Nanomaterials have been widely used in various fields due to their excellent properties. However, the long-term exposure of humans to the environment of nanomaterials may lead to some pathological and physiological changes, such as the disruption of the glomerular filtration barrier (GFB), which can lead to high permeability and filterable albumin. Glomerular albuminuria results from the failure of the GFB, which may be caused by activation of the Notch1 signaling pathway. The reduction of glomerular endocalyx results in the level of VE-cadherin being diminished between endothelial cells, leading to leakiness in the endothelium. As previously discovered, certain nanoparticles, such as negatively charged gold nanoparticles of appropriate size, can directly facilitate the onset of endothelial leakiness through disrupting connections between vascular endothelial cells, in a process known as nanomaterial-induced endothelial leakiness (NanoEL). In this study, the negatively charged Au NPs with a diameter of 20 nm were synthesized first. Through the co-incubation of 20 nm Au NPs with a single layer of continuous HUVECs, we revealed that the synthesized 20 nm Au NPs could break the tight junctions and cause endothelial cell leakage. In addition, by constructing an endothelial leakage model between glomerular vascular endothelial cells, we confirmed that 20 nm Au NPs can generate NanoEL between glomerular endothelial cells, involving downstream processes coinciding with Notch signaling activation, resulting in the increase of urinary protein. The results of this study add to the understanding of the behaviour of nanoparticles in complex biological systems.