Urinary exosome-like vesicles (ELVs) are a heterogenous mixture (diameter 
40-200 nm) containing vesicles shed from all segments of the nephron including 
glomerular podocytes. Contamination with Tamm-Horsfall protein (THP) oligomers 
has hampered their isolation and proteomic analysis. Here we improved ELV 
isolation protocols employing density centrifugation to remove THP and albumin, 
and isolated a glomerular membranous vesicle (GMV)-enriched subfraction from 7 
individuals identifying 1830 proteins and in 3 patients with glomerular disease 
identifying 5657 unique proteins. The GMV fraction was composed of 
podocin/podocalyxin-positive irregularly shaped membranous vesicles and 
podocin/podocalyxin-negative classical exosomes. Ingenuity pathway analysis 
identified integrin, actin cytoskeleton, and Rho GDI signaling in the top three 
canonical represented signaling pathways and 19 other proteins associated with 
inherited glomerular diseases. The GMVs are of podocyte origin and the density 
gradient technique allowed isolation in a reproducible manner. We show many 
nephrotic syndrome proteins, proteases, and complement proteins involved in 
glomerular disease are in GMVs and some were only shed in the disease state 
(nephrin, TRPC6, INF2 and phospholipase A2 receptor). We calculated sample sizes 
required to identify new glomerular disease biomarkers, expand the ELV proteome, 
and provide a reference proteome in a database that may prove useful in the 
search for biomarkers of glomerular disease.