Name: transcutaneous assessment of renal function in conscious rodents
Uploaded: 2016-03-26T14:00:00.000+00:00
Duration: 7 min 18 s
Description: Watch this Scientific Journal Video about Transcutaneous Assessment of Renal Function in Conscious Rodents at JoVE.com

ZHP was supported by the Marie-Curie project: NephroTools.

必要的实验开发协议是按照国家规定执行，并由当地的伦理科学委员会（Regierungspräsidium卡尔斯鲁厄）获得批准。 1. FITC的sinistrin注射溶液的制备<ol><li>溶解FITC-sinistrin在生理盐水中制备贮备液。在小鼠中的推荐剂量是7.5毫克/ 100克体重（BW），而在大鼠5毫克/ 100g的BW的FITC sinistrin是优选的。对于小鼠，以15毫克/毫升制备的FITC-sinistrin原液中的溶液，而对于大鼠以40毫克/毫升的浓度制备。 注意:FITC-sinistrin原液可以预先准备好并存储在避光-20℃。 </li></ol> 2.动物的制备<ol><li>它们在任何实验介绍之前适应动物至少一周。 </li><li>麻醉用5％异氟醚动物以5升/分钟O 2 </sub&gt;流2分钟。 </li><li>确认该动物正常通过观察呼吸，这会变慢，更深麻醉，并验证反应迟钝与缺乏脚趾反应。 </li><li>一旦动物是睡着了，减少麻醉2.5％异氟醚在2升/分钟O 2分娩率，并采取BW。 </li><li>从动物的使用电动剃须刀背面的侧面除去皮毛。剃的区域比将被双面粘合贴剂，其具有3厘米×6cm的尺寸来占据的面积稍大一点。 </li><li>随后申请脱毛霜一段短时间（2-3分钟），以除去剩余的皮毛。 </li><li>彻底清洗面积直至眼霜被完全删除，因为膏本身可能是荧光灯。 注意:避免划伤动物的皮肤，因为它可以产生刺激/水肿。如果脱毛区域显示的皮肤色素沉着，剃一个更大的区域，直到无色素斑的该装置的光学部件被发现。建议刮胡子24小时提前，以尽量减少麻醉曝光。 </li></ol> 3.设备准备<ol><li>放置光学装置来测量肾功能到双面粘合贴剂的一侧，定位在离开与保护膜的相对部分的透明窗（ 图1）中的光学部件。 注意:当与小老鼠的工作，降低中所示图2贴片的尺寸。 </li><li>附加了一块匹配大小与电池的双面粘合贴剂， 如图3。 </li></ol> 4.固定在动物的设备<ol><li>计算基于所述动物体重注射的适当体积。 </li><li>麻醉异氟烷动物如以前的章节2.1-2.3表示。 </li><li>剪下一块筒状弹性纱布包扎邻f以使用约1cm比双面粘合贴剂的宽度长。 </li><li>拉弹力纱布包扎了在它的后面动物和地方的负责人，破获留下剃光区域。 注:另外，只用胶带固定装置，但要注意在设备上不适当的压力。 </li><li>通过插入电池连接到对应端口的设备上的电池将设备连接。之后除去从片贴剂的保护膜，并装入电池的装置（ 图4A）的上表面上。确保电池正常通过检查设备开始闪烁堵塞。 注意:在大鼠中，该电池也可直接附着在粘合贴剂，旁边的设备（ 图4B）。 </li><li>从与操作装置补丁取下保护盖，并将其放置到剃光地区的动物拿着它的背面小号边缘，直到它被正确地固定的。 </li><li>盖与管状弹性纱布绷带它秉承贴剂的粘合面（ 图5）的设备。 </li><li>正确地伸展于该动物的腹部管状绷带，确保四肢可以自由移动。 </li><li>为了更好地固定和设备的保护，应用胶带的条带之后的装置的形状和覆盖所述电池的导线。 </li><li>测量1〜3分钟，无背景施加压力设备上</li></ol> 5. FITC-sinistrin管理和测量步骤<ol><li>如果需要的话，用温水喷射或沿所有的步骤的温度控制的加热板之前预热该动物的尾巴。 </li><li>通过尾静脉FITC-sinistrin原液适当的量注入。注射的体积取决于动物的BW，因此必须为每个单独的缺点计算idering所需的剂量和股票溶液的浓度，在第1.1节前述。 </li><li>确保所有的FITC sinistrin溶液静脉内，而不是皮下给药。 注意:可替换地，FITC-sinistrin可以用在颈部的在啮齿类动物中或通过在小鼠眼窝注射颈背退出预注入静脉导管施用。 </li><li>小心，动物返回到其家笼避免强烈运动和设备上的任何压力，因为这将引入移动文物。 </li><li>放置家笼在一个平静的地方，以避免动物受到干扰。 </li><li>如果与小鼠和用大鼠时2小时工作至少1小时期间执行测量。在此期间，该光学器件将通过皮肤测量由FITC的sinistrin发射的荧光。 注:在录音期间，动物必须单独主办。此外供水，以及突出结构如导线盖，必须除去，以避免损坏在电子设备和运动伪影的由于与对象的影响。 </li></ol> 6.设备删除<ol><li>一旦录音期结束后，取出无需麻醉设备;然而，如果需要的话，在此期间2分钟短暂麻醉动物5％异氟烷下，在5升/分钟的 O 2输送速率。 </li><li>小心，先拉断胶带，然后管状绷带。 </li><li>轻轻分离从皮肤双面粘合贴剂和动物返回其正常的家笼中。 </li></ol> 7.读出的数据的<ol><li>断开装置中的电池并取出粘合贴剂。 注意:直到一个新的测量开始的数据被存储在设备上。当电池连接所存储的数据将被覆盖的新记录。因此，下载从数据之前不重新安装电池设备。有用于对电池意外重接一个10秒的宽限期。 </li><li>将设备连接到使用微型USB线将PC和下载使用所提供的软件中的数据。输出文件是可以打开，并用电子表格程序修改.csv文件。 </li><li>打开与用于光学设备中的特定软件的数据文件，并使用该软件协议生成所述消除动力学曲线。 </li><li>通过以下设置有该光学装置的指示分析曲线。不久，对数据进行评估，设置背景信号测量之前的FITC sinistrin施用并标记该标记时，这通常发生15分钟，并在推注后45分钟的指数排泄阶段的开始，在小鼠和大鼠分别。 注意:软件会自动显示FITC-sinistrin半衰期（T 1/2）与R 2值一起，由1 COMPAR确定tment模式。吨1/2可以利用通过使用转换因子9,10来计算肾小球滤过率。 </li></ol>

<ol>
	<li>Stevens, L. A., Levey, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measured+GFR+as+a+confirmatory+test+for+estimated+GFR.">Measured GFR as a confirmatory test for estimated GFR.</a> J Am Soc Nephrol. 20, 2305-2313 (2009).</li><li>Katayama, R., 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=Calculation+of+glomerular+filtration+rate+in+conscious+rats+by+the+use+of+a+bolus+injection+of+iodixanol+and+a+single+blood+sample.">Calculation of glomerular filtration rate in conscious rats by the use of a bolus injection of iodixanol and a single blood sample.</a> J Pharmacol Toxicol Methods. 61, 59-64 (2010).</li><li>Reinhardt, C. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+immunoassay+technology+(FIT),+a+new+approach+for+measuring+physiological+functions:+application+of+FIT+to+measure+glomerular+filtration+rate+(GFR).">Functional immunoassay technology (FIT), a new approach for measuring physiological functions: application of FIT to measure glomerular filtration rate (GFR).</a> Am J Physiol Renal Physiol. 295, F1583-F1588 (2008).</li><li>Rieg, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-throughput+method+for+measurement+of+glomerular+filtration+rate+in+conscious+mice.">A high-throughput method for measurement of glomerular filtration rate in conscious mice.</a> J Vis Exp. , e50330 (2013).</li><li>Yu, W., Sandoval, R. M., Molitoris, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+determination+of+renal+filtration+function+using+an+optical+ratiometric+imaging+approach.">Rapid determination of renal filtration function using an optical ratiometric imaging approach.</a> Am J Physiol Renal Physiol. 292, F1873-F1880 (2007).</li><li>Wang, E., Sandoval, R. M., Campos, S. B., Molitoris, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+diagnosis+and+quantification+of+acute+kidney+injury+using+fluorescent+ratio-metric+determination+of+glomerular+filtration+rate+in+the+rat.">Rapid diagnosis and quantification of acute kidney injury using fluorescent ratio-metric determination of glomerular filtration rate in the rat.</a> Am J Physiol Renal Physiol. 299, F1048-F1055 (2010).</li><li>Colson, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Does+choice+of+the+anesthetic+influence+renal+function+during+infrarenal+aortic+surgery?.">Does choice of the anesthetic influence renal function during infrarenal aortic surgery?.</a> Anesth Analg. 74, 481-485 (1992).</li><li>Fusellier, 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=Influence+of+three+anesthetic+protocols+on+glomerular+filtration+rate+in+dogs.">Influence of three anesthetic protocols on glomerular filtration rate in dogs.</a> Am J Vet Res. 68, 807-811 (2007).</li><li>Schreiber, 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=Transcutaneous+measurement+of+renal+function+in+conscious+mice.">Transcutaneous measurement of renal function in conscious mice.</a> Am J Physiol Renal Physiol. 303, F783-F788 (2012).</li><li>Schock-Kusch, 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=Transcutaneous+measurement+of+glomerular+filtration+rate+using+FITC-sinistrin+in+rats.">Transcutaneous measurement of glomerular filtration rate using FITC-sinistrin in rats.</a> Nephrol Dial Transplant. 24, 2997-3001 (2009).</li><li>Schock-Kusch, 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=Transcutaneous+assessment+of+renal+function+in+conscious+rats+with+a+device+for+measuring+FITC-sinistrin+disappearance+curves.">Transcutaneous assessment of renal function in conscious rats with a device for measuring FITC-sinistrin disappearance curves.</a> Kidney Int. 79, 1254-1258 (2011).</li><li>Steinbach, 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=A+pilot+study+to+assess+the+feasibility+of+transcutaneous+glomerular+filtration+rate+measurement+using+fluorescence-labelled+sinistrin+in+dogs+and+cats.">A pilot study to assess the feasibility of transcutaneous glomerular filtration rate measurement using fluorescence-labelled sinistrin in dogs and cats.</a> PLoS One. 9, e111734 (2014).</li><li>Schock-Kusch, 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=Reliability+of+transcutaneous+measurement+of+renal+function+in+various+strains+of+conscious+mice.">Reliability of transcutaneous measurement of renal function in various strains of conscious mice.</a> PLoS One. 8, e71519 (2013).</li><li>Cowley, A. 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=Progression+of+glomerular+filtration+rate+reduction+determined+in+conscious+Dahl+salt-sensitive+hypertensive+rats.">Progression of glomerular filtration rate reduction determined in conscious Dahl salt-sensitive hypertensive rats.</a> Hypertension. 62, 85-90 (2013).</li><li>Giani, J. F., 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+angiotensin-converting+enzyme+is+essential+for+the+hypertension+induced+by+nitric+oxide+synthesis+inhibition.">Renal angiotensin-converting enzyme is essential for the hypertension induced by nitric oxide synthesis inhibition.</a> J Am Soc Nephrol. 25, 2752-2763 (2014).</li><li>Sadick, 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=Two+non-invasive+GFR-estimation+methods+in+rat+models+of+polycystic+kidney+disease:+3.0+Tesla+dynamic+contrast-enhanced+MRI+and+optical+imaging.">Two non-invasive GFR-estimation methods in rat models of polycystic kidney disease: 3.0 Tesla dynamic contrast-enhanced MRI and optical imaging.</a> Nephrol Dial Transplant. 26, 3101-3108 (2011).</li><li>Trudu, 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=Common+noncoding+UMOD+gene+variants+induce+salt-sensitive+hypertension+and+kidney+damage+by+increasing+uromodulin+expression.">Common noncoding UMOD gene variants induce salt-sensitive hypertension and kidney damage by increasing uromodulin expression.</a> Nat Med. 19, 1655-1660 (2013).</li><li>Zollner, F. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+measurement+of+kidney+function+by+dynamic+contrast+enhanced+MRI+and+FITC-sinistrin+clearance+in+rats+at+3+tesla:+initial+results.">Simultaneous measurement of kidney function by dynamic contrast enhanced MRI and FITC-sinistrin clearance in rats at 3 tesla: initial results.</a> PLoS One. 8, e79992 (2013).</li></ol>

NG持有生产FITC-sinistrin的专利。

本论文描述了使用小型化的设备和外源性肾标记FITC-sinistrin确定自觉啮齿类动物肾功能的过程。如前所述，这种方法在尿和血浆清除率的传统的程序相比，具有几个优点。其中之一是由血液和尿液采样的独立性这导致很大的节省的消耗品，时间方面，当然，动物应力。 如表2所示，外源性肾标记的FITC sinistrin和通过皮肤其消除动力学的测量允许肾病症对肾功能的影响进行研究9,10,14-18。评估血浆清除或肾脏疾病的生物标记物所需的血液样本的序列集合是压力为动物，麻烦和耗时的研究人员。因此，相同的动物内连续测量已廉由动物和方法的原因的福利资讯科技教育。经皮测量13的证明内动物再现性使得它用于检测肾功能由于疾病进展或老化的变化的理想方法。出于同样的原因，它也可以是研究的治疗方法的有效性非常有用的。 该技术的成功应用的两个关键步骤是在动物的光学元件和标记的正确给药的固定。该设备必须适当固定，以避免运动伪影。为了这个目的，防止双面粘合贴剂的装卸是至关重要的，因为它可以导致其粘接性能的损失，并最终不恰当的固定的动物。如通过用胶带覆盖它在协议中，固定和光学装置的保护提到是确保adequat一种有效的方法Ë固定。当执行的FITC sinistrin推注是很关键的静脉内，而不是皮下施用的所有物质。标记物在皮下组织的释放可以由黄色环盘旋由FITC的sinistrin自身颜色产生的动物的尾巴很容易识别。皮下注射一般表现为一个平坦的曲线和长期的标记排泄时间。该方法的可能的限制是更相关比使用该技术本身所使用的标记。当水肿的存在动物的皮肤上，将不被推荐的肾功能的皮评估，有可能改变的FITC sinistrin的排泄动力学的额外隔室。同样，色素沉着皮肤可以限制该技术的使用，由于FITC的sinistrin和黑色素的重叠激发和发射光谱。然而，在动物中的补丁皮肤色素沉着，该问题是通过将光学解决在一个未着色区的装置的一部分。 它是可以修改的肾功能的皮评估的不同方面，以便适应的程序不同的动物物种和/或方法。例如，推荐的FITC sinistrin剂量或测量的长度可以根据不同的物种或动物模型实验以及目标进行调整。该方法的这个适应性已经通过在不同物种啮齿动物和多种类型的具有不同的卫生条件菌株的的成功应用证实。此外，肾功能的皮评估最近已经在狗和效果极佳猫12验证。由于这些动物和啮齿动物之间的无可置疑的差异，该研究确定的FITC sinistrin为，狗和猫的适当剂量，并且它进行更长的测量时间，4个小时，而不是1或2个小时的使用d。在啮齿动物。 综合来看，使用这种技术可以帮助我们获得在肾脏病更准确的见解，包括更好地了解肾脏疾病进展和治疗的效果。肾功能的皮评估的特征宿它作为今后人类和兽医临床领域所应用的合适的技术，且应被认为是有前途的技术，以代替传统的方法。

肾小球滤过率（GFR）是评估总体肾功能的最佳参数。的黄金标准，以确定GFR依赖于外源肾脏标志物，如菊粉1的尿或血浆清除。然而，这些程序是耗时和复杂的，需要的串行血液和/或尿液样品和后实验室试验中收集的分析。此外，这些方法是侵入性的和紧张的动物，限制的时间和频率，其中测量可重复的数目。一些变通的办法已经制定，以简化古典程序，以确定GFR，但他们仍然依靠尿液和血浆采样2-4和/或需要深度麻醉5,6，这是众所周知的影响肾血流动力学和功能7， 8。代谢的终产物，如肌酸酐，也被广泛用于估计肾功能。然而，已知的是，ACCUR这些内源性标志物ACY不是最佳的，而且，对它们进行分析，尿液或血液取样也是必不可少的。 在这里，我们描述了经皮方法来评估在清醒动物肾功能。这种方法比传统的方法，以及只微创简单和快速。利用这种技术，利用一个小型化的光学装置和外源性肾标记异硫氰酸荧光素（FITC）-sinistrin，能够几乎实时地确定肾功能，而不需要为血浆或尿液取样和无外科导管为物质施用。由于这些特点，该方法适用于在相同的动物进行连续测量。 FITC-sinistrin是肾标记物通过肾小球自由过滤，用无肾小管重吸收或分泌。小型化的装置的光学部分包括两个发光二极管（LED），其激发的所施用的标记和一个光电二极管来检测通过皮肤发射的荧光。所记录的数据被存储在内部存储器集成在设备和可以使用它们来产生的FITC sinistrin消除动力学曲线。功率是由一个小的可充电锂电池供电。为设备和它的各个部件更详细的信息请参见Schreiber 等 。9。该设备在使用双面粘合贴剂携带用于光学部件的透明窗容易地安装在动物身体。结果是容易得到后的记录期结束。 用的方法和此处提供的协议中，我们引入一个有前途的技术，它可以取代目前金标准来确定的GFR，不仅在研究，而且在临床领域。

<table><tbody><tr><td>NIC-Kidney device</td><td>Mannheim Pharma &amp;amp; Diagnostics GmbH</td><td></td><td>Software, batteries and chargers are provided together with the device/s. Orderings should be done through info@mapdiagnostics.com</td></tr><tr><td>FITC-sinistrin</td><td>Mannheim Pharma &amp;amp; Diagnostics GmbH</td><td></td><td>Orderings should be done through info@mapdiagnostics.com</td></tr><tr><td>Double-sided adhesive patch</td><td>Mannheim Pharma &amp;amp; Diagnostics GmbH</td><td></td><td>Orderings should be done through info@mapdiagnostics.com</td></tr><tr><td>Isis Rodent electric shaver</td><td>Braun Aesculap&amp;nbsp;</td><td>GT420</td><td></td></tr><tr><td>Isoflurane</td><td>Abbott GmbH&amp;nbsp;</td><td>PZN4831850</td><td></td></tr><tr><td>Leukosilk adhesive tape</td><td>BSN medical</td><td>102200</td><td></td></tr><tr><td>Tubular elastic gauze bandage&amp;nbsp;</td><td>MaiMed Medical GmbH</td><td>73012</td><td>There are different sizes available. Size 1 is recommended for mice.</td></tr><tr><td>Veet depilation cream</td><td>Reckitt Benckiser</td><td>PZN7768307</td><td>Sensitive skin formulation is recommended as is more gentle with the skin of the animals</td></tr><tr><td>Micro USB cable</td><td>Samsung</td><td>APCBU10BBE</td><td></td></tr><tr><td>Deltajonin Physiologic solution</td><td>AlleMan Pharma GmbH</td><td>3366954</td><td>Alternatively, it can be used saline or PBS&amp;nbsp;</td></tr></tbody></table>

transcutaneous assessment of renal function in conscious rodents

Glomerular filtration rate (GFR) is the gold standard to assess overall kidney function. However, traditional methods to evaluate GFR are cumbersome and time-consuming. In addition, serial blood or urine samples are required, with the associated stress for the experimental animals. A recent technique significantly reduces the investment in time and resources, minimizing the invasiveness and the animal stress, but being equally valid as the traditional approaches. The method measures transcutaneously renal function. Using an optical device and the exogenous renal marker fluorescein isothiocyanate (FITC)-sinistrin, this technique is capable of measuring the elimination kinetics of the marker through the skin. With neither blood nor urine samples nor the associated laboratory assays needed, the results of the transcutaneous measurement are almost instantaneously available. The method has been already validated in different species and successfully applied in several models of renal pathology. Moreover, due to its minimally invasive characteristics, it is suitable for sequential measurements within the same animal. Here is provided a detailed protocol to carry out the transcutaneous assessment of renal function in rodents.

经皮测量的设置非常简单和快速的:设备被放置在双面粘合贴剂（ 图1），如果必要的（ 图2）的大小进行调整，在电池制备（ 图3）和连接（ 图4）。 此来评估肾功能的方法已被在不同的物种通过用血浆清除9,11,12的传统方法比较验证。继这里暴露了协议，施赖伯等人 。证实在不同小鼠模型的方法的有效性，示出了用于所有组的经皮测量和血浆清除率之间高度可比的结果的研究（ 见表1）9。在这项工作中所获得的牛逼1/2用鼠标特定的半经验转换转换为GFR因子。 肾功能的皮评估的一致性已经使用小鼠不同菌株已也证明。在相同的动物在3天内连续测量显示的3.0-6.2％13方差系数。在这项研究中，没有转化为GFR但结果表示，并直接在叔1/2来解释。 具有几乎实时的结果的可能性的这种方法的优点之一。记录周期后，其结果是立即可用于分析和所提供的软件显示的FITC sinistrin瞬时排泄曲线（ 图6）。内能够得到的FITC- sinistrin的第t 1/2相同的软件，它可以直接用作参数来评估GFR的方面转化肾功能。 图7 </st荣&gt;展示了如何看起来都与肾功能损害的动物经皮测得的FITC-sinistrin排泄曲线。当肾功能受到影响，由于该物质的减少排泄和的曲线变化的外观，FITC-sinistrin吨1/2增加。通常情况下，测得的曲线不回来背景水平，并提出了曲线下面积增加。在相同的动物，从预到损伤后测量可以由于由其减少排泄标记的累积遇到的最大荧光强度的增加。在肾功能衰竭的存在，FITC-sinistrin的经皮测曲线显示，由于严重受损功能（ 图7B）稳定状态。 利用在具有不同的健康状况啮齿类几株的小型化装置的显示，该技术是合适的和足够敏感以检测更改ðUE向肾脏疾病和老化。 表2示出迄今研究用这种方法在小鼠模型的概要。 <img alt="图1" src="/files/ftp_upload/53767/53767fig1.jpg" /> 图1.在双面粘合贴剂的设备的放置，该装置安装在粘合贴剂定位在所述透明窗口的光学部件。 <img alt="图2" src="/files/ftp_upload/53767/53767fig2.jpg" /> 图2.适应粘合贴剂为小型啮齿动物。对于小动物的使用时，建议降低贴片的尺寸。答:使用设备为指导，以适当削减补丁。 A:一旦已经获得期望的大小，该装置可以放置贴片的粘性表面上。 <a href="https://www.jove.com/files/ftp_upload/53767/53767fig2large.jpg" target="_blank">P</a>租赁点击此处查看该图的放大版本。 <img alt="图3" src="/files/ftp_upload/53767/53767fig3.jpg" /> 图3.制备电池。为了将电池安装到设备上，剪一小块双面贴片胶（A和B），并将其放置电池（C）的表面。 <a href="https://www.jove.com/files/ftp_upload/53767/53767fig3large.jpg" target="_blank">请点击这里查看更大的版本这个数字。</a> <img alt="图4" src="/files/ftp_upload/53767/53767fig4.jpg" /> 图4.在测量过程中的电池的连接和放置。（A）在小型啮齿动物例如小鼠，由于减少了空间，电池必须被放置在装置的顶部。 （ 二 ）在较大动物的电池C一个被旁边放置设备。 <a href="https://www.jove.com/files/ftp_upload/53767/53767fig4large.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/53767/53767fig5.jpg" /> 图5.放置在老鼠管状弹性纱布包扎的。管状绷带应覆盖未与四肢的动物的舒适的自由移动干扰双面胶补丁。 <a href="https://www.jove.com/files/ftp_upload/53767/53767fig5large.jpg" target="_blank">请点击此处查看大图这个数字。</a> <img alt="图6" src="/files/ftp_upload/53767/53767fig6.jpg" /> 图6的FITC sinistrin的消除曲线的代表性的图像。通过FITC标记的sinistrin产生的信号被检测到transcutaneously并存储在该装置的内部存储器中。当记录的数据被下载到个人电脑，软件生成的曲线相媲美的图像中呈现所述一个。 Y轴显示记录的荧光强度[AU]，通过注入FITC-sinistrin标记发出，而X轴表示测量时间（分钟）的时间。 <a href="https://www.jove.com/files/ftp_upload/53767/53767fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图7" src="/files/ftp_upload/53767/53767fig7.jpg" /> 图7. FITC-sinistrin的动物的肾功能受损的消除曲线的代表性图像:（A）在动物中具有降低的肾功能的FITC sinistrin消除曲线典型地示出了曲线和无力下增加的面积达到内的基线正常测量周期<html> 。 （ 二 ）严重损害动物的经皮测得的曲线可以显示表明肾功能衰竭的稳定状态。 <a href="https://www.jove.com/files/ftp_upload/53767/53767fig7large.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/53767/53767table1.jpg" /> 表1 验证通过与传统的血浆清除率比较经皮测量。肾功能的经皮测量已经在不同的小鼠模型得到验证（健康和单方面肾切除（UNX）的C57BL / 6-129 SV小鼠和nephronophthisis的小鼠模型（ PCY））与血浆清除率的比较。数值是平均值±SD。 GFR，肾小球滤过率9。 /53767/53767table2.jpg"/&gt; 表2.小鼠模型研究采用经皮测 UNX，单方面肾切除。 ACE，血管紧张素转换酶; ŧgumodwt /重量 ，转基因敲除尿调节素，SD雄性大鼠大鼠; PKD，多囊肾。

Watch this Scientific Journal Video about Transcutaneous Assessment of Renal Function in Conscious Rodents at JoVE.com

Transcutaneous Assessment of Renal Function in Conscious Rodents

Determination of glomerular filtration rate (GFR) is the gold standard to assess overall kidney function. However, traditional procedures to measure this parameter are cumbersome and require a large investment of time. Here we describe a faster and minimally invasive method to determinate GFR transcutaneously.

在鼠害自觉肾功能的评估皮

Glomerular filtration rate (GFR) is the gold standard to assess overall kidney function. However, traditional methods to evaluate GFR are cumbersome and ...

transcutaneous-assessment-of-renal-function-in-conscious-rodents

Research

JoVE Journal

Medicine

12.4K 次观看.  Medical Faculty Mannheim, University of Heidelberg. Determination of glomerular filtration rate (GFR) is the gold standard to assess overall kidney function. However, traditional procedures to measure this parameter are cumbersome and require a large investment of time. Here we describe a faster and minimally invasive method to determinate GFR transcutaneously.

视频: Transcutaneous Assessment of Renal Function in Conscious Rodents

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 ...

科研

医学

Early Detection of Drug-Induced Renal Hemodynamic Dysfunction Using Sonographic Technology in Rats

The kidney normally functions to maintain hemodynamic homeostasis and is a major site of damage caused by drug toxicity. Drug-induced nephrotoxicity is estimated to contribute to 19- 25% of all clinical cases of acute kidney injury (AKI) in critically ill patients. AKI detection has historically relied on metrics such as serum creatinine (sCr) or blood urea nitrogen (BUN) which are demonstrably inadequate in full assessment of nephrotoxicity in the early phase of renal dysfunction. Currently, there is no robust diagnostic method to accurately detect hemodynamic alteration in the early phase of AKI while such alterations might actually precede the rise in serum biomarker levels. Such early detection can help clinicians make an accurate diagnosis and help in in decision making for therapeutic strategy. Rats were treated with Cisplatin to induce AKI. Nephrotoxicity was assessed for six days using high-frequency sonography, sCr measurement and upon histopathology of kidney. Hemodynamic evaluation using 2D and Color-Doppler images were used to serially study nephrotoxicity in rats, using the sonography. Our data showed successful drug-induced kidney injury in adult rats by histological examination. Color-Doppler based sonographic assessment of AKI indicated that resistive-index (RI) and pulsatile-index (PI) were increased in the treatment group; and peak-systolic velocity (mm/s), end-diastolic velocity (mm/s) and velocity-time integral (VTI, mm) were decreased in renal arteries in the same group. Importantly, these hemodynamic changes evaluated by sonography preceded the rise of sCr levels. Sonography-based indices such as RI or PI can thus be useful predictive markers of declining renal function in rodents. From our sonography-based observations in the kidneys of rats that underwent AKI, we showed that these noninvasive hemodynamic measurements may consider as an accurate, sensitive and robust method in detecting early stage kidney dysfunction. This study also underscores the importance of ethical issues associated with animal use in research.

Early stage hemodynamic dysfunction is critical to the development of kidney disease. Yet, detection methodologies are limited. Recent advances in sonography provide a noninvasive, accurate option for early detection of kidney injury. This study outlines a step-by-step, sonographic methodology for detecting kidney dysfunction using a drug-induced nephrotoxicity rat model.

大鼠使用超声技术药物引起的肾血流动力学障碍的早期检测

The kidney normally functions to maintain hemodynamic homeostasis and is a major site of damage caused by drug toxicity. Drug-induced nephrotoxicity is ...

Novel Approach for Simultaneous Recording of Renal Sympathetic Nerve Activity and Blood Pressure with Intravenous Infusion in Conscious, Unrestrained Mice.

Renal sympathetic nerves contribute significantly to both physiological and pathophysiological phenomena. Evaluating renal sympathetic nerve activity (RSNA) is of great interest in many areas of research such as chronic kidney disease, hypertension, heart failure, diabetes and obesity. Unequivocal assessment of the role of the sympathetic nervous system is thus imperative for proper interpretation of experimental results and understanding of disease processes. RSNA has been traditionally measured in anesthetized rodents, including mice. However, mice usually exhibit very low systemic blood pressure and hemodynamic instability for several hours during anesthesia and surgery. Meaningful interpretation of RSNA is confounded by this non-physiological state, given the intimate relationship between sympathetic nervous tone and cardiovascular status. To address this limitation of traditional approaches, we developed a new method for measuring RSNA in conscious, freely-moving mice. Mice were chronically instrumented with radio-telemeters for continuous monitoring of blood pressure as well as a jugular venous infusion catheter and custom-designed bipolar electrode for direct recording of RSNA. Following a 48-72 hour recovery period, survival rate was 100% and all mice behaved normally. At this time-point, RSNA was successfully recorded in 80% of mice, with viable signals acquired up to 4 and 5 days post-surgery in 70% and 50% of mice, respectively. Physiological blood pressures were recorded in all mice (116&plusmn;2 mmHg; n=10). Recorded RSNA increased with eating and grooming, as well-established in the literature. Furthermore, RSNA was validated by ganglionic blockade and modulation of blood pressure with pharmacological agents. Herein, an effective and manageable method for clear recording of RSNA in conscious, freely-moving mice is described.

Anesthetized mice exhibit non-physiological systemic blood pressure, which precludes meaningful assessment of autonomic tone given the intimate relationship between blood pressure and the autonomic nervous system. Thus, a novel method to simultaneously record renal sympathetic nerve activity and blood pressure with intravenous infusion in conscious mice is outlined.

清醒、无拘无束的小鼠静脉输液同时记录肾交感神经活动和血压的新方法。

Renal sympathetic nerves contribute significantly to both physiological and pathophysiological phenomena. Evaluating renal sympathetic nerve activity ...

A High-throughput Method for Measurement of Glomerular Filtration Rate in Conscious Mice

The measurement of glomerular filtration rate (GFR) is the gold standard in kidney function assessment. Currently, investigators determine GFR by measuring the level of the endogenous biomarker creatinine or exogenously applied radioactive labeled inulin (3H or 14C). Creatinine has the substantial drawback that proximal tubular secretion accounts for ~50% of total renal creatinine excretion and therefore creatinine is not a reliable GFR marker. Depending on the experiment performed, inulin clearance can be determined by an intravenous single bolus injection or continuous infusion (intravenous or osmotic minipump). Both approaches require the collection of plasma or plasma and urine, respectively. Other drawbacks of radioactive labeled inulin include usage of isotopes, time consuming surgical preparation of the animals, and the requirement of a terminal experiment. Here we describe a method which uses a single bolus injection of fluorescein isothiocyanate-(FITC) labeled inulin and the measurement of its fluorescence in 1-2 &mu;l of diluted plasma. By applying a two-compartment model, with 8 blood collections per mouse, it is possible to measure GFR in up to 24 mice per day using a special work-flow protocol. This method only requires brief isoflurane anesthesia with all the blood samples being collected in a non-restrained and awake mouse. Another advantage is that it is possible to follow mice over a period of several months and treatments (i.e. doing paired experiments with dietary changes or drug applications). We hope that this technique of measuring GFR is useful to other investigators studying mouse kidney function and will replace less accurate methods of estimating kidney function, such as plasma creatinine and blood urea nitrogen.

Measurement of glomerular filtration rate (GFR) is the gold standard for kidney function assessment. Here we describe a high-throughput method which allows the determination of GFR in conscious mice by using a single bolus injection, determination of fluorescein isothiocyanate (FITC)-inulin in plasma and calculation of GFR by a two-phase exponential decay model.

高通量清醒小鼠肾小球滤过率的测定方法

The measurement of  glomerular filtration rate (GFR) is the gold standard in kidney function  assessment. Currently, investigators determine GFR by ...

生物学

Transdermal Measurement of Glomerular Filtration Rate in Mice

Transdermal analysis of glomerular filtration rate (GFR) is an established technique that is used to assess renal function in mouse and rat models of acute kidney injury and chronic kidney disease. The measurement system consists of a miniaturized fluorescence detector that is directly attached to the skin on the back of conscious, freely moving animals, and measures the excretion kinetics of the exogenous GFR tracer, fluorescein-isothiocyanate (FITC) conjugated sinistrin (an inulin analog). This system has been described in detail in rats. However, because of their smaller size, measurement of transcutaneous GFR in mice presents additional technical challenges. In this paper we therefore provide the first detailed practical guide to the use of transdermal GFR monitors in mice based on the combined experience of three different investigators who have been performing this assay in mice over a number of years.

Here we describe a protocol to measure glomerular filtration rate (GFR) in conscious, freely moving mice using a transdermal GFR monitor.

小鼠肾小球滤过率的透皮测定

Transdermal analysis of glomerular filtration rate (GFR) is an established technique that is used to assess renal function in mouse and rat models of ...

Physiology Lab Demonstration: Glomerular Filtration Rate in a Rat

Measurements of glomerular filtration rate (GFR), and the fractional excretion of sodium (Na) and potassium (K) are critical in assessing renal function in health and disease. GFR is measured as the steady state renal clearance of inulin which is filtered at the glomerulus, but not secreted or reabsorbed along the nephron. The fractional excretion of Na and K can be determined from the concentration of Na and K in plasma and urine. The renal clearance of inulin can be demonstrated in an anesthetized animal which has catheters in the femoral artery, femoral vein and bladder. The equipment and supplies used for this procedure are those commonly available in a research core facility, and thus makes this procedure a practical means for measuring renal function. The purpose of this video is to demonstrate the procedures required to perform a lab demonstration in which renal function is assessed before and after a diuretic drug. The presented technique can be utilized to assess renal function in rat models of renal disease.

The purpose of this protocol is to demonstrate the principles and techniques for measuring and calculating glomerular filtration rate, urine flow rate, and excretion of sodium and potassium in a rat. This demonstration can be used to provide students with an overall conceptual understanding of how to measure renal function.

生理学实验室演示:肾小球滤过率在一个老鼠

Measurements of glomerular filtration rate (GFR), and the fractional excretion of sodium (Na) and potassium (K) are critical in assessing renal function ...

Use of Enzymatic Biosensors to Quantify Endogenous ATP or H2O2 in the Kidney

Enzymatic microelectrode biosensors have been widely used to measure extracellular signaling in real-time. Most of their use has been limited to brain slices and neuronal cell cultures. Recently, this technology has been applied to the whole organs. Advances in sensor design have made &#160;possible the measuring of&#160;cell signaling in blood-perfused in vivo kidneys. The present protocols list the steps needed to measure ATP and H2O2 signaling in the rat kidney interstitium. Two separate sensor designs are used for the ex vivo and in vivo protocols. Both types of sensor&#160;are coated with&#160;a thin enzymatic biolayer on top of a permselectivity layer to give fast responding, sensitive and selective biosensors. The permselectivity layer protects the signal from the interferents in biological tissue, and the enzymatic layer utilizes&#160;the sequential catalytic reaction of glycerol kinase and glycerol-3-phosphate oxidase in the presence of ATP to produce H2O2. The set of sensors used for the ex vivo studies further detected analyte by oxidation of H2O2 on a platinum/iridium (Pt-Ir) wire electrode. The sensors for the&#160;in vivo studies are instead based on the reduction of H2O2 on a mediator coated gold electrode designed for blood-perfused tissue. Final concentration changes&#160;are detected by real-time amperometry followed by calibration to known concentrations of analyte. Additionally, the specificity of the amperometric signal can be confirmed by the addition of enzymes such as catalase and apyrase that break down H2O2 and ATP correspondingly. These sensors also rely heavily on accurate calibrations before and after each experiment. The following two protocols establish the study of real-time detection of ATP and H2O2 in kidney tissues, and can be further modified to extend the described method for use in other biological preparations or whole organs.

Use of Enzymatic Biosensors to Quantify Endogenous ATP or H2O2 in the Kidney

Enzymatic microelectrode biosensors enable real-time measurements of extracellular cell signaling in biologically-relevant concentrations. The following protocols extend the applications of biosensors to the ex vivo and in vivo detection of ATP and H2O2 in the kidney.

利用酶生物传感器的量化内源性ATP或H<sub&gt; 2</sub&gt; 0<sub&gt; 2</sub&gt;在肾脏

Enzymatic microelectrode biosensors have been widely used to measure extracellular signaling in real-time. Most of their use has been limited to brain ...

Long-Term Continuous Measurement of Renal Blood Flow in Conscious Rats

The kidneys play a crucial role in maintaining the homeostasis of body fluids. The regulation of renal blood flow (RBF) is essential to the vital functions of filtration and metabolism in kidney function. Many acute studies have been carried out in anesthetized animals to measure RBF under various conditions to determine mechanisms responsible for the regulation of kidney perfusion. However, for technical reasons, it has not been possible to measure RBF continuously (24 h/day) in unrestrained unanesthetized rats over prolonged periods. These methods allow the continuous determination of RBF over many weeks while also simultaneously recording blood pressure (BP) with implanted catheters (fluid-filled or by telemetry). RBF monitoring is carried out with rats placed in a circular servo-controlled rat cage that enables the unrestrained movement of the rat throughout the study. At the same time, the tangling of cables from the flow probe and arterial catheters is prevented. Rats are first instrumented with an ultrasonic flow probe placement on the left renal artery and an arterial catheter implanted in the right femoral artery. These are routed subcutaneously to the nape of the neck, and connected to the flowmeter and pressure transducer, respectively, to measure RBF and BP. Following surgical implantation, rats are immediately placed in the cage to recover for at least one week and stabilize the ultrasonic probe recordings. Urine collection is also feasible in this system. The surgical and post-surgical procedures for continuous monitoring are demonstrated in this protocol.

The present protocol describes a long-term continuous measurement of renal blood flow in conscious rats and simultaneously recording blood pressure with implanted catheters (fluid-filled or by telemetry).

意识大鼠肾血流量的长期连续测量

The kidneys play a crucial role in maintaining the homeostasis of body fluids. The regulation of renal blood flow (RBF) is essential to the vital ...

Transdermal Measurement of Glomerular Filtration Rate in Mechanically Ventilated Piglets

Transdermal measurement of glomerular filtration rate (GFR) has been used to evaluate kidney function in conscious animals. This technique is well established in rodents to study acute kidney injury and chronic kidney disease. However, GFR measurement using the transdermal system has not been validated in pigs, a species with a similar renal system to humans. Hence, we investigated the effect of sepsis on transdermal GFR in anesthetized and mechanically ventilated neonatal pigs. Polymicrobial sepsis was induced by cecal ligation and puncture (CLP). The transdermal GFR measurement system consisting of a miniaturized fluorescence sensor was attached to the pig's shaved skin to determine the clearance of fluorescein-isothiocyanate (FITC) conjugated sinistrin, an intravenously injected GFR tracer. Our results show that at 12 h post-CLP, serum creatinine increased with a decrease in GFR. This study demonstrates, for the first time, the utility of the transdermal GFR approach in determining renal function in mechanically ventilated, neonatal pigs.

Glomerular filtration rate (GFR) is the ideal marker for assessing kidney function. However, the standard measurement method using inulin injection with serial blood and urine analysis is impractical. This article delineates a practical method to measure GFR transdermally in piglets.

机械通气仔猪肾小球滤过率的透皮测量

Transdermal measurement of glomerular filtration rate (GFR) has been used to evaluate kidney function in conscious animals. This technique is well ...

Cystometric and External Urethral Sphincter Measurements in Awake Rats with Implanted Catheter and Electrodes Allowing for Repeated Measurements

Lower urinary tract function is mainly assessed by means of cystometric bladder function analysis in rodents. Conventional cystometries are usually performed as terminal analysis under urethane anesthesia. It is well known that anesthetic drugs can influence bladder function. Hence, the aim of this technique is to perform cystometric measurements of the urinary bladder and external urethral sphincter in lightly restrained awake rats. For this purpose, a bladder catheter is implanted into the bladder dome. Subsequently, two electrodes are implanted bilateral to the external urethral sphincter and a ground electrode is sutured to a non-responsive skeletal muscle. The bladder catheter and the three electrodes are finally tunneled subcutaneously to the neck region and affixed to a harness. With this technique, the lower urinary tract can be measured at multiple time points in the same animal to assess lower urinary tract function. The main application of this technique is the follow-up of simultaneous urinary bladder and external urethral sphincter function in awake healthy rats and after induction of a disease or injury. Moreover, subsequent lower urinary tract monitoring can be performed during evaluation of the disease/injury and to monitor treatment efficacy.

This protocol first describes the surgical procedure of the permanent implantation of a urinary bladder catheter combined with external urethral sphincter electrodes, and second, the measurement of the function of the urinary bladder and external urethral sphincter in implanted awake animals.

压和外尿道括约肌测量在清醒大鼠植入导管和电极允许重复测量

Lower urinary tract function is mainly assessed by means of cystometric bladder function analysis in rodents. Conventional cystometries are usually ...