作者想感谢格伦·斯洛克姆（威斯康星医学院）和科琳A.拉文（尼康仪器公司）与显微镜实验优秀的技术援助。这项研究是支持由美国国立卫生研究院授予R01 HL108880（以AS），R01 DK095029（以OPO）和K99 HL116603（以TSP），全国肾脏基金会IG1724（以TSP），美国心脏协会13GRNT16220002（以OPO）和本·J.里普斯研究奖学金由美国肾脏病学会（至DVI）。

下面描述的实验程序，在威斯康星州和德克萨斯大学健康科学中心休斯敦大学医学院获得批准的机构动物护理和使用委员会，并分别按照卫生指南实验动物的护理和使用国家研究院​​。 图1显示的组织分离和处理过程的主要步骤。简要地说，从PCK或SD大鼠的肾脏用于收集无论是从健康非拨小管或囊肿导管上皮单层的手动隔离。在这里，我们从4-16周龄PCK大鼠10,13研究肾脏。 1.肾囊肿和连接小管（CNT）/隔离集合管（CD）段<ol><li>麻醉实验动物异氟醚（5％的诱导，1.5〜2.5％的维护）/医用级O 2或其他经批准的方法。动物必须不断监控，以确保有足够的文件韦尔麻醉。稳定的呼吸频率和脚趾捏的反应是用来确定适当的麻醉。 </li><li>执行剖腹并通过腹主动脉17冲洗用磷酸缓冲盐水（PBS）的肾脏。 <ol><li>准备一个聚乙烯管导管（PE50），并把它连接到注射泵填充有PBS。切开皮肤和腹壁沿白线，然后使整个腹部横向腹部切口，从一边到另一边倒腾腹部器官用棉签以访问降主动脉与肠系膜分支和腹腔动脉。在进一步的过程滋润五脏六腑用温生理盐水，以防止其干燥。 </li><li>将一个连字（＃1）周围的肠系膜和腹腔动脉，另一结扎（＃2）周围的隔膜下主动脉（不打领带）。轻轻分开一个。腹钝性解剖结缔组织周围薄镊子把两个连写〜3左肾动脉下方毫米（＃3）和上述髂动脉分叉（＃4）。 </li><li>领带结扎＃4和附加周围左肾动脉结扎和＃3之间的主动脉的容器夹具。使钳和结扎＃4导尿主动脉之间的切口，用绷带＃3牢固地固定导管。 </li><li>释放夹紧并确保该脉是在导管可见以确保正确安装。开始灌流以6ml / min的速率，领带连字＃1和＃2，和切断左肾静脉。继续冲洗1-2分钟，直到脏器完全变白。 </li><li>切肾血管，尿道口及其周围结缔组织和脂肪组织用剪刀来收集肾脏。然后作一个简短的撕裂肾囊和剥离肾脏解封他们。将肾脏入冰 - 冷PBS。安乐死是通过开胸手术证实。 </li></ol></li><li>制备涂覆有聚-L-赖氨酸的5×5毫米玻璃盖片。切盖GLA分类用金刚石铅笔和地点大约20微升的0.01％无菌过滤聚-L-赖氨酸溶液到每玻璃芯片。准备生理盐水和两对制表师镊子解剖。 </li><li>切断与沿冠状面刀片肾脏进入1-2毫米厚度的〜切片。放置在立体显微镜下的片段之一。组织的分离应在冰冷盐水来完成。 </li><li>定位在立体显微镜下为圆形状的空腔（图2A）囊肿;他们的墙壁通常含有增生的血管突出的网络。使用细尖镊子解剖囊肿尽可能地薄，得到的单层面积的内部上皮细胞层。它附加到覆盖有聚L-赖氨酸的玻璃片。粘聚-L-赖氨酸表面允许研究者牢牢定位在玻璃内部囊肿层。暴露与顶侧上的囊肿，以提供接入。移液器（图2B）。 </li><li>使用PCK或SD大鼠的孩子内伊中央片含有乳头的非扩张的CNT / CCDsegments 18-21隔离。 注:乳头状组织比皮质和保持乳头管带用钳子和沿径向轴撕裂片可切割为薄部门更持久。个别小管是可见的，并可以拉出到被放置在盖玻璃芯片。 </li><li>识别将CNT / CCD段由分岔，比近端小管和大显着可见的细胞（图2C）更高的透明度。放置附有小管的玻璃盖片上配备有适合驱动微量显微在显微镜下。使用锋利的微量分割开管和连接它们的边缘到玻璃使顶端表面可访问（图2D）。 </li></ol> 2.单通道膜片钳电<ol><li>填充膜片钳室浴溶液和传送护罩玻璃芯片与离体组织到腔室。确保膜片钳微量具有7-10兆欧电阻为可靠的细胞（细胞连接的）测量。 </li><li>对于细胞贴附测量，将放大器的增益比20倍，并通过一个八极贝塞尔滤波器低通的电流，在300赫兹。对于ENaC的频道监测，使用一个浴溶液，以mM:150的NaCl，1的CaCl 2，2的MgCl 2，10 HEPES（pH 7.4）中;吸移管:140的LiCl，2的MgCl 2和10的HEPES（pH 7.4）中。 </li><li>进行常规的膜片钳实验中一个细胞贴附模式10。选择在上皮单层的细胞并接近吸管顶膜。形成通过施加轻柔抽吸吸管和细胞膜之间的高电阻密封。一旦高耐千兆欧姆密封形成，在保持电位的间隙 - 自由协议应启动针对感兴趣的信道的监视活动。 </li><li>从千兆存储的无间隙的单通道电流数据欧姆密封件进行后续分析。分析使用一般用膜片钳设置18提供的软件包的渠道活动。计算信道活性NPO其中N是指在贴片和P 2 O的活性通道的数量是信道的平均开放概率。 注:使用孤立囊肿或小管在膜片钳实验中不超过30分钟。 </li></ol>细胞内钙离子浓度的上皮细胞3成比例落射荧光测量<ol><li>使用5mM的的Fura-2AM溶解在DMSO中。等分试样原液成单独的500μl的锥形管中（约10微升）。从光线并存储在冰箱在-20℃下长达半年的保护。 </li><li>在PSS孵育孤立囊肿和分裂开管30-40分钟（毫米:145氯化钠，氯化钾4.5，2 氯化镁 ，氯化钙2 2和 10 HEPES pH值为7.35），含5微 ​​米的Fura-2 AM染料和0.05％聚醚酸有助于驱散乙酰氧基甲基酯。为此，含有装载鸡尾酒地方的组织在3.5厘米培养皿，避光孵育一个缓慢摇动器上在室温下进行。 </li><li>改变的Fura-2 AM含有媒体孵育后清洗PSS和放置组织，在显微镜下以进一步执行萤光成像。 </li><li>打开CCD相机和稳定的光源（单色系统）配备了滤光轮（每个过滤器的位置可以有自己的衰减水平，选择每一个过滤器被调用时，相关联）。 </li><li>查找在明视场组织。切换到检测荧光信号和调整用中性密度过滤器和灯功率，以避免信号的饱和度的光源的强度。 </li><li>监视与比率激励的组织样品中的Fura-2 AM荧光在三百八十○分之三百四十○纳米，具有0.125 Hz或更高的频率。使用40×/ NA 1.3或类似的物镜正确的解像度化形象。 </li><li>用于图像处理和计算导入图像序列。确保分裂渠道和使用hyperstack灰度模式。选择几个感兴趣的区域（单囊细胞或囊状组织选定的区域），并计算强度值每通道（340和380纳米）转换成首选数据分析软件;减法背景的强度值从每个数据点。 </li><li>对于每个时间点计算出的Fura-2 AM 340〜380信道的强度的比率。地块分散/的钙瞬变每个地区的线路点时间的变化，并计算平均值/ SE值。 </li></ol>

The authors have nothing to disclose.

我们在这里描述的传统膜片钳技术和表面荧光钙成像的应用程序从ARPKD的鼠的遗传模型导出囊性上皮单层。该协议包括三个步骤，其中最应引起重视的囊肿（协议节的步骤1.5）的隔离和电生理研究。这些关键步骤需要大量的训练和耐心，读者不应该立刻感到沮丧。 首先，最要注意的囊肿单层隔离的过程。制备的这一部分需要手工技巧和显著影响进一步的工作，作为检体和其连...

离子通道在许多生理功能，包括细胞生长和分化中发挥显著作用。常染色体显性和隐性多囊肾疾病（ADPKD和ARPKD，分别）是其特征在于，所述的肾小管上皮细胞起源的肾流体填充囊肿的发展遗传性疾病。多囊肾是由PKD1或PKD2编码polycystins 1和参与细胞增殖和分化的调节如图2所示，膜蛋白的突变引起的。 PKD2本身或作为与PKD1一个复杂也发挥作为钙 -permeable阳离子通道1。所述PKHD1基因编码fibrocystin（涉及小管和/或维持上皮极性的纤毛相关受体样蛋白）的突变是ARPKD 2的遗传动力。囊肿生长是一个复杂的现象，伴随着不安扩散3,4，5血管生成，分化和极性损失性肾小管细胞6-8。 有缺陷的再吸收和分泌增强囊性上皮有助于在管腔和囊肿膨胀9,10-流体积聚。受损的流量依赖性钙离子浓度我的信号也已PKD 11-15时挂cystogenesis。 这里，我们描述适用于单通道活性和细胞内Ca 2+水平的囊性上皮单层从PCK大鼠分离的膜片钳测量的方法。这种方法成功地被我们用于表征上皮细胞钠离子通道（ENaC的）10的活性和钙离子浓度依赖我引起的钙 -permeable TRPV4和嘌呤信号级联13处理 。 在这些研究中，我们使用的PCK大鼠，模型ARPKD引起的在PKHD1基因的自发突变。该PCK应变originallÿ从的Sprague-Dawley（SD）大鼠16从而SD大鼠被用作适当的控制用于 ​​与PCK应变比较而得。其结果是，既大鼠肾段和非扩张集合管从同一PCK大鼠分离可以作为两个不同的比较组对囊性上皮实验。

<table><tbody><tr><td>Fura-2 AM</td><td>Life Technologies</td><td>F-14185</td><td></td></tr><tr><td>Fluo-8</td><td>AAT Bioquest</td><td>21091</td><td></td></tr><tr><td>Poly-L-lysine</td><td>Sigma-Aldrich</td><td>P4707</td><td></td></tr><tr><td>Pluronic acid</td><td>Sigma-Aldrich</td><td>F-68&amp;nbsp;</td><td>solution</td></tr><tr><td>Shaker</td><td>Boekel Scientific</td><td>260350</td><td></td></tr><tr><td>Light source</td><td>Sutter Instrument Co</td><td>Lambda XL</td><td>with integrated shutter/filter wheel driver</td></tr><tr><td>Neutral density filters</td><td>Nikon</td><td></td><td>ND4, ND8</td></tr><tr><td>Objective</td><td>Nikon</td><td>SFluo&amp;nbsp;</td><td>40/1.3 DIC WD 0.22&amp;nbsp;&amp;nbsp; oil</td></tr><tr><td>Camera</td><td>Andor Technologies</td><td>Zyla sCMOS</td><td></td></tr><tr><td>Nikon&amp;nbsp; microscope (inverted)</td><td>Nikon</td><td>Nikon Eclipse TE2000-S</td><td></td></tr><tr><td>Cover Glass</td><td>Thermo Scientific</td><td>6661B52</td><td></td></tr><tr><td>Diamond pencil</td><td>Fisher Scientific</td><td>22268912</td><td></td></tr><tr><td>Image acquisition software</td><td>Nikon</td><td>Nikon NIS-Elements&amp;nbsp;</td><td></td></tr><tr><td>Image analysis software</td><td>ImageJ</td><td>http://imagej.nih.gov/</td><td>ND Utility plugin allows to import images in the native Nikon Instruments .nd2 format</td></tr><tr><td>Recording/perfusion chamber</td><td>Warner Instruments</td><td>RC-26</td><td></td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>Patch Clamp amplifier</td><td>Molecular Devices</td><td>MultiClamp 700B</td><td></td></tr><tr><td>Data Acquisition System</td><td>Molecular Devices</td><td>Digidata 1440A</td><td>Axon Digidata&amp;reg; System</td></tr><tr><td>Low Pass Filter</td><td>Warner Instruments</td><td>LPF-8</td><td>8 pole Bessel</td></tr><tr><td>Borosilicate glass capillaries</td><td>World Precision Instruments</td><td>1B150F-4</td><td></td></tr><tr><td>Micropipette Puller</td><td>Sutter Instrument Co</td><td>P-97</td><td>Flaming/Brown type micropipette puller</td></tr><tr><td>Microforge</td><td>Narishige</td><td>MF-830</td><td>Japan</td></tr><tr><td>Motorized Micromanipulator</td><td>Sutter Instrument Co</td><td>MP-225</td><td></td></tr><tr><td>Inverted microscope</td><td>Nikon</td><td>Eclipse Ti</td><td></td></tr><tr><td>Microvibration isolation table</td><td>TMC</td><td></td><td>equipped with Faraday cage</td></tr><tr><td>Multichannel valve perfusion system</td><td>AutoMake Scientific</td><td>Valve Bank II</td><td></td></tr><tr><td>Recording/perfusion chamber</td><td>Warner Instruments</td><td>RC-26</td><td></td></tr><tr><td>Software</td><td>Molecular Devices</td><td>pClamp 10 . 2</td><td></td></tr><tr><td>Temperature controlled surgical table&amp;nbsp;</td><td>MCW core</td><td></td><td>for rodents</td></tr><tr><td>Binocular stereomicroscope</td><td>Nikon</td><td>SMZ745</td><td></td></tr><tr><td>Syringe pump-based perfusion system</td><td>Harvard Apparatus</td><td></td><td></td></tr><tr><td>polyethylene tubing</td><td>Sigma-Aldrich</td><td>PE50</td><td></td></tr><tr><td>Isofluorane anesthesia</td><td>&lt;a href=&quot;http://www.vetequip.com/&quot;&gt;http://www.vetequip.com/&lt;/a&gt;</td><td>911103</td><td></td></tr><tr><td>Other basic reagents</td><td>Sigma-Aldrich</td><td></td><td></td></tr></tbody></table>

implementing patch clamp and live fluorescence microscopy to monitor functional properties of freshly isolated pkd epithelium

期间多囊肾囊肿萌生和扩展是一个复杂的过程，其特征在肾小管细胞增殖，管腔流体积累和细胞外基质的形成异常。离子通道和细胞内钙信号的活动是决定的肾小管上皮细胞功能的关键生理参数。我们开发适合于实时观察离子通道的活性与在上皮单层从肾囊肿新鲜分离的细胞内Ca 2+水平膜片钳技术和登记的方法。 PCK大鼠的常染色体隐性遗传性多囊肾病（ARPKD）遗传模型，这里被用作离子通道和钙流的体外分析。这里描述的是详细的一步一步的步骤旨在隔离囊性单层和非扩张管从PCK或正常只SD（SD）大鼠，并监视单通道活性和细胞内Ca 2+动力学。此方法不需要酶加工，并允许在分析新鲜分离的上皮单层的天然环境。此外，这种技术是将细胞内钙的变化非常敏感，并生成高分辨率的图像进行精确的测量。最后，分离的囊性上皮可进一步用于与抗体或染料，制备原代培养物的纯化和关于各种生化分析染色。

潜在的ENaC参与cystogenesis已经证明了几项研究观察到了破坏表皮生长因子（EGF）在PKD进展22-25和异常钠重吸收在ARPKD鼠模型和组织培养26-28信令。例如，Veizis 等人表明，阿米洛利敏感的 Na +吸收在光盘细胞ARPKD 29的非直系同源BPK小鼠模型下降。我们最近证明，受损的钠和水的重吸收的孢囊是在加重cystogenesis 10的一个重要因素。具体来说，我们采用电生理...

Watch this Scientific Journal Video about Implementing Patch Clamp and Live Fluorescence Microscopy to Monitor Functional Properties of Freshly Isolated PKD Epithelium at JoVE.com

Implementing Patch Clamp and Live Fluorescence Microscopy to Monitor Functional Properties of Freshly Isolated PKD Epithelium

表现在肾小管上皮细胞离子通道在多囊肾病的病理一个显著的作用。这里，我们描述用于执行囊性上皮从啮齿动物肾脏新鲜分离的膜片钳分析和细胞内钙水平的测量的实验方案。

实施膜片钳和活荧光显微镜监视新鲜分离的PKD上皮细胞的功能特性

Cyst initiation and expansion during polycystic kidney disease is a complex process characterized by abnormalities in tubular cell proliferation, luminal ...

implementing-patch-clamp-live-fluorescence-microscopy-to-monitor

Research

JoVE Journal

Biology

9.7K 次观看.  Medical College of Wisconsin. 表现在肾小管上皮细胞离子通道在多囊肾病的病理一个显著的作用。这里，我们描述用于执行囊性上皮从啮齿动物肾脏新鲜分离的膜片钳分析和细胞内钙水平的测量的实验方案。 

视频: Implementing Patch Clamp and Live Fluorescence Microscopy to Monitor Functional Properties of Freshly Isolated PKD Epithelium

Live-imaging of PKC Translocation in Sf9 Cells and in Aplysia Sensory Neurons

Protein kinase Cs (PKCs) are serine threonine kinases that play a central role in regulating a wide variety of cellular processes such as cell growth and learning and memory. There are four known families of PKC isoforms in vertebrates: classical PKCs (&alpha;, &beta;I, &beta;II and &gamma;), novel type I PKCs (&#949; and &eta;), novel type II PKCs (&delta; and &theta;), and atypical PKCs (&zeta; and &iota;). The classical PKCs are activated by Ca2+ and diacylclycerol (DAG), while the novel PKCs are activated by DAG, but are Ca2+-independent. The atypical PKCs are activated by neither Ca2+ nor DAG. In Aplysia californica, our model system to study memory formation, there are three nervous system specific PKC isoforms one from each major class, namely the conventional PKC Apl I, the novel type I PKC Apl II and the atypical PKC Apl III. PKCs are lipid-activated kinases and thus activation of classical and novel PKCs in response to extracellular signals has been frequently correlated with PKC translocation from the cytoplasm to the plasma membrane. Therefore, visualizing PKC translocation in real time in live cells has become an invaluable tool for elucidating the signal transduction pathways that lead to PKC activation. For instance, this technique has allowed for us to establish that different isoforms of PKC translocate under different conditions to mediate distinct types of synaptic plasticity and that serotonin (5HT) activation of PKC Apl II requires production of both DAG and phosphatidic acid (PA) for translocation 1-2. Importantly, the ability to visualize the same neuron repeatedly has allowed us, for example, to measure desensitization of the PKC response in exquisite detail 3. In this video, we demonstrate each step of preparing Sf9 cell cultures, cultures of Aplysia sensory neurons have been described in another video article 4, expressing fluorescently tagged PKCs in Sf9 cells and in Aplysia sensory neurons and live-imaging of PKC translocation in response to different activators using laser-scanning microscopy.

In this video, we demonstrate visualization of PKC translocation in living cells using fluorescently tagged PKCs.

在Sf9细胞，并在海兔感觉神经元PKC移位实时成像

Protein kinase Cs (PKCs) are serine threonine kinases that play a central role in regulating a wide variety of cellular processes such as cell growth and ...

科研

神经科学

Use of pHluorin to Assess the Dynamics of Axon Guidance Receptors in Cell Culture and in the Chick Embryo

During development, axon guidance receptors play a crucial role in regulating axons sensitivity to both attractive and repulsive cues. Indeed, activation of the guidance receptors is the first step of the signaling mechanisms allowing axon tips, the growth cones, to respond to the ligands. As such, the modulation of their availability at the cell surface is one of the mechanisms that participate in setting the growth cone sensitivity. We describe here a method to precisely visualize the spatio-temporal cell surface dynamics of an axon guidance receptor both in vitro and in vivo in the developing chick spinal cord. We took advantage of the pH-dependent fluorescence property of a green fluorescent protein (GFP) variant to specifically detect the fraction of the axon guidance receptor that is addressed to the plasma membrane. We first describe the in vitro validation of such pH-dependent constructs and we further detail their use in vivo, in the chick spinal chord, to assess the spatio-temporal dynamics of the axon guidance receptor of interest.

We describe here the use of a pH-sensitive green fluorescent protein variant, pHluorin, to study the spatio-temporal dynamics of axon guidance receptors trafficking at the cell surface.&#160;The pHluorin-tagged receptor is expressed both in cell culture and in vivo, using electroporation of the chick embryo.

使用普洛林来评估阿克森指导受体在细胞培养和小鸡胚胎中的动力学

During development, axon guidance receptors play a crucial role in regulating axons sensitivity to both attractive and repulsive cues. Indeed, activation ...

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

异源表达在哺乳动物细胞离子通道的突变和功能分析

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

生物学

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) ...

医学

Analysis of Nephron Composition and Function in the Adult Zebrafish Kidney

The zebrafish model has emerged as a relevant system to study kidney development, regeneration and disease. Both the embryonic and adult zebrafish kidneys are composed of functional units known as nephrons, which are highly conserved with other vertebrates, including mammals. Research in zebrafish has recently demonstrated that two distinctive phenomena transpire after adult nephrons incur damage: first, there is robust regeneration within existing nephrons that replaces the destroyed tubule epithelial cells; second, entirely new nephrons are produced from renal progenitors in a process known as neonephrogenesis. In contrast, humans and other mammals seem to have only a limited ability for nephron epithelial regeneration. To date, the mechanisms responsible for these kidney regeneration phenomena remain poorly understood. Since adult zebrafish kidneys undergo both nephron epithelial regeneration and neonephrogenesis, they provide an outstanding experimental paradigm to study these events. Further, there is a wide range of genetic and pharmacological tools available in the zebrafish model that can be used to delineate the cellular and molecular mechanisms that regulate renal regeneration. One essential aspect of such research is the evaluation of nephron structure and function. This protocol describes a set of labeling techniques that can be used to gauge renal composition and test nephron functionality in the adult zebrafish kidney. Thus, these methods are widely applicable to the future phenotypic characterization of adult zebrafish kidney injury paradigms, which include but are not limited to, nephrotoxicant exposure regimes or genetic methods of targeted cell death such as the nitroreductase mediated cell ablation technique. Further, these methods could be used to study genetic perturbations in adult kidney formation and could also be applied to assess renal status during chronic disease modeling.

The zebrafish adult kidney is an excellent system for renal regeneration and disease studies. An essential aspect of such research is the assessment of nephron structure and function. This protocol describes several methodologies that can be implemented to assess nephron tubule composition and to evaluate renal reabsorption.

肾单位的组成与功能的成年斑马鱼肾脏分析

The zebrafish model has emerged as a relevant system to study kidney development, regeneration and disease. Both the embryonic and adult zebrafish kidneys ...

Single-channel Analysis and Calcium Imaging in the Podocytes of the Freshly Isolated Glomeruli

Podocytes (renal glomerular epithelial cells) are known to regulate glomerular permeability and maintain glomerular structure; a key role for these cells in the pathogenesis of various renal diseases has been established since podocyte injury leads to proteinuria and foot process effacement. It was previously reported that various endogenous agents may cause a dramatic overload in intracellular Ca2+ concentration in podocytes, presumably leading to albuminuria, and this likely occurs via calcium-conducting ion channels. Therefore, it appeared important to study calcium handling in the podocytes both under normal conditions and in various pathological states. However, available experimental approaches have remained somewhat limited to cultured and transfected cells. Although they represent a good basic model for such studies, they are essentially extracted from the native environment of the glomerulus. Here we describe the methodology of studying podocytes as a part of the freshly isolated whole glomerulus. This preparation retains the functional potential of the podocytes, which are still attached to the capillaries; therefore, podocytes remain in the environment that conserves the major parts of the glomeruli filtration apparatus. The present manuscript elaborates on two experimental approaches that allow 1) real-time detection of calcium concentration changes with the help of ratiometric confocal fluorescence microscopy, and 2) the recording of the single ion channels activity in the podocytes of the freshly isolated glomeruli. These methodologies utilize the advantages of the native environment of the glomerulus that enable researchers to resolve acute changes in the intracellular calcium handling in response to applications of various agents, measure basal concentration of calcium within the cells (for instance, to evaluate disease progression), and assess and manipulate calcium conductance at the level of single ion channels.

Changes in the intracellular calcium levels in the podocytes are one of the most important means to control the filtration function of glomeruli. Here we explain a high-throughput approach that allows detection of real-time calcium handling and single ion channels activity in the podocytes of the freshly isolated glomeruli.

单通道分析和钙成像在新鲜分离的肾小球足细胞

Podocytes (renal glomerular epithelial cells) are known to regulate glomerular permeability and maintain glomerular structure; a key role for these cells ...

Optical Quantification of Intracellular pH in Drosophila melanogaster Malpighian Tubule Epithelia with a Fluorescent Genetically-encoded pH Indicator

Epithelial ion transport is vital to systemic ion homeostasis as well as maintenance of essential cellular electrochemical gradients. Intracellular pH (pHi) is influenced by many ion transporters and thus monitoring pHi is a useful tool for assessing transporter activity. Modern Genetically Encoded pH-Indicators (GEpHIs) provide optical quantification of pHi in intact cells on a cellular and subcellular scale. This protocol describes real-time quantification of cellular pHi regulation in Malpighian Tubules (MTs) of Drosophila melanogaster through ex vivo live-imaging of pHerry, a pseudo-ratiometric GEpHI with a pKa well-suited to track pH changes in the cytosol. Extracted adult fly MTs are composed of morphologically and functionally distinct sections of single-cell layer epithelia, and can serve as an accessible and genetically tractable model for investigation of epithelial transport. GEpHIs offer several advantages over conventional pH-sensitive fluorescent dyes and ion-selective electrodes. GEpHIs can label distinct cell populations provided appropriate promoter elements are available. This labeling is particularly useful in ex vivo, in vivo, and in situ preparations, which are inherently heterogeneous. GEpHIs also permit quantification of pHi in intact tissues over time without need for repeated dye treatment or tissue externalization. The primary drawback of current GEpHIs is the tendency to aggregate in cytosolic inclusions in response to tissue damage and construct over-expression. These shortcomings, their solutions, and the inherent advantages of GEpHIs are demonstrated in this protocol through assessment of basolateral proton (H+) transport in functionally distinct principal and stellate cells of extracted fly MTs. The techniques and analysis described are readily adaptable to a wide variety of vertebrate and invertebrate preparations, and the sophistication of the assay can be scaled from teaching labs to intricate determination of ion flux via specific transporters.

Optical Quantification of Intracellular pH in Drosophila melanogaster Malpighian Tubule Epithelia with a Fluorescent Genetically-encoded pH Indicator

Cellular ion transport can often be assessed by monitoring intracellular pH (pHi). Genetically Encoded pH-Indicators (GEpHIs)&#160;provide optical quantification of intracellular pH in intact cells. This protocol details the quantification of intracellular pH through cellular ex vivo live-imaging of Malpighian tubules of Drosophila melanogaster with pHerry, a pseudo-ratiometric genetically encoded pH-indicator.

用荧光基因编码的 ph 指示剂对果蝇氏小管上皮细胞内 ph 值的光学定量

Epithelial ion transport is vital to systemic ion homeostasis as well as maintenance of essential cellular electrochemical gradients. Intracellular pH ...

Isolation, Characterization, And High Throughput Extracellular Flux Analysis of Mouse Primary Renal Tubular Epithelial Cells

Mitochondrial dysfunction in the renal tubular epithelial cells (TECs) can lead to renal fibrosis, a major cause of&#160;chronic kidney disease (CKD). Therefore, assessing mitochondrial function in primary TECs may provide valuable insight into the bioenergetic status of the cells, providing insight into the pathophysiology of CKD. While there are a number of complex protocols available for the isolation and purification of proximal tubules in different species, the field lacks a cost-effective method optimized for&#160;tubular cell isolation without the need for purification. Here, we provide an isolation protocol that allows for studies focusing on both primary mouse proximal and distal renal TECs. In addition to cost-effective reagents and minimal animal procedures required in this protocol, the isolated cells maintain high energy levels after isolation and can be sub-cultured up to four&#160;passages, allowing for continuous studies. Furthermore, using a high throughput extracellular flux analyzer, we assess the mitochondrial respiration directly in the isolated TECs in a 96-well plate for which we provide recommendations for the optimization of cell density and compound concentration. These observations suggest that this protocol can be used for renal tubular ex vivo studies with a consistent, well-standardized production of renal TECs. This protocol may have broader future applications to study mitochondrial dysfunction associated with renal disorders for drug discovery or drug characterization purposes.

This protocol provides a cost-effective approach to isolate and characterize mouse primary renal tubular cells that can subsequently be sub-cultured to assess renal biological functions ex vivo, including mitochondrial bioenergetics.

小鼠原发性肾管上皮细胞的分离、鉴定及高通量的细胞外流量分析

Mitochondrial dysfunction in the renal tubular epithelial cells (TECs) can lead to renal fibrosis, a major cause of&#160;chronic kidney disease (CKD). ...

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, ...

A Fluorescence-Based Assay of Membrane Potential for High-Throughput Functional Study of Two Endogenous Ion Channels in Two Epithelial Cell Lines

Fluorescence-based studies are suitable for high-throughput plate reader assays of cells in culture. They have been commonly employed for drug discovery campaigns targeting recombinant ion channel proteins overexpressed in cells such as HEK-293 cells. However, there is increasing emphasis on the use of tissue-relevant cell lines for studying the effects of small molecule interventions. The following protocol describes the adaptation of a fluorescence-based membrane potential assay for the study of ion channels endogenously expressed in epithelial cell lines. The membrane potential assay details a high-throughput assay for chloride channel activity of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) in two commonly studied epithelial cell lines, Caco-2 and Calu-3. In addition, this paper describes a novel application of this system to measure the activity of the Epithelial Sodium Channel (ENaC) in a high-throughput format in the same epithelial cell lines. Together, these fluorescence-based assays provide a robust and flexible platform for studying small molecule modulators, targeting two epithelial channels in a relevant cellular context.

This protocol describes a method for the study of electrogenic membrane proteins by measuring changes in membrane potential. This assay provides a platform for the functional readout of multiple ion channels endogenously expressed in epithelial cell lines.

基于荧光的膜电位测定，用于两个上皮细胞系中两个内源性离子通道的高通量功能研究

Fluorescence-based studies are suitable for high-throughput plate reader assays of cells in culture. They have been commonly employed for drug discovery ...

实施膜片钳和活荧光显微镜监视新鲜分离的PKD上皮细胞的功能特性

摘要

探索更多视频

此视频中的章节

在Sf9细胞，并在海兔感觉神经元PKC移位实时成像

使用普洛林来评估阿克森指导受体在细胞培养和小鸡胚胎中的动力学

异源表达在哺乳动物细胞离子通道的突变和功能分析

免疫标记肾组织的光学清除与成像

肾单位的组成与功能的成年斑马鱼肾脏分析

单通道分析和钙成像在新鲜分离的肾小球足细胞

用荧光基因编码的 ph 指示剂对果蝇氏小管上皮细胞内 ph 值的光学定量

小鼠原发性肾管上皮细胞的分离、鉴定及高通量的细胞外流量分析

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

基于荧光的膜电位测定，用于两个上皮细胞系中两个内源性离子通道的高通量功能研究

实施膜片钳和活荧光显微镜监视新鲜分离的PKD上皮细胞的功能特性

摘要

探索更多视频

此视频中的章节

在Sf9细胞，并在海兔感觉神经元PKC移位实时成像

使用普洛林来评估阿克森指导受体在细胞培养和小鸡胚胎中的动力学

异源表达在哺乳动物细胞离子通道的突变和功能分析

免疫标记肾组织的光学清除与成像

肾单位的组成与功能的成年斑马鱼肾脏分析

单通道分析和钙成像在新鲜分离的肾小球足细胞

用荧光基因编码的 ph 指示剂对果蝇氏小管上皮细胞内 ph 值的光学定量

小鼠原发性肾管上皮细胞的分离、鉴定及高通量的细胞外流量分析

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

基于荧光的膜电位测定，用于两个上皮细胞系中两个内源性离子通道的高通量功能研究

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