Molekulare Grundlagen der Podozytenschädigung verursacht durch Mutationen in Genen des tRNA modifizierenden KEOPS Komplexes.
Zusammenfassung der Projektergebnisse
Recessive mutations in genes that encode subunits of the evolutionarily highly conserved KEOPS complex cause Galloway-Mowat syndrome, a disease characterized by the combined occurrence of severe renal and neurodevelopmental abnormalities. The aim of this study was to investigate the molecular pathogenesis of KEOPS-related renal disease. Due to technical difficulties in generating mice with conditional knockout of the gene Osgep, which encodes the catalytic subunit of the KEOPS complex, Garland cell nephrocytes of Drosophila melanogaster were used as an in vivo model. Nephrocytes with knockdown of Tcs3, the Osgep orthologue showed increased apoptosis and defects of the nephrocyte slit diaphragm, a structure was high similarity to the human renal filtration barrier. In nephrocytes and in cultured human podocytes (the relevant cell type in this disease) with knockdown of OSGEP/Tcs3, we observed an activation of the ATF pathway, a central mediator of the unfolded protein response (UPR) that is induced in the presence of endoplasmic reticulum (ER) stress. Interestingly, we found that UPR-mediated ATF4 activation was not only responsible for mediating apoptosis in nephrocytes, but also for the observed slit diaphragm defects, which are central to the pathogenesis in this type of renal disease. While UPR-mediated apoptosis is a well-known principle, a direct link between UPR activation and defects in slit diaphragm formation or maintenance has not been described yet. In genetic interaction studies and by using a pharmacological inhibitor, we demonstrate that an inhibition of the ATF pathway mitigates the observed phenotypes and we thereby establish this pathway as a novel and potentially druggable therapeutic target for KEOPS-related kidney disease. The beneficial effects of the ATF4 inhibitor were seen in Tcs3 knockdown nephrocytes and in cultured human podocytes with chemically-induced ER stress, thus suggesting that this principle may have implications beyond KEOPS-related renal disease, which is a rare condition. As seen in flies, an RNASeq evaluation in OSGEP knockdown podocytes revealed an upregulation of functional gene clusters related to apoptosis and even though we identified evolutionary differences in ATF4 regulation in flies as compared to human cells, the data in both organisms highlighted the central role of the ATF4 signaling pathway. As an interesting result for follow-up studies that has not yet been described in the pathogenesis of renal diseases, the RNASeq analysis yielded a downregulation of gene clustered related to biosynthetic processes. In summary, our study provides novel insights into the pathogenesis of KEOPS-related renal disease, highlights the central role of the UPR and most prominently, the UPR-related stress signaling protein ATF4, and suggests ATF4 signaling as a novel and potentially druggable therapeutic target for certain kidney diseases.
