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Biologie

Implementering Patch Clamp og live Fluorescens mikroskopi til Monitor funktionelle egenskaber af frisk isolerede PKD epitel

Published: September 01, 2015
doi:
10.3791/53035
, , , , ,
1Department of Physiology,Medical College of Wisconsin, 2Department of Integrative Biology & Pharmacology,University of Texas Health Science Center at Houston

Summary

Ionkanaler udtrykt i tubulær epitel spiller en væsentlig rolle i patologien af ​​polycystisk nyresygdom. Her beskriver vi forsøgsprotokoller bruges til at udføre patch-clamp analyse og intracellulært calcium niveau målinger i cystisk epitel frisk isoleret fra gnaver nyrerne.

Abstract

Cyst initiering og ekspansion i polycystisk nyresygdom er en kompleks proces kendetegnet ved abnormiteter i rørformede celleproliferation, luminal væskeophobning og ekstracellulær matrix formation. Aktivitet af ionkanaler og intracellulært calcium signalering er centrale fysiologiske parametre, som betinger funktioner af rørformet epitel. Vi udviklede en metode egnet til realtid observation af ionkanaler aktivitet med patch-clamp-teknik og registrering af intracellulær Ca2 + niveau epitel monolag frisk isolerede fra renale cyster. PCK rotter, en genetisk model for autosomal recessiv polycystisk nyresygdom (ARPKD), blev her anvendt til ex vivo analyse af ionkanaler og calcium flux. Beskrevet her er der en detaljeret trin-for-trin procedure designet til at isolere cystisk monolag og ikke-forstørrede tubuli fra PCK eller normale Sprague Dawley (SD) rotter, og overvåge enkelt kanal aktivitet og intracellulære Ca 2 + dynamik.Denne metode kræver ikke enzymatisk behandling og tillader analyse i en nativ indstilling af frisk isoleret epitelial monolag. Desuden er denne teknik er meget følsom over for intracellulære calcium ændringer og genererer høj opløsning billeder for nøjagtige målinger. Endelig kan isoleret cystisk epitel yderligere kan anvendes til farvning med antistoffer eller farvestoffer, fremstilling af primære kulturer og oprensning af forskellige biokemiske assays.

Introduction

Ionkanaler spiller en væsentlig rolle i mange fysiologiske funktioner, herunder cellevækst og differentiering. Autosomale dominante og recessive polycystiske nyresygdomme (ADPKD og ARPKD henholdsvis) er genetiske sygdomme karakteriseret ved udviklingen af ​​renale væskefyldte cyster i den rørformede epitelcelle oprindelse. ADPKD er forårsaget af mutationer af pKD1 eller PKD2 gener, der koder polycystins 1 og 2, membranproteiner er involveret i reguleringen af ​​celledeling og differentiering. PKD2 sig selv eller som et kompleks med pKD1 også fungere som en Ca2 + -permeable kationkanal 1. Mutationer af PKHD1 gen, der koder fibrocystin (a cilia-associeret receptor-lignende protein involveret i tubulogenesis og / eller vedligeholdelsen af polariteten af epitel) er den genetiske indflydelse af ARPKD 2. Cyste vækst er et komplekst fænomen ledsaget med forstyrret spredning 3,4, angiogenese 5, dedifferentiation og tab af polæreity af rørformede celler 6-8.

Defekt reabsorption og udvidet sekretion i cystisk epitel bidrage til væskeophobning i hulrummet og cyste ekspansion 9,10. Nedsat flow-afhængige [Ca2 +] i-signalering er også knyttet til cystogenesis under PKD 11-15.

Her beskriver vi en fremgangsmåde egnet til patch-clamp målinger af enkelt kanal aktivitet og intracellulære Ca2 + niveauer ved cystisk epiteliale monolag isoleret fra PCK rotter. Denne metode blev anvendt med succes af os at karakterisere aktivitet af epitel Na + kanal (ENaC) 10 og [Ca2 +] i -afhængige påført af Ca 2+ -permeable TRPV4 og purinergisk signaleringskaskade 13.

I disse undersøgelser anvendte vi PCK rotter, en model af ARPKD forårsaget af en spontan mutation i PKHD1 genet. Den PCK-stammen var originally afledt fra Sprague-Dawley (SD) rotter 16 derved SD rotter anvendes som en passende kontrol for sammenligning med PCK stamme. Som følge heraf kan både SD rotter nephron segmenter og ikke-dilateret samlekanalerne isoleret fra samme PCK rotter tjene som to forskellige sammenligningsgrupper for forsøg på cystisk epitel.

Protocol

De eksperimentelle procedurer beskrevet nedenfor, blev godkendt af Institutional Animal Care og brug Udvalg på Medical College of Wisconsin og University of Texas Health Science Center på Houston og var i overensstemmelse med National Institutes of Health Guide til Pleje og anvendelse af forsøgsdyr. Figur 1 viser de vigtigste trin i vævet isolation og behandlingsprocedure. Kort fortalt nyrerne fra PCK eller SD-rotter anvendes til manuel isolering af epitelceller monolag af samlekanalerne enten fra r…

Representative Results

Potentiale ENaC involvering i cystogenesis er blevet påvist ved flere undersøgelser, der observerede forstyrret epidermal vækstfaktor (EGF), signalering i PKD progression 22-25 og unormal natrium reabsorption i ARPKD murine modeller og vævskulturer 26-28. For eksempel Veizis et al. Viste, at amilorid-følsomme Na + absorption er faldet i cd-celler fra den ikke-ortologe BPK musemodel for ARPKD 29. Vi har for nylig vist, at nedsat natrium og vand reabsorption i cyst…

Discussion

Vi beskrev her anvendelser af konventionelle patch-clamp-teknik og epifluorescens calcium billeddannelse til cystisk epitel monolag afledt af en murin genetisk model af ARPKD. Protokollen består af tre trin, hvoraf skal betales mest opmærksomhed til isolering af cyster (trin 1.5 i protokoller sektionen) og til de elektrofysiologiske undersøgelser. Disse centrale procedurer kræver omfattende uddannelse og tålmodighed, og læseren bør ikke være frustreret på én gang.

Først og fremmes…

Divulgations

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. Denne undersøgelse blev støttet af National Institutes of Health giver R01 HL108880 (AS), R01 DK095029 (til OPO) og K99 HL116603 (til TSP), National Kidney Foundation IG1724 (til TSP), American Heart Association 13GRNT16220002 (til OPO) og Ben J. Lipps Research Fellowship fra American Society of Nefrologisk (til DVI).

Materials

Fura-2 AM Life Technologies F-14185
Flou-8 AAT Bioquest 21091
Poly-L-lysine Sigma-Aldrich P4707
Pluronic acid Sigma-Aldrich F-68  solution
Shaker Boekel Scientific 260350
Light source Sutter Instrument Co Lambda XL with integrated shutter/filter wheel driver
Neutral density filters Nikon ND4, ND8
Objective Nikon SFluo  40/1.3 DIC WD 0.22   oil
Camera Andor Technologies Zyla sCMOS
Nikon  microscope (inverted) Nikon Nikon Eclipse TE2000-S
Cover Glass Thermo Scientific 6661B52
Diamond pencil Fisher Scientific 22268912
Image acquisition software Nikon Nikon NIS-Elements 
Image analysis software ImageJ http://imagej.nih.gov/ ND Utility plugin allows to import images in the native Nikon Instruments .nd2 format
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
Temperature controlled surgical table  MCW core for rodents
Binocular stereomicroscope 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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Tags

Keywords: Patch ClampLive Fluorescence MicroscopyPolycystic Kidney DiseasePKDARPKDIon ChannelsCalcium SignalingEpithelial MonolayerEx Vivo AnalysisPCK RatSprague Dawley RatNative SettingEpithelial IsolationImmunostainingPrimary CultureBiochemical Assays
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Pavlov, T. S., Ilatovskaya, D. V., Palygin, O., Levchenko, V., Pochynyuk, O., Staruschenko, A. Implementing Patch Clamp and Live Fluorescence Microscopy to Monitor Functional Properties of Freshly Isolated PKD Epithelium. J. Vis. Exp. (103), e53035, doi:10.3791/53035 (2015).
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