Enkanalig Analys och Kalcium Imaging i podocyter av nyisolerade Glomeruli
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
Njurar upprätthålla homeostatiska balansen för olika substanser och reglera blodvolymen på ett sätt som bestämmer den totala blodtryck. Störningar i njur filtrering reabsorption eller utsöndring leder till eller följa patologiska tillstånd, allt från hyper- eller hypotension njursjukdom i slutskedet som så småningom behöver njurtransplantation. Njurfiltreringsenhet (glomerulus) består av tre lager – kapillärendotelet, basalmembran och encelliga lager av epitelceller – podocyter, som spelar en viktig roll i upprätthållandet av slitsmembranet integritet och funktion 1. Dysfunktion i permselektiva glomerulär filtrering orsakar urin förlust av makromolekyler, såsom proteinuri. Olika medel kan påverka strukturen av podocyter och deras fot processer, vilka bestämmer integriteten hos glomeruli filtreringsbarriären.
De podocyter är involverade i upprätthållandet av glomEruli filtreringsfunktion. Det har konstaterats att felaktig kalcium hantering av podocyte leder till cellskada och spelar en viktig roll i utvecklingen av olika former av nefropatier 2,3. Därför, utveckling av en modell som möjliggör direkt mätning av intracellulära förändringar kalciumkoncentration kommer att vara avgörande för studier av podocyte funktion. Isolerade glomeruli har tidigare använts i ett stort antal studier, inklusive mätning av albumin reflektionskoefficienten förändras 4 och bedömning av integralcellströmmar i hela celler elektro patch-clamp mätningar 5,6. I föreliggande dokument beskriver vi protokoll som gör det möjligt för forskare att mäta intracellulära förändringar kalciumkoncentration som svar på tillämpningar av farmakologiska medel, uppskatta basala nivåer av kalcium i cellerna, och bedöma enskilda kalciumkanaler aktivitet. Ratometric mätningar kalciumkoncentration och patch-clamp electrophysiology användes för att bestämma förändringar i den intracellulära kalciumkoncentrationen inom podocyte och kanalaktiviteten, respektive.
Protocol
Representative Results
Discussion
Tillvägagångssättet beskrivs här möjliggör analys av kalcium hantering av podocyter av gnagare glomeruli. Denna teknik medger tillämpning av patch-clamp enda kanal elektro och fluorescens ratiometrisk konfokal avbildning. Däremot kan båda metoderna användas separat, på egen hand. Den föreslagna protokollet har flera relativt enkla steg, inklusive 1) njure flush; 2) isolering av glomeruli genom differentiell siktning; 3) utföra patch-clamp elektrofysiologiska experiment eller inkubation av glomeruli med fluo…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
Författarna vill tacka Glen Slocum (Medical College of Wisconsin) och Colleen A. Lavin (Nikon Instruments, Inc.) för utmärkt tekniskt bistånd med mikroskopi experiment. Gregory Blass är känd för kritiskt korrekturläsning av manuskriptet. Denna forskning stöds av National Institutes of Health bidrag HL108880 och American Diabetes Association bevilja 1-15-BS-172 (AS), och Ben J. Lipps forskartjänst från 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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