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Mäusen Und Adrenokortikalen Zellen Mit KCNJ5‐ Mutation

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Mäusen Und Adrenokortikalen Zellen Mit KCNJ5‐ Mutation Untersuchungen zur Pathogenese von primärem Hyperaldosteronismus in neugeborenen Task3‐/‐ Mäusen und adrenokortikalen Zellen mit KCNJ5‐ Mutation DISSERTATION ZUR ERLANGUNG DES DOKTORGRADES DER NATURWISSENSCHAFTEN (DR. RER. NAT.) DER NATURWISSENSCHAFTLICHEN FAKULTÄT III – BIOLOGIE UND VORKLINISCHE MEDIZIN DER UNIVERSITÄT REGENSBURG vorgelegt von Philipp Tauber aus Straubing im Jahr 2013 Die vorliegende Arbeit entstand im Zeitraum von Dezember 2010 bis Dezember 2013 unter der Anleitung von Herrn Prof. Dr. med. Richard Warth am Lehrstuhl für Medizinische Zellbiologie/Institut für Physiologie der Universität Regensburg. Promotionsgesuch eingereicht am: 25.11.2013 Die Arbeit wurde angeleitet von: Herr Prof. Dr. med. Richard Warth Prüfungsausschuss: Frau Prof. Dr. Charlotte Wagner (Vorsitzender) Herr Prof. Dr. Richard Warth (1. Gutachter/Prüfer) Herr Prof. Dr. Stephan Schneuwly (2. Gutachter/Prüfer) Herr Prof. Dr. Jens Schlossmann (3. Prüfer) Unterschrift _______________________ (Philipp Tauber) Meinem Großvater Emil Tauber Inhaltsverzeichnis INHALTSVERZEICHNIS 1. Einleitung ................................................................................................ 1 1.1 Die Nebenniere ........................................................................................................................ 1 1.1.1 Aufbau und Funktion der Nebenniere .............................................................................. 1 1.1.2 Entwicklung der Nebenniere ............................................................................................. 2 1.2. Systemische Regulation und Wirkung von Aldosteron ........................................................... 3 1.2.1 Das Renin‐Angiotensin‐Aldosteron‐System (RAAS) .......................................................... 3 1.2.2 Wirkungen von Aldosteron ............................................................................................... 5 1.2.3 Syntheseweg von Aldosteron ............................................................................................ 7 1.3 Pathophysiologie: Aldosteronbedingte Krankheitsbilder ........................................................ 9 1.3.1 Ursachen und Folgen von Hyperaldosteronismus ............................................................ 9 1.3.2 Hypoaldosteronismus ..................................................................................................... 10 1.4 Zelluläre Regulation der Aldosteronsekretion ....................................................................... 10 1.4.1 Regulation durch Angiotensin II ...................................................................................... 10 1.4.2 Regulation durch extrazelluläres Kalium ......................................................................... 12 1.4.3 Calcium‐induzierte CYP11B2‐Transkription .................................................................... 13 1.5 Kaliumkanäle in der Nebenniere ............................................................................................ 14 1.6 Hyperaldosteronismus durch Störungen der zellulären Regulation ...................................... 15 1.6.1 Hyperaldosteronismus durch Deletion von Task‐Kanälen in Mäusen ............................ 15 1.6.2 KCNJ5‐Mutationen als Ursache für Hyperaldosteronismus beim Menschen ................. 17 1.7 Zielsetzung .............................................................................................................................. 20 2. Material und Methoden ........................................................................ 21 ‐/‐ 2.1 Methoden zur Charakterisierung neugeborener Task3 Mäuse ........................................... 21 2.1.1 Task3‐/‐ Mausmodell ........................................................................................................ 21 2.1.2 Bestimmung der Plasma‐Na+‐Konzentration und des Hämatokrits ................................ 21 2.1.3 Steroidhormonmessungen .............................................................................................. 21 2.1.4 Nebennierenlysate zur Aldosteronbestimmung ............................................................. 22 2.1.5 Immunfluoreszenzfärbung .............................................................................................. 22 Inhaltsverzeichnis 2.1.6 Reninmessung in Plasmaproben und Gewebelysaten .................................................... 23 2.1.7 RNA‐Isolation und quantitative Real‐time PCR ............................................................... 23 2.1.8 Genechip‐Analyse des adrenalen RNA‐Expressionsprofils ............................................. 24 2.1.9 Statistik ............................................................................................................................ 25 2.2 Methoden zur Charakterisierung von KCNJ5‐Mutationen ..................................................... 26 2.2.1 Zellkultur und Transfektion ............................................................................................. 26 2.2.2 Patch clamp Experimente ............................................................................................... 27 2.2.3 Ca2+‐Messung an transfizierten NCI‐Zellen ..................................................................... 27 2.2.4 RNA‐Isolation und quantitative Real‐time PCR ............................................................... 28 2.2.5 Substanzen zur pharmakologischen Charakterisierung von KCNJ5 L168R ..................... 28 2.2.6 Statistik ............................................................................................................................ 28 3. Ergebnisse ............................................................................................. 29 3.1 Adrenaler Phänotyp der neonatalen Task3‐/‐ Maus ............................................................... 29 3.1.1 Lokalisation von Task3 und Cyp11b2 in Nebennieren neugeborener Mäuse ................ 29 3.1.2 Adrenales Steroidhormonprofil neugeborener Task3‐/‐ Mäuse ...................................... 30 3.1.3 Adrenale mRNA‐Expression steroidogener Enzyme in Task3‐/‐ Mäusen ......................... 31 3.1.4 Plasma‐Na+‐Level und Hämatokrit in neugeborenen Task3‐/‐ Mäusen ........................... 32 3.1.5 Microarray‐Analyse: Adrenales Expressionsprofil 1 und 12 Tage alter Mäuse .............. 33 3.1.6 Reninexpression in Nebennieren neugeborener Task3‐/‐ Mäuse .................................... 35 3.1.7 Reninkonzentration in Plasma und Nierenlysaten von Task3‐/‐ Mäusen ........................ 37 3.1.8 Adrenaler Phänotyp der Task3‐/‐ Maus während der fötalen Entwicklung ..................... 38 3.1.9 Weitere Phänotyp‐assoziierte, regulierte Gene in Task3‐/‐ Mäusen ............................... 39 3.2 Funktionelle und pharmakologische Untersuchung APA‐assoziierter KCNJ5‐Mutationen ... 40 3.2.1 Verlust der Ionenselektivität durch Mutationen in KCNJ5 .............................................. 41 3.2.2 Gesteigerte Ca2+‐Aktivität in NCI‐Zellen mit KCNJ5‐Mutation ........................................ 44 3.2.3 Expression von NCX und NCKX in humanem Nebennierengewebe ................................ 47 3.2.4 Pharmakologische Eigenschaften von KCNJ5 L168R ....................................................... 48 Inhaltsverzeichnis 4. Diskussion ............................................................................................. 52 4.1 Hyperaldosteronismus in neonatalen Task3‐/‐ Mäusen ......................................................... 52 4.1.1 Störung der adrenalen Steroidhormonproduktion in neugeborenen Task3‐/‐ Mäusen .. 52 4.1.2 Adrenale Entwicklungsstörungen ursächlich für Hyperaldosteronismus in neonatalen Task3‐/‐ Mäusen? ............................................................................................................. 54 4.1.3 Adrenale Reninexpression in neugeborenen Task3‐/‐ Mäusen ....................................... 54 4.2 Pathophysiologie und Pharmakologie von APA‐assoziierten KCNJ5‐Mutanten .................... 57 4.2.1 Pathologischer Na+‐Einstrom und Depolarisation in KCNJ5‐mutierten Zellen ................ 57 4.2.2 Erhöhte Ca2+‐Aktivität in NCI‐Zellen mit KCNJ5‐Mutation .............................................. 59 4.2.3 Pharmakologie des mutierten KCNJ5‐Kanals (L168R) ..................................................... 60 4.3 Bedeutung von Kaliumkanälen für die Pathophysiologie von primärem Hyperaldosteronismus ........................................................................................................... 62 5. Zusammenfassung ................................................................................. 64 6. Literaturverzeichnis ............................................................................... 66 7. Anhang .................................................................................................. 79 7.1 Arbeitsbezogene Daten .......................................................................................................... 79 7.1.1 Primersequenzen für Real‐time PCR ............................................................................... 79 7.1.2 Adrenales mRNA‐Expressionsprofil von Wildtyp und Task3‐/‐ Mäusen .......................... 79 7.1.3 Plasmidkarte pIRES‐CD8 .................................................................................................. 83 7.1.4 Inhibitoren des KCNJ5 L168R‐Kanals ............................................................................... 84 7.2 Personenbezogene Daten .....................................................................................................
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