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

超滤是维持体内平衡的关键功能，也是遗传性肾脏疾病的主要目标。虽然肾脏过滤器的结构成分-内皮细胞，肾小球基底膜和狭缝膈(SD) -多年前已经被描述，但SD的复杂超微结构直到最近才通过我们的多物种平台进一步阐明，结合遗传模型和冷冻电镜分析。与目前的概念相反，我们发现NEPH1和NEPHRIN在邻近的足突之间形成多层的两部分支架，只有很少的NEPH1/NEPHRIN反式相互作用。结构建模和与其他已知免疫球蛋白超家族蛋白（如TITIN）的比对表明，SD是一种高度灵活、不堵塞的网状物。范式转换，我们的模型没有将SD视为白蛋白的关键屏障，而是将其视为足突形成、维持和功能的看门人。由于我们的工作假设对影响SD的遗传性和获得性肾脏疾病具有广泛的意义，因此我们建议通过以下方法来完善当前的SD模型：1)对SD通透性进行功能分析，结合诱导成人NEPH1/NEPHRIN KO模型和单肾小球微穿刺进行初级尿蛋白质组分析；2)采用综合结构生物学方法对NEPH1/NEPHRIN进行高分辨率结构分析，结合x射线晶体学，3)基因分析，基于结构数据建立人类致病NEPHRIN突变模型，并在三维细胞培养模型中筛选突变分子的功能。总的来说，这些研究将对SD的结构和功能提供新的见解，从而促进改善遗传性和获得性肾小球疾病的治疗方法。

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.