Podocytes are glomerular epithelial cells that form the slit diaphragm (SD), an important component of the multi-layered renal filter and a common target of hereditary diseases. Conceptually, the SD is a specialized cell-cell junction established in between the foot processes of neighboring cells by a zipper-like arrangement of protein complexes anchored in the podocyte plasma membrane. Several membrane molecules associated with hereditary nephrotic syndrome localize to the SD. However, direct investigations of the molecular composition and architecture of SD complexes have not been possible, mostly because of technical limitations precluding comprehensive proteomic analyses. Likewise, there is no information on the time-dependent changes of the SD-complexes generally thought to occur in response to various stimuli associated with genetic diseases.
The central goal of this proposal is to achieve a comprehensive understanding of the composition and the architecture of the protein complex(es) reconstituting the podocyte slit diaphragm and their disease-induced dynamics. For this goal we will perform the following experiments: (i) comprehensive proteomic analyses of the SD from rat and mice (wild-type, knock-out models for congenital nephrotic syndrome) by multi-epitope affinity-purifications and quantitative high-resolution mass spectrometry, (ii) investigation of the functional significance of the identified SD constituents, and (iii) proteomic analysis of the dynamics of SD complexes initiated by common confounding factors such as hypertension and diabetes. For this purpose we will use a multidisciplinary and interactive approach comprising complex biochemical and proteomic techniques, functional analyses as well as fluorescence-based and electron microscopy.