Differentielle Proteinexpression in Nierenepithelzellen unter hypoosmolaren Kulturbedingungen

Tubolopathies with renal loss of electrolytes, e.g. antenatale bartter-syndrome, are specifically caused by epithelial ion-transport deficiancies. A primary cause for this syndrome, alternativly called furosemid-type tubulopathy (FSLT), is suspected to be caused by a compromised resorption in the rising part of the henle-loop. Goal of this thesis was to do a in vitro study of the molecular changes caused by intracellular changes of electrolyt. The study was exercised in primary cultivated kidney-epithelcells. These were isolated from the renal medulla of pig kidneyes. Patients who suffer of antenatal bartter-syndrome experience a deprivation of sodium and chloride partially because of a loss of function of the Na/K/2Cl-co-transporter NKCC2. This situation was mimicked in the in vitro model by changes of the extracellular ion-concentration. The incubation of the in vitro cells in a hypoosmalar medium caused a intracellular deprivation of electrolytes. Main target in this study was to detect the so affected proteinexpression, to identify them and to map the so affected intracellular pathways. The proteinexpression in the in vitro tissue was studied by using a 2-dimensonial gel-electrophoresis with a basis of high-sensitive silver-staining. The differentally expressed proteins were detected by a mass-spectrometrical investigation with a specified software and the analysis of these proteins was performed by a peptide-mass-fingerprint method. Doing so there was a significantly change of intensity-changes of the proteins vimentin and cytoceratin 8. These Proteins belong to the group of intermediar filaments of the cell skeleton. The identified two proteins showed a high level of sequence-homologies in the NCBI databanks of the species Cricetulus griseus (Acession Number: gi/860908) for vimentin and the species Bos taurus (Acession Number: gi/125109) for cytokeratin 8. Compared to the control the in vitro cells exposed to the hypoosomolaric medium showed a change of intensitiy for vimentin by 12 times and for cytokeratin 8 the intensity was doubled. Further the western-blot study using vimentin- and cytokeratin-specific antibodies confirmed the altered proteinexpression in the 1-dimensional gel. In conclusion these investigations showed that renal epithel cells undergo alternated proteinexpression when exposed to a hypoosmolaric medium. How these results can be transferred to the in-vivo antenatale bartter-syndrome, has to be proven in further investiations.