Deciphering interaction and collaborative function of basement membrane and slit diaphragm in Drosophila.

• Applicant: Privatdozent Dr. Tobias Hermle
• Subject Area: Nephrology
• Term: since 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 539635640

Glomerular disease underlies the majority of chronic kidney failure. Targeted therapies are frequently lacking due to limited understanding of the complex pathogenesis. The critical layers of the filtration barrier are the glomerular basement membrane and the slit diaphragm. Both structures also represent signaling platforms. However, the collaborative function and exchange of signaling cues between both layers remain poorly understood. Approximately 10% of chronic kidney disease has a genetic cause. Recent studies suggest that variants in the three type IV collagens of the basement membrane account for about 30% of these, even in adult cohorts. Surprisingly, these DNA variants frequently manifest as focal segmental glomerulosclerosis, i.e. as a podocytopathy. This highlights the relevance of the interaction of podocytes with the extracellular matrix. Slit diaphragms and basement membranes are lacking in cultured cells and mouse models are hampered by low throughput and difficult accessibility of the filtration barrier. The Drosophila nephrocyte combines a functional, molecularly conserved slit diaphragm and a basement membrane in a genetically tractable model organism. Nephrocytes are therefore ideal to explore the dynamic interaction between the slit diaphragm and the extracellular matrix. We first want to establish the nephrocytes as model for the glomerular basement membrane (Aim 1) by identification of the matrix components, their localization, origin and loss-of-function phenotypes. Fluorescence Recovery After Photobleaching (FRAP) analysis will reveal the dynamics of the basement membrane. Then, we want to unravel the signaling cues exchanged between basement membrane and slit diaphragm (Aim 2). We will study the role of the basement membrane for nephrin trafficking and slit diaphragm establishment, identity matrix receptors, and examine a putative role for excessive autophagy and defective lysosomal degradation. We further will compare transcriptional profiles using RNA sequencing using fine dissection of nephrocytes to obtain RNA exclusively from this cell type for an unbiased trancriptomic analysis. To establish a personalized platform for the glomerular collagen IV genes, we will employ overexpression of the human protein in comparison to the mutant for rescue and gain-of-function. Taken together, the proposed work is suitable to elucidate the collaborative function of slit diaphragm and the glomerular basement membrane.

• DFG Programme: Research Grants