| Popis: |
Autosomal recessive polycystic kidney disease (ARPKD) is a monogenetic hereditary disease during the childhood with an incidence of 1:20.000. It is caused by mutations in the gene PKHD1. Phenotypically it is characterized in mainly small-cystic kidneys, congenital hepatic fibrosis, portal hypertension and respiratory insufficiency. The gene PKHD1 has one of the longest open reading frames in the human genome and consists of 66 exons. The gene-product fibrocystin (FPC) has a size of 4074 amino acids and it is a very big protein in comparison to other proteins of the human organism. It is known that FPC contains a transmembrane domain, a very big N-terminal domain and a very small C-terminal domain. The highest expression of FPC exists in kidney, liver and pancreas. Especially, it is detectable in the cortical collecting duct cells of the kidney. Pathologic variations at the primary cilia have been identified as the aetiological factor of ARPKD. This is the reason why ARPKD is a member of the ciliopathies. In the kidney the primary cilia is working as a mechano-, chemo- and osmosensor. The exact pathomechanism of ARPKD has not yet been identified. The aim of this dissertation was therefore to identify further interaction partners of FPC. Furthermore, a method should be established to obtain primary cells of kidney epithel cells which are affected by ARPKD in order to establish cell lines of these primary cells. In this dissertation RACK1 (Receptor for Activated C Kinase 1) could be identified as an interaction partner of FPC. Because of the seven WD40 repeats (formed a beta-propeller structure) it is involved in many signaling pathways like MAPK-kinase, cell migration pathways and cell proliferation pathways. ADAM12 is another interaction partner of FPC. ADAM12 is a zinc depending membrane anchored metalloprotease. Treating the kidney epithel cells with collagenase B and trypsin has been shown as the most efficient method to gain primary cells from kidney epithel cells. For separation and isolation of the cell suspension (fibroblast and kidney epithel cells) the supported Mini-MACS-System (company Miltenyi) were used. For the specific separation of the two types of cells it is possible to use the surface antigen CD40. It is known that FPC starts a Notch-similar pathway by an ectodomain shedding of its N-terminal part. By this mechanism the N-terminal part is separated and binds to the rest of the FPC again and within it activates an intramembrane proteolysis. As a consequence the C-terminal part is separated and translocated to the nucleus and cytosol. ADAM12 is identified as the probable protease which is responsible for the separation of the N-terminal part from FPC. Probably RACK1 is responsible for the correct transport and position of the two proteins during that process. |
