Single-kanal Analyse og Kalsium Imaging i Podocytes av nyisolerte 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
Nyrene opprett homeostatisk balanse for forskjellige stoffer og regulere blodvolum på en måte som bestemmer det totale blodtrykket. Forstyrrelser i nyrefiltrasjon, reabsorpsjon eller sekresjon føre til eller følge patologiske tilstander, alt fra hyper- eller hypotensjon å ende nyresykdom som etter hvert trenger nyretransplantasjon. Nyre filtrering enhet (glomerulus) består av tre lag – kapillære endotel, kjeller membran og en encellet lag av epitelceller – podocytes, som spiller en viktig rolle i vedlikehold av spaltegulvet integritet og funksjon 1. Dysfunksjon i det permselektive glomerulær filteret forårsaker tap av urin makromolekyler, slik som proteinuri. Forskjellige midler kan påvirke strukturen av podocytes og deres fot prosesser, som bestemmer integriteten av glomeruli filtreringsbarriere.
De podocytes er involvert i vedlikehold av glomeruli filtrering funksjon. Det har blitt fastslått at kalsium uriktig håndtering av podocyte leder til celleskade og spiller en viktig rolle i progresjonen av forskjellige former av nefropatier 2,3. Derfor er utvikling av en modell som tillater direkte måling av intracellulære kalsiumkonsentrasjonen endringer vil være medvirkende for studier av podocyte funksjon. Isolerte glomeruli ble tidligere brukt i en rekke studier, inkludert måling av albumin refleksjonskoeffisienten endrer 4 og vurdering av integrerte mobil strømninger i hel-celle elektro patch-clamp målinger 5,6. I den foreliggende beskrivelsen beskriver vi den protokoll som tillater forskeren til å måle intracellulære kalsiumkonsentrasjonen endres som reaksjon på anvendelser av farmakologiske midler, estimere basale nivåer av kalsium i cellene og vurdere individuell kalsiumkanaler aktivitet. Ratometric kalsium konsentrasjonsmålinger og patch-clamp electrophysiology ble anvendt for å bestemme endringer i den intracellulære kalsiumkonsentrasjon innenfor podocyte og kanalaktivitet, respektivt.
Protocol
Representative Results
Discussion
Den metode som er beskrevet her gjør det mulig for analyse av kalsium håndtering av podocytes av gnager glomeruli. Denne teknikken tillater bruk av patch-clamp enkelt kanal elektrofysiologi og fluorescens proporsjonal confocal bildebehandling. Imidlertid kan begge tilnærminger brukes separat, på egen hånd. Den foreslåtte protokollen har flere relativt enkle grep, inkludert 1) nyre flush; 2) isolering av glomeruli ved differensial sikting; 3) utføre patch-clamp elektrofysiologiske eksperimenter, eller inkubasjon a…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Forfatterne ønsker å takke Glen Slocum (Medical College of Wisconsin) og Colleen A. Lavin (Nikon Instruments, Inc.) for utmerket teknisk assistanse med mikroskopi eksperimenter. Gregory Blass er anerkjent for kritisk korrekturlesing av manuskriptet. Denne forskningen ble støttet av National Institutes of Health tilskudd HL108880 og American Diabetes Association gi 1-15-BS-172 (til AS) og Ben J. Lipps Stipendiat fra American Society of Nefrologi (til 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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