Der Einfluss von MAGED2 auf NHE3 in humanen embryonalen Nierenzellen (HEK293)

Das transiente antenatale Bartter-Syndrom (taBS, OMIM # 300971) ist eine seltene X-chromosomal vererbte Erkrankung, die durch fetale Polyurie, Polyhydramnion, Frühgeburtlichkeit und transienten postnatalen renalen Salzverlust charakterisiert ist. Verantwortlich sind Mutationen des Maged2-Gens, welch...

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Bibliographic Details
Main Author: Ruttkowski, Lars Lennart
Contributors: Kömhoff, Martin (Prof. Dr. med.) (Thesis advisor)
Format: Doctoral Thesis
Published: Philipps-Universität Marburg 2021
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The transient antenatal Bartters-Syndrome (taBS) is a rare, x-chromosomal genetic disease, which presents with polyuria, polyhydramnios, prematurity and transient postnatal salt wasting. Mutations of Maged2 were found to be responsible for the phenotype, most likely, by influencing the posttranslational modification of ion channel proteins in the distal kidney tubule. Laghmani et al. were able to show, that NKCC2- and NCC-expression was lowered in the presence of mutant MAGED2. This might be due to an altered interaction of MAGED2, Hsp40 and Gs-α. The severe pehontype suggests, that other ion channel proteins might be affected as well. NHE3 is a Na+/H+-antiporter, which is responsible for most of the Na+-reabsorption in the proximal tubule. However, MAGED2 is expressed in the proximal tuble only during the fetal period. NHE3 regulation is not well known. A lowered activity would present in lower urine concentration. The aim of this study is to find an influence of MAGED2 on NHE3. HEK293-cells were transfected with NHE3 and MAGED2 (wildtype and mutant). For analyzing the effect of MAGED2 on NHE3 protein stability cycloheximide chase (CC) experiments were performed, where protein synthesis was inhibited with cycloheximide in MAGED2 overexpressing cells, as well as MAGED2-knock-down cells, at different timepoints. The influence of MAGED2 on the expression of NHE3 was investigated by co-transfecting NHE3 with increasing doses of MAGED2 (WT and MT). The effect of MAGED2 on the cell surface integration of NHE3 was examined using cell surface biotinylation (CSB) studies. The results of the CoIP and PLA corresponded with one another and showed, that NHE3 and MAGED2 (WT and MT) may co-exist in a protein complex. In the CC, MAGED2 related changes in the halftime of NHE3 could not be observed. The dose response experiments showed no influence of MAGED2 on the levels of intracellular NHE3. Also the CSB could not show any changes in the cell surface integration of NHE3, when transfected with MAGED2 (WT or MT). We were able to show, that MAGED2 and NHE3 form a complex at some point in the cell and might be interacting through other proteins. Other than NCC and NKCC2, MAGED2 does not seem to have an influence on protein expression, protein stability and cell surface integration of NHE3. These studies however do not rule out the possibility that MAGED2 may influence NHE3 function. Further functional studies are required to study the effects of MAGED2 on NHE3 channel function. So far NHE3 can be used as a negative control in MAGED2-based experiments.