单通道分析和钙成像在新鲜分离的肾小球足细胞
Summary
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.
Abstract
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.
Introduction
肾脏维持自我平衡各种物质和的方式，确定总血压调节血容量。干扰在肾过滤，重吸收或分泌导致或伴随的病理状态，从高血糖或低血压结束末期肾病，最终需要肾移植。肾过滤单元（肾小球）由三层-毛细血管内皮，基底膜和上皮细胞的单细胞层-足细胞，从而起到的狭缝隔膜的完整性和功能1的维持起主要作用。功能障碍的选择性渗透肾小球过滤导致大分子，如蛋白的尿中丢失。各种试剂可以影响足细胞和它们的足突，这确定肾小球滤过屏障的完整性的结构。
足细胞都参与了格莱姆教授的维护eruli过滤功能。它已经确定，不当钙处理由足细胞导致细胞损伤和起着各种形式肾病2,3的发展中起重要作用。因此，一个模型，允许对细胞内钙离子浓度的变化直接测量将有助于对足细胞功能研究的发展。孤立肾小球在许多研究，包括白蛋白反射系数测量变化4，并在全细胞电生理膜片钳测量5,6-积分蜂窝电流的评估以前使用。在本论文中，我们描述的协议，该协议允许研究者测量响应于药理学试剂的应用细胞内钙离子浓度的变化，估算出细胞内钙的基础水平，和评估个体的钙离子通道的活性。 Ratometric钙离子浓度的测量和膜片钳electrophysiology分别用来确定足细胞和信道活动中的变化，在细胞内钙离子浓度。
Protocol
Representative Results
Discussion
这里所描述的方法允许的钙处理由啮齿类肾小球的足细胞的分析。这种技术允许应用膜片钳单通道电生理和荧光比率共焦成像。然而，这两种方法可以单独使用，在他们自己的。该协议有几个相对简单的步骤，包括：1）肾脏冲洗; 2）肾小球通过差筛分分离; 3）进行膜片钳电生理实验，或用荧光钙标记染料更改胞内钙的比例共焦成像的肾小球的孵化。
为了分离肾小球，基于Gloy…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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。格雷戈里·布拉斯是公认的手稿校对至关重要。这项研究是支持的健康补助HL108880和美国糖尿病协会的国家机构给予1-15-BS-172（AS到），以及本J.里普斯研究奖学金由美国肾脏病学会（至DVI）。
Materials
| Fluo4 AM | Life Technologies | F14217 | 500µl in DMSO |
| FuraRed AM | Life Technologies | F-3020 | |
| Poly-L-lysine | Sigma-Aldrich | P4707 | |
| Pluronic acid | Sigma-Aldrich | F-68 | solution |
| Ionomycin | Sigma-Aldrich | I3909-1ML | |
| Tube rotator | Miltenyi Biotec GmbH | 130-090-753 | Germany |
| Nikon confocal microscope (inverted) | Nikon | Nikon A1R | Laser exitation 488nm. Emission filters 500-550nm and 570-620nm |
| Objective | Nikon | Plan Apo 60x/NA 1.4 Oil | |
| Cover Glass | Thermo Scientific | 6661B52 | |
| High vacuum grease | Dow Corning | Silicone Compound | |
| Software | Nikon | Nikon NIS-Elements | |
| Recording/perfusion chamber | Warner Instruments | RC-26 | |
| Patch Clamp amplifier | Molecular Devices | MultiClamp 700B | |
| Data Acquisition System | Molecular Devices | Digidata 1440A | Axon Digidata® System |
| Low Pass Filter | Warner Instruments | LPF-8 | 8 pole Bessel |
| Borosilicate glass capillaries | World Precision Instruments | 1B150F-4 | |
| Micropipette Puller | Sutter Instrument Co | P-97 | Flaming/Brown type micropipette puller |
| Microforge | Narishige | MF-830 | Japan |
| Motorized Micromanipulator | Sutter Instrument Co | MP-225 | |
| Inverted microscope | Nikon | Eclipse Ti | |
| Microvibration isolation table | TMC | equipped with Faraday cage | |
| Multichannel valve perfusion system | AutoMake Scientific | Valve Bank II | |
| Recording/perfusion chamber | Warner Instruments | RC-26 | |
| Software | Molecular Devices | pClamp 10 . 2 | |
| Nicardipine | Sigma-Aldrich | N7510 | |
| Iberiotoxin | Sigma | I5904-5UG | |
| Niflumic acid | Sigma-Aldrich | N0630 | |
| DIDS | Sigma-Aldrich | D3514-25MG | |
| TEA chloride | Tocris | T2265 | |
| RPMI 1640 | Life Technologies | 11835030 | without antibiotics |
| BSA | Sigma-Aldrich | A8327 | 30% albumin solution |
| Temperature controlled surgical table | MCW core | for rodents | |
| Steel sieves: | #100 (150 μm), 140 (106 μm) | ||
| Gilson, Inc SIEVE 3 SS FH NO200 | Fisher Sci | 50-871-316 | |
| Gilson, Inc SIEVE 3 SS FH NO270 | Fisher Sci | 50-871-318 | |
| Gilson, Inc SIEVE 3 SS FH NO400 | Fisher Sci | 50-871-320 | |
| mesh 200 | Sigma-Aldrich | s4145 | screen for CD-1 |
| Binocular microscope | Nikon | Eclipse TS100 | |
| Binocular microscope | Nikon | SMZ745 | |
| Syringe pump-based perfusion system | Harvard Apparatus | ||
| polyethylene tubing | Sigma-Aldrich | PE50 | |
| Isofluorane anesthesia | http://www.vetequip.com/ | 911103 | |
| Other basic reagents | Sigma-Aldrich |
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