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Mühlenhardt, Petra (2006): Funktionelle Untersuchungen zur Bedeutung des Kidney Injury Molecule-1 (Kim-1) bei der Differenzierung der Tubulusepithelzelle von Säugetieren. Dissertation, LMU München: Faculty of Veterinary Medicine
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Abstract

Kim-1 (kidney injury molecule-1), a member of the TIM (T-cell immunglobulin mucin) family is a glykosylated type 1 transmembrane protein, which is markley upregulated during the repair of renal epithel cells after ischemia and in malignant and cystic kidney disease, while expressed at low levels in normal kidneys. In previous experiments Kim-1 has been shown to localize in primary cilia of cultured renal epithelial cells in-vitro. Primary cilia have been shown to play a role in development and recently cilial dysfunction has been shown to be the unifying defect causing polycystic kidney disease. Kim-1 interacts with Inversin, a ciliary protein, which is mutated in Type 2 nephronophthisis, and which has been shown to modulate the Wnt pathway by targeting Dishevelled (Dvl) for proteasomal degradation. The expression of Wnt proteins is necessary for many fundamental processes in differentiation, proliferation, polarity and adhesion. Wnts play an important role in the early embryonic development. There is only limited knowledge on the physiological role of Kim-1 during differentiation, proliferation and survival of tubular epithel cells after renal injury. The aim of this study was to characterize the role of Kim-1 in epithelial polarization and ciliogenesis of tubular epithel cells outside of pathological injury models. Additionally the role of Kim-1 in the Wnt signaling pathway was examined. Renal tubular cells were analyzed phenotypically with immunfluorescence staining. Protein expression levels were detected by SDS PAGE and Western blot. Signaltransduction activity was measured in luciferase reporter assays. Protein overexpression was achieved with transient transfection of plasmid DNA in cultured cells after calcium chloride precipitation. Gene reduction was achieved through lentivirus-mediated transduction of short interfering RNAs. Examination of immunfluorescence stained cryosections showed, that Kim-1 is localized in-vivo in primary cilia of tubular epithelial cells in mouse kidneys. Kim-1 expression levels correlate with the polarization of tubular epithel cells in-vitro. Reporter assays using TOP FLASH luciferase constructs revealed that Kim-1 reduced the Dvl-induced TOP FLASH activation and decreased steady-state levels of ß-Catenin in HEK 293t cells, suggesting that Kim-1 antagonizes the canonical Wnt pathway. Conversely Kim-1 expression increased AP-1 activity, which has been linked to the non-canonical Wnt pathway. Phenotypical characterization of two different immortalized renal epithelial cell lines after Kim-1 knock-down showed no changes in epithelial polarization, or ciliogenesis. These results suggest that Kim-1 is a ciliary protein that probably plays no active role in cell polarization or ciliogenesis, but may participate in the Wnt signaling pathway. Kim-1 may act as a molecular switch between the canonical and non-canonical Wnt pathway and thus may play a role in epithelial differentiation.