Single-channel Analyse og Calcium Imaging i podocytterne af frisk isolerede 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
Nyrer opretholde homeostatiske balance for forskellige stoffer og regulere blodvolumen på en måde, der bestemmer den samlede blodtryk. Forstyrrelser i renal filtration, reabsorption eller sekretion fører til eller ledsage patologiske tilstande, der spænder fra hyper- eller hypotension at ende nyresygdom, som til sidst kræver nyretransplantation. Den renale filtreringsenhed (glomerulus) består af tre lag – den kapillære endotel, basalmembran og en enkelt-celle lag af epitelceller – podocytter, som spiller en vigtig rolle i opretholdelsen af slidsen-membran integritet og funktion 1. Dysfunktion i permselektive glomerulære filter forårsager urin tab af makromolekyler, såsom proteinuri. Forskellige midler kan påvirke strukturen af podocytterne og deres fodprocesser, som er bestemmende integriteten af glomeruli filtrering barriere.
Podocytterne er involveret i opretholdelsen af glomeruli filtrering funktion. Det er blevet fastslået, at forkert calcium håndtering af podocyte fører til celle skade og spiller en vigtig rolle i udviklingen af forskellige former for nefropatier 2,3. Derfor, udvikling af en model, der giver mulighed for direkte måling af intracellulære calciumkoncentration ændringer vil være medvirkende til studier af podocyte funktion. Isolerede glomeruli blev tidligere anvendt i en lang række undersøgelser, herunder måling af albumin refleksion koefficient ændrer 4 og vurdering af integrerede cellulære strømninger i de hel-celle elektrofysiologiske patch-clamp målinger 5,6. I nærværende dokument beskriver vi den protokol, der gør det muligt for forskeren at måle intracellulære calciumkoncentration ændringer i respons på anvendelser af farmakologiske midler, anslår basale niveauer af calcium i cellerne, og vurdere individuelle calciumkanaler aktivitet. Ratometric calciumkoncentration målinger og patch-clamp electrophysiology blev anvendt til at bestemme ændringer i den intracellulære calciumkoncentration i podocyte og kanal aktivitet hhv.
Protocol
Representative Results
Discussion
Den her beskrevne fremgangsmåde giver mulighed for analyse af calcium håndtering af podocytterne af gnaver glomeruli. Denne teknik tillader anvendelse af patch-clamp enkelt kanal elektrofysiologi og fluorescens ratiometrisk konfokal billeddannelse. Dog kan begge tilgange anvendes separat, på egen hånd. Den foreslåede protokol har flere relativt enkle trin, herunder 1) nyre flush; 2) isolering af glomeruli ved differentiel sigtning; 3) kan udføre patch-clamp elektrofysiologiske eksperimenter eller inkubering af glo…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Forfatterne vil gerne takke Glen Slocum (Medical College of Wisconsin) og Colleen A. Lavin (Nikon Instruments, Inc.) for fremragende teknisk bistand med mikroskopi eksperimenter. Gregory Blass er anerkendt for kritisk korrekturlæsning af manuskriptet. Denne forskning blev støttet af National Institutes of Health tilskud HL108880 og American Diabetes Association giver 1-15-BS-172 (AS) og Ben J. Lipps Research Fellowship fra American Society of Nefrologisk (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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