Single-channel Analyse et Imagerie de calcium dans les podocytes des glomérules fraîchement isolés
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
Les reins maintiennent l'équilibre homéostatique pour diverses substances et de réguler le volume sanguin d'une manière qui détermine la pression sanguine totale. Des perturbations dans la filtration rénale, la réabsorption ou la sécrétion d'entraîner ou d'accompagner des états pathologiques, allant de l'hyper ou hypotension pour mettre fin à l'insuffisance rénale qui nécessite éventuellement une transplantation rénale. L'unité de filtration rénale (glomérule) se compose de trois couches – l'endothélium capillaire, la membrane basale et une couche mono-cellulaire des cellules épithéliales – podocytes, qui jouent un rôle majeur dans le maintien de l'intégrité et de la fonction 1 fente diaphragme. Dysfonction dans le filtre glomérulaire provoque une perte urinaire à perméabilité sélective de macromolécules, telles que la protéinurie. Différents agents peuvent affecter la structure des podocytes et leurs procédés de pied, qui déterminent l'intégrité de la barrière de filtration de glomérules.
Les podocytes sont impliqués dans le maintien de la glomfonction de filtration Eruli. Il a été établi que la manipulation de calcium impropre à l'podocytes conduit à une lésion de la cellule et joue un rôle important dans la progression de diverses formes de néphropathies 2,3. Par conséquent, le développement d'un modèle qui permet de mesure directe des changements de concentration de calcium intracellulaire sera déterminante pour les études de la fonction des podocytes. Glomérules isolés ont été précédemment utilisés dans de nombreuses études, y compris une mesure du coefficient de réflexion albumine change 4 et l'évaluation des courants cellulaires intégraux dans les cellules entières électrophysiologiques mesures de patch-clamp 5,6. Dans le présent article, nous décrivons le protocole qui permet au chercheur de mesurer intracellulaires changements de concentration de calcium en réponse aux demandes d'agents pharmacologiques, estimer les niveaux de calcium dans les cellules basales, et d'évaluer l'activité des particuliers canaux calciques. Mesures de la concentration de calcium Ratometric et patch-clamp electrophysiology ont été utilisés pour déterminer respectivement des changements dans la concentration de calcium intracellulaire au sein de l'activité de canal et podocytes.
Protocol
Representative Results
Discussion
L'approche décrite ici permet l'analyse de manipulation de calcium par les podocytes des glomérules chez les rongeurs. Cette technique permet l'application de patch-clamp électrophysiologie à canal unique et imagerie confocale à fluorescence ratiométrique. Cependant, les deux approches peuvent être utilisés séparément, sur leur propre. Le protocole proposé comporte plusieurs étapes relativement simples, y compris 1) Kidney Flush; 2) l'isolement des glomérules par tamisage différentiel; 3) e…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Les auteurs tiennent à remercier Glen Slocum (Medical College of Wisconsin) et Colleen A. Lavin (Nikon Instruments, Inc.) pour une excellente assistance technique avec des expériences de microscopie. Gregory Blass est reconnu pour la relecture critique du manuscrit. Cette recherche a été financée par les Instituts nationaux de la santé HL108880 de subvention et American Diabetes Association accorder 1-15-BS-172 (AS), et Ben J. Lipps bourse de recherche de l'American Society of Nephrology (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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