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Titel:Funktionelle und pathophysiologische Untersuchung neuer Mutationen des Natrium-Chlorid-Cotransporters der Niere beim Gitelman Syndrom
Autor:Knop, Caroline
Weitere Beteiligte: Waldegger, Siegfried (Prof. Dr.)
Veröffentlicht:2010
URI:https://archiv.ub.uni-marburg.de/diss/z2010/0582
DOI: https://doi.org/10.17192/z2010.0582
URN: urn:nbn:de:hebis:04-z2010-05823
DDC: Medizin
Titel(trans.):Functional and pathophysiological assesments of novel mutations of the NACl cotransporter in Gitelman disease
Publikationsdatum:2010-10-26
Lizenz:https://rightsstatements.org/vocab/InC-NC/1.0/

Dokument

Schlagwörter:
Nierenkrankheit, Gitelman Syndrom, Nierenfunktion, NCCT, NCCT, Gitelman Syndrome

Zusammenfassung:
Das Gitelman Syndrom ist eine autosomal rezessiv vererbte renale Tubulopathie, die durch eine hypokalämische Alkalose, eine Hypomagnesiämie und eine Hypocalciurie charakterisiert ist. Die Erkrankung basiert auf Mutationen im SCL12A3 Gen, das für den Natrium-Chlorid-Transporter (NCCT) im distalen Abschnitt des Tubulussystems der Niere kodiert. Funktionelle Untersuchungen mittel Cl-Uptakes sowie Untersuchungen zur Proteinbiochemie mittels Western Blot und Immunfluoreszenz wurden durchgeführt, um den Effekt von acht neuen Mutationen auf die NCCT-Funktion zu analysieren. Nach heterologer Expression des Wildtyps in Eizellen des südafrikanischen Krallenfrosches zeigten die Mutationen keine signifikant gesteigerte Chloridaufnahme. Bei keiner der Mutationen konnte eine Restaktivität nachgewiesen werden. In den Western Blot Untersuchungen konnte kein Unterschied zwischen dem Wildtyp und den Mutationen gesehen werden: alle, bis auf die Mutation T180R, wiesen eine spezifische Bande bei 110kD auf. Die Oberflächenexpression wurde mit Hilfe von Immunfluoreszenzanfärbungen untersucht. Die Mutation T180R konnte passend zum Ergebnis der Western Blot Analyse nicht mit Hilfe der Immunfluoreszenz angefärbt werden. Die Mutation R83W konnte nur im Cytoplasma der Zelle nachgewiesen werden. Die Mutationen G731R, R887Q und V1015M zeigten sowohl Signale im Cytoplasma als auch in der Plasmamembran. Nicht zu unterscheiden vom WT und fast ausschließlich in der Plasmamembran anzufärben, waren die Mutationen R135C, D486N und S614P. Weitere funktionelle Untersuchungen mit Hilfe der WNK1 zeigten, dass im Gegensatz zum Wildtyp die mutierten NCCT Proteine nicht aktiviert wurden. Zusammenfassend stellt diese Studie dar, dass der Grund für den Funktionsverlust der mutierten NCCT Transporter in unterschiedlichen Mechanismen zu suchen ist: in der gestörten Proteinsynthese, in der gestörten Proteinweiterverarbeitung, in dem gestörten Einbau in die Plasmamembran oder in der intrinsischen Aktivität des Transporters. Eine Korrelation zwischen dem Genotyp und der Krankheitsausprägung bei den Gitelman Patienten kann mit dieser Studie nicht gezeigt werden.

Summary:
Gitelmans syndrome is an autosomal recessiv renal tubular dissorder characterized by hypokalemic, metabolic alkalosis, hypomagnesemia and hypocalciuria. This disorder results from mutations in the NaCl cotransporter. To elucidate the functional implications of the mutations, Cl-uptakes were determined in Xenopus laevis oocytes expressing wild-typ or mutant NCCT. All mutants exhibited no significant Cl uptake. In Western blot all mutant proteins were represented. Only one mutation (T180R) was not represented in the Western blot analysis. Immunocytochemical analysis showed the lokalisation of the mutant proteins. One mutant protein (R83W) was found only below the plasma membrane, three (G731R, R887Q und V1015M) could be detected at and below the oocyte plasma membrane and three (R135C, D486N und S614P) could only detected at the membrane like the wild typ. In conclusion, this study substantiates NCCT processing defects as the underlying pathogenic mechanism in Gitelmansyndrome. A correlation between the functional analysis and the varity of the symptomes in Gitelman patient could not shown with this study.

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