The development and use of new methods and strategies for the monitoring of nephrotoxicity and the study of renal physiology [Elektronische Ressource] : proteomics and metabonomics based studies / vorgelegt von Jost Klawitter

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• Publié par: universitat_bremen
• Publié le: 01 janvier 2006
• Nombre de lectures: 84
• Langue: Deutsch
• Poids de l'ouvrage: 3 Mo

Extrait

The Development and Use of New Methods and
Strategies for the Monitoring of Nephrotoxicity
and the Study of Renal Physiology:
Proteomics- and Metabonomics-based Studies

DISSERTATION
Zur Erlangung des Grades eines
Doktors der Naturwissenschaften
-Dr. rer. nat.-

Dem Fachbereich Biologie/Chemie der
Universität Bremen
Vorgelegt von

Jost Klawitter
Bremen
2006


Universität Bremen
University of Colorado


















Für meine Eltern















Mein besonderer Dank gilt Prof. Dr. Dieter Leibfritz und Prof. Dr. Uwe Christians,
welche durch die Themenstellung, ihre Ratschläge und Unterstützung diese Arbeit
ermöglicht haben.

Bei Dr. Volker Schmitz bedanke ich mich für die gute Zusammenarbeit. Ohne seine
Hilfe und der Bereitstellung von Proben aus den Tierexperimenten wäre die Arbeit in
diesem Umfang nicht möglich gewesen.

Bei Dr. Manuel Haschke und Kristine Kahle möchte ich mich fuer die
Bereitstellung der Proben aus der humanen Studie und fuer die Messung der
Isoprostankonzentrationen bedanken.

Bei Johannes Stelten und Dr. Wiland Willker möchte ich mich für die Hilfe bei der
Einstellung von 2D-NMR Experimenten und für die Tips, die mir zu einem besseren
Verständnis der Methode verhelfen konnten, bedanken.

Allen Mitarbeitern der Arbeitsgruppe Leibfritz danke ich für ihre Hilfe und
Diskussionsbereitschaft. Ihre Ratschläge halfen mir sehr weiter.

I would like to thank Jamie Bendrick-Peart, who measured the drug concentrations
and helped with the Western blot analysis.

Thanks to all colleagues of the University of Colorado DACR&D for their
continuous help. I also would like to thank Prof. Dr. Larry Chan and the collaborators in
Prof. Dr. Tomas Berl’s lab for the supply of cell samples and the helpful tips.




Danke Jelena ♥
Table of Contents
1A. ZUSAMMENFASSUNG....................................................................................................................... 6
1B. SUMMARY.......................................................................................................................................... 10
2. INTRODUCTION .................................................................................................................................. 13
2.1 GENERAL INTRODUCTION........................................................................................................................ 13
2.2 THEORETICAL BACKGROUND.................................................................................................................. 15
2.2.1 History of Immunosuppressants................................................................................................. 15
2.2.2 Mechanisms of Immunosuppression .......................................................................................... 16
2.2.3 Brief overview: kidney physiology and function ........................................................................ 18
2.2.4 Immunosuppressant toxicity and chronic allograft dysfunction ................................................ 22
2.2.5 Proteomic Profiling ................................................................................................................... 24
2.2.6 Metabolic Profiling.............. 26
2.2.7 Organ conservation solutions for transplantation ..................................................................... 30
2.3 AIMS OF THIS STUDY ............................................................................................................................... 31
2.4 RESEARCH STRATEGIES ........................................................................................................................... 32
3. RESULTS AND DISCUSSION....... 34
3.1 LC/LC-ESI/MS METHOD DEVELOPMENT ................................................................................................ 34
3.2 I/MS METHOD VALIDATION (FOLLOWING FDA GUIDELINES) ................................................. 36
3.3 INFLUENCE OF ISCHEMIA ON NUCLEOTIDE METABOLISM IN THE KIDNEY 39
3.4 ENERGY CHARGE AS A QUALITY CONTROL OF ORGAN CONSERVATION SOLUTIONS ................................. 40
3.5 DRUG CONCENTRATIONS AND RENAL FUNCTION AFTER TREATMENT WITH IMMUNOSUPPRESSANTS ALONE
AND IN COMBINATION (A1) ........................................................................................................................... 42
3.6 ANALYSIS OF URINE METABOLITES IN ANIMAL MODEL (A1).................................................................... 46
3.7 AURINE METABOLITEMAL MODEL (A2) 51
3.8 ENERGY METABOLISM AFTER TRANSPLANTATION AND TREATMENT (B)................................................. 54
3.9 8-ISO-PROSTAGLANDIN-F LEVELS IN URINE (FORMATION OF ROS) ...................................................... 55 2Α
3.10 NMR SPECTROSCOPY FOR WHOLE BLOOD METABOLITE PROFILING....................................................... 56
3.11 NMR BLOOD EXTRACT PATTERN ANALYSIS AFTER IMMUNOSUPPRESSANT TREATMENT AND
TRANSPLANTATION (B) ................................................................................................................................. 57
3.12 ANALYSIS OF URINE METABOLITES IN THE TRANSPLANT RAT MODEL (B).............................................. 60
3.13 METABOLITE ANALYSIS AFTER A SINGLE DOSE OF CYCLOSPORINE IN HUMANS ..................................... 63
3.14 PROTEOME ANALYSIS AFTER IMMUNOSUPPRESSANT TREATMENT ( A-1 AND A-2) ................................ 68
3.14.1 Proteins involved in the calcium homeostasis: Regucalcin and Calbindin.............................. 73
3.14.2 Endothelial dysfunction: Dimethylarginine Dimethylaminohydrolase (DDAH) ..................... 76
3.14.3 Cytoskeleton proteins: vimentin, caldesmon, actin binding protein 1 (ABP1) , actin related
protein 3 ARP3, plastin 3T isoform..................................................................................................... 77
3.14.4 Hypoxia, HIF and mitochondrial dysfunction: prolyl 4-hydroxylase, proteasome, NADH
dehydrogenase and pyruvate kinase ................................................................................................... 80
3.14.5 Important proteins for cell metabolism: Arginine:glycine-amidinotransferase (AGAT) and
kidney aminoacylase (KA), pyruvate kinase (PK) and fructose-1,6-bisphospatase (F-1,6-BP) ......... 83
3.15 METABOLIC PROFILING OF IMCD3 CELLS EXPOSED TO ACUTE HYPEROSMOTIC CONDITIONS ............... 86
3.16 MIC PROFILING OF IMCD3 CELLS CHRONICALLY ADAPTED TO HYPEROSMOTIC CONDITIONS ... 91
3.16.1 Cellular adaptation to hypertonicity and phenotypic changes................................................. 91
3.16.2 Hypertonicity induced changes in osmolytes, amino acids, and metabolites........................... 92
3.16 CONCLUSIONS AND PERSPECTIVES ...................................................................................................... 100
4. MATERIALS AND METHODS......................................................................................................... 106
4.1 NUCLEOTIDE HPLC-MS ....................................................................................................................... 106
4.2 COMPARISON OF EXTRACTION PROCEDURES ........................................................................................ 108
4.3 HPLC/MS METHOD VALIDATION PROCEDURES..................................................................................... 108
4.4 INFLUENCE OF ISCHEMIA TIME ON KIDNEY ENERGY STATE.................................................................... 109
4.5 ANIMAL PROTOCOLS AND PROCEDURES ................................................................................................ 110
4.5.1 Animal model for the quality control of conservation solutions .............................................. 110
4.5.2 Animal models: Model A-1 and A-2 (non-transplant, IS treatment) ........................................ 111
4.5.3 Animal models: Model B (transplanted rats, IS treatment) ..................................................... 112
4.6 CELL CULTURE ...................................................................................................................................... 113
4.7 DETERMINATION OF KIDNEY HIGH ENERGY PHOSPHATE LEVELS ........................................................... 114
4.7.1 High energy phosphate levels for the comparison of conservation solutions .......................... 114
4.7.2 High energy phosphate levels in transplanted rats (Model B)................................................. 114
4.8 THE DETERMINATION OF GLOMERULAR FILTRATION RATE (MODEL A-1 AND A-2) ............................... 115
4.9 CREATININE, BLOOD UREA NITROGEN (BUN) AND DRUG CONCENTRATIONS ........................................ 116
4.10 METABONOME ANALYSIS .................................................................................................................... 116
4.10.1 Sample handling (all studies)..............................................................