型是研究肾血管性高血压发病机制的成熟实验模型17，18，19，20。该模型通过使用夹子17，20，21的肾动脉收缩而生成，因此产生肾动脉阻塞，导致肾素表达增加和高血压17，19，20，21。然而，没有可用的技术报告，其中描述了在动物模型中产生肾动脉狭窄的分步过程。
传统的U形银夹，聚氨酯管和其他夹子已被用于收缩肾动脉以诱导肾动脉狭窄。一些研究表明，夹子的设计和材料对于获得2K1C动物模型的可靠和可重复数据至关重要。根据Lorenz等人的说法，使用传统的U型设计银夹诱导高血压的成功率较低（40-60%）21。由于夹子设计，肾动脉被横向压压，引发一些收缩和更大的概率从肾动脉中移出。银的延展性和延展性可能允许夹子宽度的变化;因此，在小鼠中引起不同的高血压水平。夹子上的二氧化银可引起血管周围炎症、内膜增殖和组织肉芽形成，改变肾动脉直径22。由于用常规U-design银夹获得的高血压水平的可变性，Warner等人和Lorenz等人已经成功地使用圆形设计的聚氨酯管来启动小鼠的肾动脉狭窄，从而产生更可靠和一致的诱导两肾一夹动物模型20，21。
在这份报告中，我们描述了一种手术方案，以在小鼠中产生实验性RAStenosis，使用聚氨酯管来收缩肾动脉。聚氨酯圆形设计袖口是一种更具可重复性，可靠性和低成本的夹子，可在小鼠中产生狭窄。该实验模型的目标是研究和定义肾动脉狭窄期间肾素表达控制的分子和细胞机制。我们通过测量肾素表达和肾损伤标志物中性粒细胞明胶酶相关脂质运载蛋白（N-GAL）来确认RAS狭窄小鼠模型的成功。

<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

肾动脉狭窄是冠状动脉或外周血管疾病患者的常见病症，其中肾素血管紧张素醛固酮系统（RAAS）过度活化。在这种情况下，肾动脉变窄，刺激肾素（RAAS中的限速蛋白酶）表达和释放的增加。由此产生的肾素表达升高是肾血管性高血压的已知驱动因素，通常与肾损伤和终末器官损伤有关。因此，人们对开发针对这种情况的新疗法非常感兴趣。肾动脉狭窄中肾素控制的分子和细胞机制尚不完全清楚，需要进一步研究。为了诱导小鼠肾动脉狭窄，开发了一种修饰的2肾1夹（2K1C）Goldblatt小鼠模型。野生型小鼠右肾被冷冻，假手术小鼠用作对照。肾动脉狭窄后，我们确定了肾素表达和肾损伤。收获肾脏，并使用新鲜皮质来确定肾素的蛋白质和mRNA表达。该动物模型是可重复的，可用于研究参与肾血管性高血压和肾损伤的病理生理学反应，分子和细胞途径。

肾动脉收缩增加狭窄肾中的肾素表达，同时抑制对侧肾的表达。狭窄的两个肾一夹（2K1C）或Goldblatt模型诱导肾素表达增加和肾损伤。这被认为是人类单侧肾动脉狭窄的最佳代表性模型。
使用免疫印迹法测量肾素和原脯素（肾素的前体）的表达。数据显示，与对侧肾和假肾相比，狭窄肾中的肾素和原脯素表达增加，表明袖带收缩肾动脉，导致肾灌注改变（图1</s...

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

使用聚氨酯管开发改良的2肾1夹（2K1C）Goldblatt小鼠模型以启动肾动脉狭窄，诱导肾素表达增加和肾损伤。在这里，我们描述了准备袖带并将其置于肾动脉上以产生可重复且一致的2K1C小鼠模型的详细程序。

肾动脉狭窄肾素调节的改良双肾单夹小鼠模型

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

a-modified-two-kidney-one-clip-mouse-model-renin-regulation-renal

Research

JoVE Journal

Medicine

4.5K 次观看.  Vanderbilt University Medical Center. 使用聚氨酯管开发改良的2肾1夹（2K1C）Goldblatt小鼠模型以启动肾动脉狭窄，诱导肾素表达增加和肾损伤。在这里，我们描述了准备袖带并将其置于肾动脉上以产生可重复且一致的2K1C小鼠模型的详细程序。

视频: 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.

一个悬重系统使用隔离肾动脉闭塞

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 ...

科研

医学

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.

大脑中动脉闭塞的小鼠模型

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.

血管功能在慢性肾脏疾病的评估

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.

肾缺血再灌注损伤：损伤的小鼠模型和再生

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.

小鼠肾脏移植技术

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.

应用改良袖套技术的小鼠胰腺移植模型

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.

小鼠肾小球疾病模型肾脏功能的评价

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.

用线 Myography 测定小鼠肠系膜动脉的等轴收缩力

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.

一种从成年大鼠肾脏中分离完整型肾小球的有效筛分方法

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.

通过肾脏动脉直接向肾脏输送药物

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