The Drosophila Nephrocyte - Modeling Podocyte Function and Disease

In modern industrialized nations, chronic non-communicable diseases have become the main reason of mortality. During the last years, the global health burden of chronic kidney diseases (CKD) has grown considerably and in many of these cases, no targeted or personalized therapies are available. To keep up with the rising challenges, the understanding of the causes and mechanisms underlying CKD needs to be improved and novel approaches must be found and refined to support and enrich conventional research methods. This dissertation encompasses four manuscripts concerned with various aspects of utilizing Drosophila nephrocytes for modeling mammalian kidney function and disease and contributing valuable insights, in addition to established kidney models, in a quick and cost-effective way. In general, the morphological characterization of a biological sample via electron microscopy strongly depends on the degree of sample preservation. In the first project, the ultrastructural preservation of nephrocytes after preparation for transmission electron microscopy could be greatly improved by using both an optimized dissection saline and cryofixation techniques. Also, the initially challenging immunogold detection of endogenous Crumbs protein could be realized by an indirect approach detecting GFP-tagged Crumbs using an antibody directed against the tag. The 3D ultrastructure of the complex subcellular structures in nephrocytes was further characterized by creating a 3D model of the outlying network of cell membrane invaginations based upon STEM tomography data. In conventional two-dimensional TEM, only a limited assessment of the spatial arrangement of cellular structures in possible. The question whether foot processes of mammalian podocytes interact with other foot processes of the same cell therefore remained unanswered. The second manuscript arrives at the conclusion, that the interaction exclusively happens between foot processes of different podocytes, by thorough investigation of a 3D model, which was based on a whole podocyte, sectioned and imaged using FIB-SEM technology. The cell-cell contacts between foot processes were further characterized via dual-axis tomography in human and mouse podocytes, as well as Drosophila nephrocytes, leading to the realization that there is both one quasiplanar slit diaphragm spanning the whole slit diameter between two processes and additionally several filamentous contacts, which are locally limited. Additional studies are necessary to identify the proteins these two types of cell-cell contacts are composed of. Podocytes are highly complex polarized epithelial cells. For a better understanding of the molecular fundamentals of development and maintenance of normal podocyte function, the role of Pals1, which acts as scaffolding protein and key factor necessary for apicobasal polarization, was more closely investigated in podocytes in the third manuscript. Therein, Pals1 was found to be an essential, specific and dose-dependent regulator of nephrogenesis, being a negative regulator of Hippo and TGF-β signaling in mouse, which was then also confirmed in the Drosophila model. These findings indicate a connection between renal diseases, especially renal cyst diseases, and apical polarity proteins. The pathogenesis of these diseases needs to be better understood by studying the networks regulated by Pals1. In the course of the last project, the role of Drosophila Crumbs (DmCrb) in nephrocytes was closer examined. DmCrb is an interaction partner of Stardust, the Drosophila homolog of Pals1, and a key determinant of apicobasal polarity. It was found to be involved in the regulation of early endocytosis via interaction with Moesin through its FERM-binding domain, located on its C-terminus. It further contributes to nephrocyte diaphragm assembly and maintenance via its extracellular domain. The functions of its different protein domains and the evolutionary conservation within the Crumbs family was examined by conducting a series of rescue experiments with various deletion constructs of DmCrb as well as its human orthologs. The herein describes mechanisms of nephrocyte development and function are likely also conserved in mammalian podocytes.