High-resolution proteomics and functional analyses of the podocyte slit diaphragm and its disease-induced dynamics

• Applicants: Professor Dr. Bernd Fakler; Professor Dr. Florian Grahammer
• Subject Area: Anatomy and Physiology
• Term: since 2020
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 442759790

The renal slit diaphragm (SD) is a proteinaceous layer that extends between the foot processes of neighboring podocytes and contains proteins Nephrin and Podocin; mutations in either protein cause hereditary nephrotic syndrome. Functionally, the SD is thought to either serve as the crucial barrier in the renal filtration process and/or as a ‘molecular sensor’ for dynamic control of formation and function of podocyte foot processes. In the last funding period, by de-novo determination of the interactomes of the core constituents Nephrin Neph1 and Podocin, we identified the native SD as a network of proteins encompassing quite a number of previously unknown constituents including proteins primarily known for their signaling properties, such as receptors, kinases and phosphatases, as well as GTPases and cell adhesion molecules. Knock-out and knock-down of a selected set of core and novel SD constituents in mice and flies demonstrated the fundamental importance of both the structural integrity/stability, and the dynamics of the SD networks for filtration. Moreover, this work fundamentally changed the molecular picture of the SD and strongly argues for its operation as a ‘sensor and effector’ for adapting and adjusting renal filtration to the ever changing conditions in the mammalian organism. In the second period, we will build on the newly identified interactomes of the SD core components for (1) deciphering the molecular structure and assembly of the SD networks by re-building them in cellular expression systems, (2) determine their stability and (3) analyze their dynamics (in structure and function) under physiological and patho-physiological conditions using mice with targeted deletion of newly identified network constituents, as well as mice challenged with induced hypertension and immunogenic nephrotic syndrome. Together, this work program is expected to provide unprecedented insights into function and dynamics of the SD under normal and pathological conditions and to uncover marker proteins for detection of impaired renal filtration.

• DFG Programme: Research Grants