Combined high-resolution structural and functional characterization of the multi-layered bipartite NEPH-NEPHRIN based slit diaphragm

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Ultrafiltration is a key function to maintain body homeostasis, and a major target of hereditary kidney diseases. While the structural components of the kidney filter - endothelial cells, glomerular basement membranes and slit diaphragms (SD) - have been described many years ago, the complex ultrastructure of the SD was only recently further elucidated by our multispecies platform, combining genetic modeling and cryo-EM analysis. Contrary to current concepts, we discovered that NEPH1 and NEPHRIN form a multi-layered bipartite scaffold between adjacent foot processes with only very few NEPH1/NEPHRIN trans-interactions. Structural modeling and alignment with other known immunoglobulin superfamily proteins such as TITIN suggests that the SD acts as a highly flexible, non-clogging mesh. Paradigm shifting, our model does not view the SD as a critical barrier for albumin, but rather as the gatekeeper for foot process formation, maintenance and function. Since our working hypothesis has extensive implications for hereditary and acquired kidney diseases affecting the SD, we propose to refine the current SD model by 1) functional analysis of SD permeability, combining inducible adult NEPH1/NEPHRIN KO models and single-glomeruli micro-puncture for primary urine proteome analysis, 2) high-resolution structural analysis of NEPH1/NEPHRIN using an integrative structural biology approach, by combining X-ray crystallography, electron microscopy and complementary methods, 3) genetic analysis, by modeling of human disease-causing NEPHRIN mutations based on the structural data and by screening the functionality of mutated molecules in 3D cell culture models. Collectively, these studies will provide novel insight into the structure and function of the SD, thereby facilitating therapeutic approaches to ameliorate hereditary and acquired glomerular diseases.