Name: assessment of mitochondrial functions and cell viability in renal cells overexpressing protein kinase c isozymes
Uploaded: 2013-01-07T12:00:00
Description: Watch this Scientific Journal Video about Assessment of Mitochondrial Functions and Cell Viability in Renal Cells Overexpressing Protein Kinase C Isozymes at JoVE.com

Dette arbejde blev støttet af en bevilling fra National Institutes of Health, National Institute of Diabetes og Digestive og nyresygdomme, 2R01DK59558 (til GN). UAMS Translationel Research Institute støttet af National Institutes of Health National Center for Forskning Ressourcer tilskud UL1 RR029884 giver delvis finansiering af Flowcytometri Core på UAMS. Vi takker Dr. Peipei Ping (University of California i Los Angeles, Los Angeles, CA) for at tilvejebringe en alikvot af adenovirus bærer cDNA kodende den dominerende negative (inaktive) mutant af PKC-ε og Dr. Allen Samarel (Loyola University Medical Center; Maywood, IL) til tilvejebringelse af en alikvot af adenoviral vektor, der koder den konstitutivt aktive mutant af PKC-ε. Vi takker også Drs. Peter Parker og Peter Sugden (Imperial College London, London, UK) for at tilvejebringe cDNA kodende konstitutivt aktive PKC-ε.

1. Isolering af nyre proksimale tubuli til primær kultur <ol><li> Anesthetize kanin, punktafgifter begge nyrer og placere dem i en petriskål fyldt med steril prep buffer (DMEM/F12-medium) i en laminar strømning hætte. </li><li> Perfundere hver nyre med 50 ml prep-puffer efterfulgt af 50 ml steril phosphatbufret saltvand, pH 7,4 (PBS), og PBS-jernoxid opløsning (45 ml + 5 ml). De-capsulate nyrer og overføre dem til prep buffer indeholdende deferoxamin. </li><li> Høste cortex af hver nyre. </li><li> Homogenisere ...

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Den fremgangsmåde, præsenteres her tillader overekspression af individuelle isozymer af PKC i primær kultur af renale proximale tubulære celler. Der er flere styrker af denne fremgangsmåde: 1. Den gør det muligt at undersøge reguleringsmekanismer i en homogen population af celler (nyre proximale tubulære celler), som er det primære mål for forskellige fornærmelser (iskæmi, hypoxi, oxidativ stress), narkotika og nephrotoxicants i nyren. 2. Mitokondrielle funktioner i denne in vitro model af RPTC dyrk...

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assessment of mitochondrial functions and cell viability in renal cells overexpressing protein kinase c isozymes

Proteinkinasen C (PKC) familie af isozymer er involveret i mange fysiologiske og patologiske processer. Vores seneste data viser, at PKC regulerer mitokondriefunktionen og cellulære energi status. Talrige rapporter vist, at aktiveringen af ​​PKC-a og PKC-ε forbedrer mitokondriefunktion i iskæmisk hjertesygdom og medierer kardiobeskyttelse. I modsætning hertil har vi vist, at PKC-α-og PKC-ε er involveret i nephrotoxicant-induceret mitokondriel dysfunktion og celledød i nyreceller. Derfor er målet med denne undersøgelse var at udvikle en in vitro model af nyreceller opretholde aktive mitochondriale funktioner, hvor PKC-isozymer kan selektivt aktiveres eller inhiberes at bestemme deres rolle i regulering af oxidativ phosphorylering og celleoverlevelse. Primære kulturer af renale proximale tubulære celler (RPTC) blev dyrket i forbedrede forhold resulterer i mitokondriel respiration og aktiviteten af ​​Mitochondrial enzymer svarende til dem i RPTC in vivo. Fordi traditionelle transfektionsteknikker (Lipofectamin, elektroporering) er ineffektive i primære kulturer og har negative virkninger for mitokondriefunktion blev PKC-ε mutant cDNA&#39;er leveret til RPTC gennem adenovirusvektorer. Denne fremgangsmåde resulterer i transfektion af over 90% dyrket RPTC. Her præsenteres metoder til at vurdere den rolle, som PKC-ε i: 1. regulering af mitochondrial morfologi og som er forbundet med ATP-syntese, og 2. overlevelse af RPTC i primær kultur. PKC-ε aktiveres ved overekspression af konstitutivt aktive PKC-ε mutant. PKC-ε inhiberes af overekspression den inaktive mutant af PKC-ε. Mitokondriefunktion vurderes ved at undersøge respiration, integritet respirationskæden, aktiviteter af respiratoriske komplekser og F 0 F 1-ATPase, ATP produktionshastigheden, og ATP indhold. Respiration er enssessed i digitonin-permeabiliserede RPTC som tilstand 3 (maksimal respiration i nærvær af overskydende substrater og ADP) og ukoblede respirations. Integritet af respirationskæden vurderes ved måling af aktiviteter af alle fire komplekser af respirationskæden i isolerede mitokondrier. Kapaciteten af oxidativ phosphorylering vurderes ved måling af mitokondriemembranspændingen, ATP produktionshastigheden, og aktiviteten af F 0 F 1-ATPase. Energi status RPTC vurderes ved at bestemme den intracellulære ATP indhold. Mitochondrial morfologi i levende celler er visualiseret under anvendelse MitoTracker Red 580, et fluorescerende farvestof, som specifikt akkumuleres i mitokondrierne, og levende monolag undersøges under et fluorescensmikroskop. RPTC levedygtighed vurderes ved anvendelse af annexin V / propidiumiodid-farvning efterfulgt af flowcytometri til at bestemme apoptose og oncosis. Disse fremgangsmåder giver mulighed for en selektiv aktivering / inhibering af individuelle PKC isozymerat vurdere deres rolle i cellulære funktioner i en række fysiologiske og patologiske tilstande, der kan reproduceres in vitro.

Figur 1 viser, adenoviral levering af cDNA, der koder de konstitutivt aktive (caPKC-ε) og inaktive (dnPKC-ε) mutanter af PKC-ε resulterer i signifikant forøgede proteinniveauer af PKC-ε i ​​RPTC og i mitochondria. Celler inficeret med cDNA, der bærer caPKC-ε vektor overudtrykkes den phosphorylerede (aktive) form af PKC-ε hvorimod celler inficeret med cDNA kodende dnPKC-ε overudtrykte PKC-ε, som var inaktivt (ikke phosphoryleret) (figur 1). Tilstedeværelsen af aktivt PKC-?...

Watch this Scientific Journal Video about Assessment of Mitochondrial Functions and Cell Viability in Renal Cells Overexpressing Protein Kinase C Isozymes at JoVE.com

Assessment of Mitochondrial Functions and Cell Viability in Renal Cells Overexpressing Protein Kinase C Isozymes

Virkningerne af aktivering af proteinkinase C (PKC) isozymer på mitochondriale som er forbundet med åndedræt og oxidativ phosphorylering og på cellelevedygtighed, er beskrevet. Den fremgangsmåde tilpasser adenoviral teknik til selektivt at overudtrykke PKC-isozymer i primær cellekultur og en række assays til bestemmelse mitochondriale funktioner og energi status af cellen.

Vurdering af Mitochondriale funktioner og cellelevedygtighed i Renal celler, der overudtrykker protein kinase C isozymer

The protein kinase C (PKC) family of isozymes is involved in numerous physiological and pathological processes. Our recent data demonstrate that PKC ...

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