The authors would like to thank Hans-Joachim Schurek for invaluable scientific advice. The authors would like to thank Monique Carrel and Mich&#232;le Heidemeyer for excellent technical assistance, David Penton Ribas and Nourdine Faresse for a critical reading of the manuscript and Carsten Wagner and J&#252;rg Biber for the NaPi-2a antibody. This work was supported by the Swiss National Centre for Competence in Research "Kidney.CH" and by a project grant (310030_143929/1) from the Swiss National Science Foundation.

所有涉及这个手稿中描述动物的程序进行了根据瑞士法律和瑞士苏黎世州的兽医行政管理部门批准。 1.缓冲液配制<ol><li>制备溶液1 - 4和抗利尿激素（ADH）溶液（ 表1）。 </li><li>制备透析缓冲液（ 表1）。 注意:这是用作灌注在透析缓冲的缓冲器。以后，红细胞将在这个缓冲液稀释以形成最终灌洗液。 </li><li>红细胞制剂。 <ol><li>稀释250毫升人红细胞浓缩至500毫升透析缓冲液（从当地血库获得测试材料）。离心在2000 xg离心8分钟。删除缓冲区，注意不要删除任何红细胞。重复3倍。 </li></ol></li><li>制备白蛋白（牛血清白蛋白; BSA）的缓冲液中。 <ol><li>在200毫升dialysi的的缓冲器中，用搅拌棒溶解44克BSA的。过滤用滤纸将溶液。 </li></ol></li><li>准备灌注液。 <ol><li>从步骤1.3.1通过滤纸进入BSA缓冲过滤红细胞。填充到800毫升的透析缓冲液的总体积。 注意:这是最后的灌注液。血细胞比容现在应该在8和12％之间。灌注液可以储存于4℃多达12小时下进行。 </li></ol></li></ol> 2.起爆透析和氧合<ol><li>打开周围较大缓冲储水，较小的缓冲液贮存和湿室（一个小的，双壁腔带到37℃和100％湿度稍后握住肾）至37℃的水浴（ 图2） 。 </li><li>填充透析缓冲液的较大的缓冲区贮存器和与灌注液较小贮存器。 </li><li> 2/95％O 2的气体流入转动5％CO到透析缓冲液。 </li><li>打开对透析缓冲灌注液的连续透析。小心使用低通量透析管。继续执行步骤3。 </li></ol><img alt="图2" src="/files/ftp_upload/54712/54712fig2.jpg" /> 图2:灌注回路的示意图。方案示出了灌注回路的主要组成部分和缓冲液流的方向。由深蓝色包围所有部件都保持在37℃的水浴/恒温器。 1:灌注缓冲液中的至少3倍体积的透析缓冲液连续地用95％O 2/5％CO 2鼓泡。 2:透析缓冲液和灌注缓冲液被连续地对在透析管彼此通过一个滚子泵透析。 3:由于这种透析，在灌注缓冲液用9％氧气富集2/5％CO 2和电解质水平保持恒定throughoUT灌注。 4:滚子泵推动朝肾脏灌注缓冲区。 5:删除的Windkessel蠕动波，并作为一个泡沫陷阱。 6:压力传感器（连接到4（滚子泵），以保持连续的压力，同时允许自由交替流）。 7:在整个灌注，肾保持在100％的空气湿度和37℃肾温度的潮湿室中。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54712/54712fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 3.手术过程第1部分（适用于所有结扎的示意图，见图3） 注意:请用5-0外科缝线结扎所有。 <ol><li>麻醉通过腹膜内注射的小鼠（体重20毫克/毫升氯胺酮和1毫克/毫升甲苯噻嗪溶解在0.9％NaCl中的10微升/克）。确认anesth足够的深度ESIA通过测试后无足反射的。 </li><li>固定鼠标在湿室仰卧位置。保护眼睛与兽医药膏。放置脊柱低于1ml注射器来升高腰椎血管。 </li><li>从耻骨嵴到胸骨开幕先将患处，然后腹肌，用剪刀执行中位数剖腹探查术。 </li><li>除去肠并将其放置在从腹部鼠标侧的左侧。 </li><li>无结缔组织，膀胱，探索两侧输尿管和尿道。 </li><li>周围放置左侧输尿管（结扎I）的结扎。关闭它。 </li><li>将尿道周围（结扎II）连字。关闭它。 </li><li>将围绕整个膀胱（结扎III）一个"套索"连字。 </li><li>切开膀胱1毫米。 </li><li>导管插入与2厘米PE管材50开。 </li><li>关闭结扎III管道周围。 </li><li>剪切从结扎左侧输尿管和尿道远端。该BLAD德现在连接到右输尿管仅自由移动。 </li><li>清除结缔组织和脂肪的腹主动脉。 </li><li>将腹部中期主动脉结扎（结扎IV）。 </li><li>周围放置肠系膜上动脉和腹腔干（结扎Ⅴ）之间的隔膜下方的主动脉结扎。 </li><li>周围放置肠系膜动脉（结扎VI）的结扎。 </li><li>放置一个主动脉结扎正下方的右侧和左侧肾动脉（结扎Ⅶ）以上。 </li><li>周围放置尾静脉包（静脉）（结扎Ⅷ）结扎。继续执行步骤4。 </li></ol><img alt="图3" src="/files/ftp_upload/54712/54712fig3.jpg" /> 图3:手术时放置在连字的示意图。查看剖腹手术后开腹部。肠道移出到左侧。 L和R指示的左右肾。该黑线显示各个结扎的面积。连字放在前面，然后关闭，在文中给出的顺序排列。 X标记为主动脉插管切口的位置。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54712/54712fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 4.灌注回路的引发<ol><li>启动回转泵和填充灌注液管路。小心从它清空所有气泡。 </li><li>该设备的Windkessel填写大约级中旬灌流。 </li><li>校准压力传感器为0毫米汞柱时所有管路被填充和流量为0保持在肾脏水平灌注针在这段时间。 </li><li>保持流动在一个恒定的最低水平（0.6ml /分钟），并继续执行步骤5。 </li></ol> 5.手术过程第2部分<ol><li>放置结扎IV和左RENA的分支之间的夹紧升动脉。 </li><li>使结扎IV的主动脉尾一小切口，注意不要切背墙。 </li><li>用扩张血管扩张主动脉开幕。 </li><li>导管插入用针主动脉（2厘米长，拉PE 50），推尖端正好夹。 </li><li>打开夹子。 </li><li>推针颅的前端，直到它到达右肾动脉和主动脉的连接处。 </li><li>关闭结扎七。 </li><li>关闭结扎IV。 </li><li>通过解剖隔膜打开用剪刀胸部。通过单一切口，分离主动脉，腔静脉，心脏和植物神经。与此步骤中，该动物是通过在连续深度麻醉迅速放血处死。 </li><li>启动灌注泵的压力控制。保持80至100毫米汞柱之间的平均压力。 </li><li>关闭结扎V. </li><li>关闭结扎VI。 </li><li>关闭结扎八。 </li><li>从结缔组织Tissu酒店免费右肾e和它嵌入到用剪刀脂肪囊。 </li><li>切主动脉近端至结扎线V. </li><li>切肠系膜上动脉远端，以结扎线VI。 </li><li>切肾血管配套捆绑了，小心不要切入血管自己。 </li><li>割肝在连接到肾脏。照顾到释放肾，但将肝粘附的一小部分到它，因此，腔静脉通过它保持打开。 </li><li>取肾捆绑了鼠标。从湿室中取出鼠标。 </li><li>周围放置肝脏和肾脏（结扎IX）的连接的"套索"连字。 </li><li>导管插入与静脉线路（2毫升的PE 50）的下腔静脉。 </li><li>关闭结扎九。通过静脉行静脉流出应立即启动。 </li><li>关闭湿室。 </li></ol> 6.下游分析<ol><li>在以下小时，连续监测血流量和intravasculAR压力15。收集静脉流出，它可以被用于分析，例如，肾肾素释放7。收集尿液的电解质浓度及肾小球滤过率14分析。灌注1小时后，肾脏可以管理单元冷冻western印迹或固定成像方法16。 </li></ol>

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The authors have no competing financial interests and nothing else to disclose.

离体灌注肾脏鼠标是用于在受控环境中的体外研究肾功能1小时，架桥在完整的动物体内实验，这可以通过许多全身性因素的影响，是有缺陷的之间的间隙的一个工具，并在体外实验中分离的肾段或培养细胞，这必然忽视完整器官结构对功能的影响。还有就是，以作者的知识，没有替代技术，用以执行这一特定的任务。对肾组织的生化研究，然而，可以在使用肾脏切片17上的更简单的方式执行。而提出的技术目前主要用于研究肾脏生理，许多新的应用正在讨论之中。例如，它可以灌注脱细胞和recellularization 12中用于肾生物工程。此外，现有的肾脏的长期常温灌注到TRansplantation，高剂量抗排斥或基因组编辑药物进入灌流，正在研究中的管理;即使在临床设置8，9，10，11。鼠标离体灌注肾脏是可以用于这些应用中，特别是阐明各个基因的同基因敲除模型中的作用的优秀工具。 三个步骤是一个成功的实验关键。本文提供了重现的程序向有关实验室。首先，缓冲需要的肾接收最必要可重复的条件下长期生存能力的营养素这样的方式来制备。的平衡必须充分控制的条件下（合成缓冲液）和本地灌注状态（全血）之间找到。因此，白蛋白和哺乳动物红细胞至10％的血细胞比容都被包括在内。这血细胞比容表示，在作者的经验，足以t的最佳平衡问题氧合和低粘度的缓冲液（增加红细胞压积下降压力不断灌注过程中显着流量（比较无红细胞18肾脏灌注本文提出将值值））。红细胞的质量和新鲜度是至关重要的。在洗涤步骤，红细胞需要小心处理以防止溶血。缓冲器需要在实验的当天新鲜制备。第二，手术需要由受过训练的医生在最短的时间内进行。外科医生需要执行肾脏周围的连接到身体的其他部分的主要血管6连字，同时保持动物的血液损失降到最低。为获得最佳结果，手术干预应在30分钟内完成。如果动物的平均动脉血压下降，长时间手术期间或由于麻醉，低于60毫米汞柱，肾灌注将已经是最低限度和组织就容易坏死。为了排除这一点，左肾可以在手术后可采取，当右肾灌注已经开始，并用作未处理的对照。第三，在灌注的灌注装置的正确功能，由一个标准的小动物灌注回路的时间，需要确保。当计算机执行其本身的灌注，人力监督是必要的，例如，以检查在灌注回路气泡。 所提供的具有代表性的数据允许肾脏存活率后离体灌注的1小时，并且此方法在本文中描述的方法的任何执行的技术成功的比较评估。在至少1小时，肾血流量，血管阻力，静脉流出和肾小球滤过率的血液气体应保持稳定。 有该技术，其中之一必须记住某些限制。即使きdney从最成功的手术发出只能在有限的时间保持存活。作者发现，1小时是在其停止灌注的最佳时间点。另外，虽然分离肾灌流允许在深度分离的整个器官的研究，这是难以区分在一个细胞类型特异性效应。固定1小时后肾脏可能会显示在近端小管和升支粗细胞的S3段的缺陷，而肾小球，S1段的DCT和CD应该保持不变。强制性使用红细胞，特别是来自人，也与对实验者潜在风险（ 例如 ，感染）。 总之，小鼠离体灌注肾脏是一种有用的技术，用以研究整个器官离体。像的任何技术，它具有一定的优点和局限性。笔者认为，无论是多方面的新的应用所产生的目前这个协议可以提供sufficien其他实验室牛逼势头重现这一努力。

器官的隔离灌注一直生理学家中不断努力的主题几十年1。该技术使器官的功能，而没有全身的影响，如血压，激素，或神经，进行研究。卡尔·爱德华Loebell被认为是第一个所描述的一个孤立肾灌注成功，于1849年2。此后，灌注设备发生了显著细化。弗雷和格鲁伯引入了氧合搏动泵人工肺的连续灌流2。虽然早期的研究人员重点研究大型哺乳动物-即，猪2和狗3使用大鼠肾脏的-the第一份报告，魏斯等人的肾脏。 ，是小型哺乳动物器官灌注4研究的一个里程碑。 Schurek 等。报道添加哺乳动物红细胞到灌流如果有足够的肾小管的必要性氧合要达到5。临界长期实验是由相同的研究组6引进缓冲器的连续透析。在2003年，Schweda 等。是第一个报告功能的鼠标离体灌注肾（MIPK）7，后来被Rahgozar 等精制而成。 18和林德尔等。 14。 虽然在技术上不是孤立肾灌注大鼠更具挑战性，使用MIPK负有能够使用各种各样基因改造的小鼠中的优势。本文介绍了作者的方法的详细信息隔离灌注小鼠肾1小时。该方法允许对肾流量，血管阻力，激素释放，血气分析，尿液分析，以及药物的应用程序的连续评估。以下的方法，肾脏可以用于分子和生化分析进行处理，是固定的显微镜，或移植到受体小鼠（ 图1）。 <img alt="图1" src="/files/ftp_upload/54712/54712fig1.jpg" /> 图1:可能的输入/输出到离体肾的概述。 BGA:血气分析。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54712/54712fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 这种技术可能会得到更多的关注，未来数年，多创新应用正在与长期常温肾脏血流灌注的曙光移植前的讨论（含或不含抗排斥或基因组编辑药物的应用）8，9，10 11，从脱细胞支架12整个肾脏的生物工程，剂量和高剂量荧光染料对多光子成像13应用</sup&gt;。这也是与研究急性肾损伤14中特定基因的作用的理想模型。 一个一步一步的协议，是考虑到让其他实验室成功地执行孤立的小鼠肾脏灌注。首先，组合物和制剂的缓冲液中被指定。然后，手术中详细描述和示出的关键步骤。第三，数据呈现的是，用于展示成功制备的:肾血流量，血管阻力，肾小球滤过率和分数电解质排泄-所有灌注肾脏的不同肾段的形态的生存能力和透射电子显微照片的功能性测量灌注后的1小时固定。

<table><tbody><tr><td>&lt;strong&gt;Perfusion Circuit:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Moist chamber 834/8</td><td>Harvard Apparatus/Hugo Sachs Elektronik GmbH</td><td>73-2901</td><td></td></tr><tr><td>Cannular with basket and side port</td><td>Harvard Apparatus/Hugo Sachs Elektronik GmbH</td><td>73-2947</td><td></td></tr><tr><td>Thermostat TC120-ST5&amp;nbsp;</td><td>Harvard Apparatus/Hugo Sachs Elektronik GmbH</td><td>73-4544</td><td></td></tr><tr><td>ISM 827/230V Roller Pump Reglo Analogue</td><td>Harvard Apparatus/Hugo Sachs Elektronik GmbH</td><td>73-0114</td><td></td></tr><tr><td>Reservoir jacketed for buffer solution 1 L</td><td>Harvard Apparatus/Hugo Sachs Elektronik GmbH</td><td>73-3438</td><td></td></tr><tr><td>Reservoir jacketed for buffer solution 0.5 L</td><td>Harvard Apparatus/Hugo Sachs Elektronik GmbH</td><td>73-3436</td><td></td></tr><tr><td>Pressure Transducer APT300&amp;nbsp;</td><td>Harvard Apparatus/Hugo Sachs Elektronik GmbH</td><td>73-3862</td><td></td></tr><tr><td>TAM-D Plugsys Transducer</td><td>Harvard Apparatus/Hugo Sachs Elektronik GmbH</td><td>73-1793</td><td></td></tr><tr><td>SCP Plugsys servo controller</td><td>Harvard Apparatus/Hugo Sachs Elektronik GmbH</td><td>73-2806</td><td></td></tr><tr><td>Windkessel&amp;nbsp;</td><td>Harvard Apparatus/Hugo Sachs Elektronik GmbH</td><td>73-3717</td><td></td></tr><tr><td>HSE-USB data acquisition</td><td>Harvard Apparatus/Hugo Sachs Elektronik GmbH</td><td>73-3330</td><td></td></tr><tr><td>Low-Flux Dialysator Diacap Polysulfone</td><td>B.Braun</td><td>7203525</td><td></td></tr><tr><td>PE-Tubing for aorta cannulation 1.19 mm I.D. x 1.70 mm O.D.</td><td>Scientific Commodities Inc.</td><td>BB31695-PE/8</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Buffer reagents:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Aminoplasmal 10%</td><td>B.Braun</td><td>134518064</td><td></td></tr><tr><td>Sodium pyruvate</td><td>Sigma-Aldrich</td><td>P2256-25G</td><td></td></tr><tr><td>L-Glutamic acid monosodium salt hydrate</td><td>Sigma-Aldrich</td><td>G1626-100G</td><td></td></tr><tr><td>L-(-)-Malic acid sodium salt</td><td>Sigma-Aldrich</td><td>M1125-25G</td><td></td></tr><tr><td>Sodium-L-Lactate</td><td>Sigma-Aldrich</td><td>L7022-10G</td><td></td></tr><tr><td>alpha-Ketoglutaric acid sodium salt</td><td>Sigma-Aldrich</td><td>K1875-25G</td><td></td></tr><tr><td>NaCl</td><td>Sigma-Aldrich</td><td>31434-1KG-R</td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>S5761-5KG</td><td></td></tr><tr><td>KCl</td><td>Sigma-Aldrich</td><td>60130-1KG</td><td></td></tr><tr><td>Urea</td><td>Sigma-Aldrich</td><td>U5378-500G</td><td></td></tr><tr><td>Creatinine</td><td>Sigma-Aldrich</td><td>C4255-10G</td><td></td></tr><tr><td>Ampicillin</td><td>Roche</td><td>10835242001</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt; * 6H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma-Aldrich</td><td>M2393-500G</td><td></td></tr><tr><td>D-Glucose</td><td>Sigma-Aldrich</td><td>G8270-1KG</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt; * 6H&lt;sub&gt;2&lt;/sub&gt;O&amp;nbsp;</td><td>Riedel-de-Ha&amp;euml;n</td><td>12074</td><td></td></tr><tr><td>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>S9638-500G</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>S0876-500G</td><td></td></tr><tr><td>Antidiuretic Hormone dDAVP</td><td>Sigma-Aldrich</td><td>V2013-1MG</td><td></td></tr><tr><td>FITC-Inulin</td><td>Sigma-Aldrich</td><td></td><td></td></tr><tr><td>Filter used for erythrocyte filtration</td><td>Macherey-Nagel</td><td>MN 615</td><td></td></tr><tr><td>BGA Analysis:</td><td></td><td></td><td></td></tr><tr><td>ABL 80 flex</td><td>Radiometer Medical ApS</td><td></td><td></td></tr><tr><td>Electron Microscope:</td><td></td><td></td><td></td></tr><tr><td>Philips CM100 TEM</td><td>FEI</td><td></td><td></td></tr></tbody></table>

the mouse isolated perfused kidney technique

鼠标离体灌注肾（MIPK）是用于灌注和1小时官能体外条件下保持一个小鼠肾脏的技术。这是研究分离的器官，并为许多创新的应用程序，可能会在未来成为可能，包括灌注脱细胞肾生物工程或抗排斥或基因组编辑药物在高剂量黄金肾脏的管理生理学的一个先决条件用于移植。在灌注时，肾脏可以被操纵，肾功能可以评估，和给药的各种药物。手术后，肾脏可以移植或用于分子生物学，生物化学分析，或显微处理。 本文介绍了灌流，需要鼠标肾脏的体外灌注的手术技术。灌注装置的细节给出并且呈现表示V数据肾脏的制剂iability:肾血流量，血管阻力，和尿的数据作为不同肾段作为形态学读数，和不同肾段作为分子读出的转运蛋白的蛋白质印迹的官能，透射电子显微镜照片。

与所描述的方法，分离小鼠肾可为至少1小时保持存活。我们后官能（肾血流量和血管阻力，静脉流出，肾小球滤过率，尿分数Na +和K +排泄血气分析和尿液摩尔渗透压浓度）和形态（透射电子连续灌注的1小时测试的组织活力显微镜，TEM）的方法，在野生型C57BL / 6小鼠四个肾脏。此外，进行了不同的小管的根尖标记蛋白的蛋白质印迹，隔离灌注肾脏比较同一动物unperfused肾脏。在所有的实验中，使用恒压灌注，保持灌注压力在约100毫米汞柱（ 图4）。在那些肾脏，灌流液流量和血管阻力位在肾重和3的15.6±0.4毫升/分钟* G超过55分钟时间保持稳定5±1.0毫米汞柱*分钟/毫升，分别为（ 图4 A）。灌注液流动或增加血管阻力的减少是与问候减小组织活力并应在整个小心灌注监视一个敏感的参数。当动脉流入物相比，静脉流出（ 表2）血液气体和灌注发作后取60分钟100μl的样品电解质分析表明肾的 CO 2生产和O 2消耗量。其它参数保持动脉和静脉（ 表2）之间保持不变。特别强调要抓好不变静脉释放钾，如组织损伤过程中可能出现的增加。肾小球滤过率，通过FITC标记的菊粉清除14评估，倾向于随时间而增加（ 图4 B）中 ，但这种趋势没有达到显着性。钠和钾的排泄分数55分钟灌注后评估相比， 在体内的情况（ 图4℃）增加。尿渗透压没有后灌注的55分钟（ 图4 D）的静脉流出的渗透压以上升高。 形态学研究，肾脏中的3％多聚甲醛灌注和0.1％戊二醛后在透析缓冲液，如前所述17固定。肾超微结构与TEM评估。近端小管的肾小球和S1段出现灌注（ 图5 A和B）后基本上保持不变。有些，但不是近端小管的所有S2 / S3段和厚升序肢体的未收到维管束被示出器官损伤的迹象，如在大鼠离体灌注肾脏5如前所述（ 图5 C和D）。即使在仔细检查，远曲小管和集合管没有表现出坏死明显的迹象（图5 E，F和G）。总之，在小鼠肾脏形态学结果反映在大鼠离体灌注肾脏5中发现的情况。 相比同动物unperfused对侧肾脏在离体肾脏的不同肾段顶标志蛋白印迹（后灌注1小时快速冷冻）（灌注来临之前快速冷冻）没有发现显著的改变。我们研究NAPI-IIA（位于近端小管），NKCC2（厚升序肢体），NCC（远曲小管），和ENaC的（连接管/集合管）（ 图6 A和B）。 <img alt="图4" src="/files/ftp_upload/54712/54712fig4.jpg" /> 图4: 灌注55分钟肾脏的功能可行性评估。 A）&lt;/ STRONG&gt;在恒压灌注，血管阻力及灌液流55分钟保持稳定。前15分钟被排除由于高变异性在此期间，总是看到。 B）的肾小球滤过率的后25和55分钟进行评估4肾脏（FITC-菊粉）显示随时间轻微但不显著增加。 C）的钠和钾的排泄分数后灌注的55分钟进行评估。 D）的静脉血浆和尿液后灌注的55分钟渗透压。 n = 4时在所有实验中。数据表示为平均值±SEM表示。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54712/54712fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/54712/54712fig5.jpg" /> 图5: 透射电子显微镜。的D代表形态<html>灌流肾脏ifferent肾路段在1小时的终点。 A）肾小球都完好无损。在肾小球足细胞一个细节。一个近曲小管的B）S1段。 S1段都完好无损。一个近曲小管C）S2 / S3段。有些，但不是近端小管的所有S2 / S3的片段是高度坏死。细胞出现浮肿，并显示大量细胞内的损伤。管状轮廓仅由基底膜保持。 D）升支粗（TAL）。这些技援贷款是远离维管束显示水样变性的第一个迹象。管腔膜出现变薄，脆弱。 E）远曲小管（DCT）。 DCT的都完好无损。 F），一个DCT单元的基底infoldings的详细。窗孔内皮细胞，基底膜，基底infoldings和线粒体都完好无损。 G）收集管道（CD）。光盘都完好无损。e.jove.com/files/ftp_upload/54712/54712fig5large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图6" src="/files/ftp_upload/54712/54712fig6.jpg" /> 图6: 根尖标记蛋白质的蛋白质印迹在离体肾脏相比从相同的动物Unperfused肾脏。 A）肾脏的Western印迹灌注1小时，unperfused肾脏。 NAPI-IIA是近端肾小管代表。 NKCC2为厚上边缘代表。 NCC和NCC磷酸在苏氨酸53（pT53 NCC）在远曲小管中只找到。 ENaC的在其全长和它们各自的蛋白水解切割产物的α和γ亚单位示为连接小管/集合管代表。 B A）的量化）并未透露肾脏灌注F的显著差异或1小时，unperfused肾脏。 n = 3的所有实验。数据表示为平均值±SEM和单个数值被示出。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54712/54712fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="表格1" src="/files/ftp_upload/54712/54712tbl1.jpg" /> 表1:解决方案和透析缓冲液的组成。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54712/54712tbl1large.jpg" target="_blank">请点击此处查看该表的放大版本。</a> <img alt="表2" src="/files/ftp_upload/54712/54712tbl2.jpg" /> 表2: 血气和动脉流入的电解质分析和灌注发病后的灌注小鼠肾脏60分钟静脉流出（N = 4）。数据表示为平均值±SEM。

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The Mouse Isolated Perfused Kidney Technique

鼠标离体灌注肾（MIPK）是用于灌注和1小时官能体外条件下保持一个小鼠肾脏的技术。的缓冲液和外科技术进行详细说明。

鼠标离体肾技术

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. This is a ...

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Nanyang Technological University

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21.7K 次观看.  University of Zürich. 鼠标离体灌注肾（MIPK）是用于灌注和1小时官能体外条件下保持一个小鼠肾脏的技术。的缓冲液和外科技术进行详细说明。 

视频: The Mouse Isolated Perfused Kidney Technique

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

Isolation of Glomeruli and In Vivo Labeling of Glomerular Cell Surface Proteins

Proteinuria results from the disruption of the glomerular filter that is composed of the fenestrated endothelium, glomerular basement membrane, and podocytes with their slit diaphragms. The delicate structure of the glomerular filter, especially the slit diaphragm, relies on the interplay of diverse cell surface proteins. Studying these cell surface proteins has so far been limited to in vitro studies or histologic analysis. Here, we present a murine in vivo biotinylation labeling method, which enables the study of glomerular cell surface proteins under physiologic and pathophysiologic conditions. This protocol contains information on how to perfuse mouse kidneys, isolate glomeruli, and perform endogenous immunoprecipitation of a protein of interest. Semi-quantitation of glomerular cell surface abundance is readily available with this novel method, and all proteins accessible to biotin perfusion and immunoprecipitation can be studied. In addition, isolation of glomeruli with or without biotinylation enables further analysis of glomerular RNA and protein as well as primary glomerular cell culture (i.e., primary podocyte cell culture).

Isolation of Glomeruli and In Vivo Labeling of Glomerular Cell Surface Proteins

Here we present a protocol for murine in vivo labeling of glomerular cell surface proteins with biotin. This protocol contains information on how to perfuse mouse kidneys, isolate glomeruli, and perform endogenous immunoprecipitation of the protein of interest.

肾小球的分离及肾小球细胞表面蛋白的活体标记

Proteinuria results from the disruption of the glomerular filter that is composed of the fenestrated endothelium, glomerular basement membrane, and ...

生物化学

Quantifying Glomerular Permeability of Fluorescent Macromolecules Using 2-Photon Microscopy in Munich Wistar Rats

Kidney diseases involving urinary loss of large essential macromolecules, such as serum albumin, have long been thought to be caused by alterations in the permeability barrier comprised of podocytes, vascular endothelial cells, and a basement membrane working in unison. Data from our laboratory using intravital 2-photon microscopy revealed a more permeable glomerular filtration barrier (GFB) than previously thought under physiologic conditions, with retrieval of filtered albumin occurring in an early subset of cells called proximal tubule cells (PTC)1,2,3.
Previous techniques used to study renal filtration and establishing the characteristic of the filtration barrier involved micropuncture of the lumen of these early tubular segments with sampling of the fluid content and analysis4. These studies determined albumin concentration in the luminal fluid to be virtually non-existent; corresponding closely to what is normally detected in the urine. However, characterization of dextran polymers with defined sizes by this technique revealed those of a size similar to serum albumin had higher levels in the tubular lumen and urine; suggesting increased permeability5.
Herein is a detailed outline of the technique used to directly visualize and quantify glomerular fluorescent albumin permeability in vivo. This method allows for detection of filtered albumin across the filtration barrier into Bowman's space (the initial chamber of urinary filtration); and also allows quantification of albumin reabsorption by proximal tubules and visualization of subsequent albumin transcytosis6. The absence of fluorescent albumin along later tubular segments en route to the bladder highlights the efficiency of the retrieval pathway in the earlier proximal tubule segments. Moreover, when this technique was applied to determine permeability of dextrans having a similar size to albumin virtually identical permeability values were reported2. These observations directly support the need to expand the focus of many proteinuric renal diseases to included alterations in proximal tubule cell reclamation.

A technique utilizing high resolution intavital 2-photon microscopy to directly visualize and quantify gloemrular filtration in surface glomeruli. This method allows for direct determination of permeability characteristics of macromolecules in both normal and diseased states.

利用双光子显微镜在慕尼黑Wistar大鼠量化肾小球通透性荧光大分子

Kidney  diseases involving urinary loss of large essential macromolecules, such as  serum albumin, have long been thought to be caused by alterations in ...

Reliable and High Efficiency Extraction of Kidney Immune Cells

Immune system activation occurs in multiple kidney diseases and pathophysiological processes. The immune system consists of both adaptive and innate components and multiple cell types. Sometimes, the cell type of interest is present in very low numbers among the large numbers of total cells isolated from the kidney. Hence, reliable and efficient isolation of kidney mononuclear cell populations is important in order to study the immunological problems associated with kidney diseases. Traditionally, tissue isolation of kidney mononuclear cells have been performed via enzymatic digestions using different varieties and strengths of collagenases/DNAses yielding varying numbers of viable immune cells. Recently, with the development of the mechanical tissue disruptors for single cell isolation, the collagenase digestion step is avoided and replaced by a simple mechanical disruption of the kidneys after extraction from the mouse. Herein, we demonstrate a simple yet efficient method for the isolation of kidney mononuclear cells for every day immune cell extractions. We further demonstrate an example of subset analysis of immune cells in the kidney. Importantly, this technique can be adapted to other soft and non-fibrous tissues such as the liver and brain.

Techniques that are reliable and efficient for the isolation of kidney immune cells are needed for downstream applications. This requires surface antibody labeling of a small number of kidney immune cells. Herein, we describe a concise method for isolation of kidney immune cells that seemingly achieves this goal.

肾脏免疫细胞的可靠和高效率的提取

Immune system activation occurs in multiple kidney diseases and pathophysiological processes.  The immune system consists of both adaptive and innate ...

免疫与感染

Microdissection of Primary Renal Tissue Segments and Incorporation with Novel Scaffold-free Construct Technology

Kidney transplantation is now a mainstream therapy for end-stage renal disease. However, with approximately 96,000 people on the waiting list and only one-fourth of these patients achieving transplantation, there is a dire need for alternatives for those with failing organs. In order to decrease the harmful consequences of dialysis along with the overall healthcare costs it incurs, active investigation is ongoing in search of alternative solutions to organ transplantation. Implantable tissue-engineered renal cellular constructs are one such feasible approach to replacing lost renal functionality. Here, described for the first time, is the microdissection of murine kidneys for isolation of living corticomedullary renal segments. These segments are capable of rapid incorporation within scaffold-free endothelial-fibroblast constructs which may enable rapid connection with host vasculature once implanted. Adult mouse kidneys were procured from living donors, followed by stereoscope microdissection to obtain renal segments 200 - 300 &#181;m in diameter. Multiple renal constructs were fabricated using primary renal segments harvested from only one kidney. This method demonstrates a procedure which could salvage functional renal tissue from organs that would otherwise be discarded.

Tissue-engineered renal constructs provide a solution for the organ shortage and deleterious effects of dialysis. Here, we describe a protocol to micro dissect murine kidneys for isolation of cortico-medullary segments. These segments are implanted into scaffold-free cellular constructs, forming renal organoids.

原发性肾组织段的显微解剖与新型无支架结构技术的结合

Kidney transplantation is now a mainstream therapy for end-stage renal disease. However, with approximately 96,000 people on the waiting list and only ...

生物工程

Optical Clearing and Imaging of Immunolabeled Kidney Tissue

Optical clearing techniques render tissue transparent by equilibrating the refractive index throughout a sample for subsequent three-dimensional (3-D) imaging. They have received great attention in all research areas for the potential to analyze microscopic multicellular structures that extend over macroscopic distances. Given that kidney tubules, vasculature, nerves, and glomeruli extend in many directions, which have been only partially captured by traditional two-dimensional techniques so far, tissue clearing also opened up many new areas of kidney research. The list of optical clearing methods is rapidly growing, but it remains difficult for beginners in this field to choose the best method for a given research question. Provided here is a simple method that combines antibody labeling of thick mouse kidney slices; optical clearing with cheap, non-toxic and ready-to-use chemical ethyl cinnamate; and confocal imaging. This protocol describes how to perfuse kidneys and use an antigen-retrieval step to increase antibody- binding without requiring specialized equipment. Its application is presented in imaging different multicellular structures within the kidney, and how to troubleshoot poor antibody penetration into tissue is addressed. We also discuss the potential difficulties of imaging endogenous fluorophores and acquiring very large samples and how to overcome them. This simple protocol provides an easy-to-setup and comprehensive tool to study tissue in three dimensions.

The combination of antibody labeling, optical clearing, and advanced light microscopy allows three-dimensional analysis of complete structures or organs. Described here is a simple method to combine immunolabeling of thick kidney slices, optical clearing with ethyl cinnamate, and confocal imaging that enables visualization and quantification of three-dimensional kidney structures.

免疫标记肾组织的光学清除与成像

Optical clearing techniques render tissue transparent by equilibrating the refractive index throughout a sample for subsequent three-dimensional (3-D) ...

A Quantitative Detection Method for MicroRNAs in the Kidney of an Ischemic Kidney Injury Mouse Model

MicroRNAs (miRNAs) are involved in various disease states and are effective biomarkers for the early diagnosis of diseases and treatment in mice. However, standard protocols for the purification of miRNAs and detection of their expression in the kidneys of acute kidney injury (AKI) mice have not been well established. This study developed an effective and simple protocol to purify and quantify miRNAs in the kidneys of an AKI mouse model induced by renal ischemia-reperfusion using quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR). This protocol comprises five steps: 1) induction of AKI by renal ischemia-reperfusion, 2) harvesting of kidneys, 3) purification of total RNA, including miRNAs, from kidneys, 4) cDNA synthesis by reverse transcription of miRNA, and 5) qRT-PCR to detect miRNA expression. Using this protocol, the renal ischemia-reperfusion injury model can be generated with mild to severe forms of AKI. Additionally, if the procedure is followed properly, a consistent AKI model with minimal individual differences can be obtained. This qRT-PCR assay shows a very wide dynamic range and enables the discrimination of mature miRNAs, which can be accurately quantified with high specificity. This protocol can be used to study the miRNA expression profile in AKI kidneys.

<meta charset="utf8"></head><body>缺血性肾损伤小鼠模型肾脏中 MicroRNAs 的定量检测方法

This article presents a protocol for detecting microRNA expression in the kidneys of an acute kidney injury mouse model using quantitative real-time reverse-transcription polymerase chain reaction. This protocol emphasizes an ischemic kidney injury mouse model and the careful extraction of microRNA samples.

缺血性肾损伤小鼠模型肾脏中 MicroRNAs 的定量检测方法

MicroRNAs (miRNAs) are involved in various disease states and are effective biomarkers for the early diagnosis of diseases and treatment in mice. However, ...

Isolating and Imaging Live, Intact Pacemaker Regions of Mouse Renal Pelvis by Vibratome Sectioning

The renal pelvis (RP) is a funnel-shaped, smooth muscle structure that facilitates normal urine transport from the kidney to the ureter by regular, propulsive contractions. Regular RP contractions rely on pacemaker activity, which originates from the most proximal region of the RP at the pelvis-kidney junction (PKJ). Due to the difficulty in accessing and preserving intact preparations of the PKJ, most investigations on RP pacemaking have focused on single-cell electrophysiology and Ca2+ imaging experiments. Although important revelations on RP pacemaking have emerged from such work, these experiments have several intrinsic limitations, including the inability to accurately determine cellular identity in mixed suspensions and the lack of in situ imaging of RP pacemaker activity. These factors have resulted in a limited understanding of the mechanisms that underlie normal rhythmic RP contractions. In this paper, a protocol is described to prepare intact segments of mouse PKJ using a vibratome sectioning technique. By combining this approach with mice expressing cell-specific reporters and genetically encoded Ca2+ indicators, investigators may be able to more accurately study the specific cell types and mechanisms responsible for peristaltic RP contractions that are vital for normal urine transport.

The goal of this protocol is to isolate intact pacemaker regions of the mouse renal pelvis using vibratome sectioning. These sections can then be used for in situ Ca2+ imaging to elucidate Ca2+ transient properties of pacemaker cells and other interstitial cells in vibratome slices.

隔离和成像现场， 通过颤音分割老鼠肾佩尔维斯的精致起搏器区域

The renal pelvis (RP) is a funnel-shaped, smooth muscle structure that facilitates normal urine transport from the kidney to the ureter by regular, ...

生物学

Glomeruli Isolation: A Technique to Obtain Intact Glomeruli from Murine Kidney

Source: Eymael, J. et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/video/60324" target="_blank">Glomerular Outgrowth as an Ex Vivo Assay to Analyze Pathways Involved in Parietal Epithelial Cell Activation.</a> J. Vis. Exp. (2020)
This video describes the method to isolate intact glomeruli from mouse kidneys. The encapsulated and decapsulated glomeruli thus obtained can further be used for culturing parietal epithelial cell outgrowths from the murine kidney.

肾小球分离:一种从小鼠肾脏获得完整肾小球的技术

Source: Eymael, J. et al. Glomerular Outgrowth as an Ex Vivo Assay to Analyze Pathways Involved in Parietal Epithelial Cell Activation. J. Vis. Exp. ...

鼠标离体肾技术

摘要

探索更多视频

一个悬重系统使用隔离肾动脉闭塞

肾小球的分离及肾小球细胞表面蛋白的活体标记

利用双光子显微镜在慕尼黑Wistar大鼠量化肾小球通透性荧光大分子

肾脏免疫细胞的可靠和高效率的提取

原发性肾组织段的显微解剖与新型无支架结构技术的结合

免疫标记肾组织的光学清除与成像

缺血性肾损伤小鼠模型肾脏中 MicroRNAs 的定量检测方法

缺血性肾损伤小鼠模型肾脏中 MicroRNAs 的定量检测方法

隔离和成像现场，通过颤音分割老鼠肾佩尔维斯的精致起搏器区域

肾小球分离:一种从小鼠肾脏获得完整肾小球的技术

鼠标离体肾技术

摘要

探索更多视频

一个悬重系统使用隔离肾动脉闭塞

肾小球的分离及肾小球细胞表面蛋白的活体标记

利用双光子显微镜在慕尼黑Wistar大鼠量化肾小球通透性荧光大分子

肾脏免疫细胞的可靠和高效率的提取

原发性肾组织段的显微解剖与新型无支架结构技术的结合

免疫标记肾组织的光学清除与成像

缺血性肾损伤小鼠模型肾脏中 MicroRNAs 的定量检测方法

缺血性肾损伤小鼠模型肾脏中 MicroRNAs 的定量检测方法

隔离和成像现场， 通过颤音分割老鼠肾佩尔维斯的精致起搏器区域

肾小球分离:一种从小鼠肾脏获得完整肾小球的技术

隔离和成像现场，通过颤音分割老鼠肾佩尔维斯的精致起搏器区域