mRNA Expression elektrolytregulierender Proteine an der Maus

Das Hyperprostaglandinsyndrom (HPS), auch antenatales Bartter-Syndrom genannt, stellt eine Salzverlusttubulopathie dar, die durch Mutationen in den Genen für die Proteine NKCC2, ROMK, Barttin oder ClC-Kb hervorgerufen werden kann. Klinisch ist ein massiver Salzverlust zu beobachten, der einhergeht m...

Full description

Saved in:
Bibliographic Details
Main Author: Ludewig, Christoph Michael
Contributors: Nüsing, Rolf (Prof. Dr.) (Thesis advisor)
Format: Dissertation
Published: Philipps-Universität Marburg 2007
Zentrum für Kinder- und Jugendmedizin
Online Access:PDF Full Text
Tags: Add Tag
No Tags, Be the first to tag this record!
Table of Contents: The hyperprostaglandin E syndrome (HPS), which is also known as antenatal Bartter syndrome, is a salt loss tubulopathy, evoked by mutations in the genes of the proteins NKCC2, ROMK, Barttin or ClC-Kb. Clinically, a massive salt loss can be seen, accompanied by enormously increased renin activity (activated RAAS) and high prostaglandine E2 (PGE2) excretion. If not treated, this disease leads to the death of the affected child. By lifelong administration of a prostaglandin synthesis inhibitor, like Indomethacin, and sufficient salt and water intake, considerable suppression of the symptoms can be achieved. Different papers point out a pathogenetic role of the enzyme COX-2 (responsible for the generation of PGE2) and the PGE2 receptor-subtype EP4. In this study, the HPS was simulated in an animal model, by administering the NKCC2-inhibitor furosemide, a loop diuretic. It was mixed into the drinking water for ten days. The aim of this dissertation was to investigate the modifications of expression of different proteins, which are involved in the transport of electrolytes in the kidney. These investigations were carried out on C57Bl6 wildtype mice compared to COX-2 and EP4 knockout mice. The kidneys of treated and untreated animals (n = 12) were extracted and the RNA was isolated. Afterwards, the mRNA of the proteins NKCC2, ROMK, KCC1, 3, 4, ClC-K1, NCCT, Na+-K+-ATPase, EnaC β and γ, SGK and Nedd-4 was examined using the PCR-technique. The relative expression of mRNA was standardized to that of GAPDH. The results of the analysis show that NKCC2, ROMK, as well as the potassium-chloride-channels KCC1, 3, 4 and NCCT were downregulated by administering furosemid to C57Bl6-mice, while the mRNA concentrations of the COX-2-/- mice were not decreased and in the case of ROMK even increased. These observations indicate that the expression is regulated by Cyclooxygenase-2 and PGE2 and that this could be one of the reasons for the clinically seen salt and water loss. The investigations of the EP4-/- mice allow the assumption that this PGE2 related regulation could be mediated by the EP4 receptor, with the exception of KCC1, 3, 4. The results are different in terms of the ENaC channel. The investigated subunits β and γ show an increase of the mRNA-concentrations in the wildtype mice when furosemide is given, however there were no significant differences for the COX-2-/-mice. Astonishingly, a significant β subunit decrease of EP4-receptor-/- mice could be noticed whereas the γ-subunit expression remained stable. It is postulated that there is an increased water and salt loss caused by the deficit or the inhibition of NKCC2 or ROMK which leads to inadequate ability for urine to concentrate in the thick ascending limb of Henle. Furthermore, it is suspected that, for the same reason, the expression of COX-2 and renin in the juxtaglomerular apparatus is increased by a decreased chloride concentration. If the already decreased or inhibited exprimated channels were additionally downregulated by COX-2, as could be supposed according to the previous results, there would be a circulus vitiosus of decreased channel concentration