Identification and proteomic profiling of exosomes in human urine

Abstract

Urine provides an alternative to blood plasma as a potential source of disease biomarkers. One urinary biomarker already exploited in clinical studies is aquaporin-2. However, it remains a mystery how aquaporin-2 (an integral membrane protein) and other apical transporters are delivered to the urine. Here we address the hypothesis that these proteins reach the urine through the secretion of exosomes [membrane vesicles that originate as internal vesicles of multivesicular bodies (MVBs)]. Low-density urinary membrane vesicles from normal human subjects were isolated by differential centrifugation. ImmunoGold electron microscopy using antibodies directed to cytoplasmic or anticytoplasmic epitopes revealed that the vesicles are oriented "cytoplasmic-side inward," consistent with the unique orientation of exosomes. The vesicles were small (<100 nm), consistent with studies of MVBs and exosomes from other tissues. Proteomic analysis of urinary vesicles through nanospray liquid chromatography-tandem mass spectrometry identified numerous protein components of MVBs and of the endosomal pathway in general. Full liquid chromatography-tandem MS analysis revealed 295 proteins, including multiple protein products of genes already known to be responsible for renal and systemic diseases, including autosomal dominant polycystic kidney disease, Gitelman syndrome, Bartter syndrome, autosomal recessive syndrome of osteopetrosis with renal tubular acidosis, and familial renal hypomagnesemia. The results indicate that exosome isolation may provide an efficient first step in biomarker discovery in urine.

Figures

Fig. 1.

Immunoelectron microscopy of urinary vesicles. ImmunoGold labeling using antibodies to membrane proteins targeted to external epitopes (APN and CD9) or cytoplasmic epitopes (AQP2 and NCC). Insets show selected fields at increased magnification.

Fig. 2.

Size distribution of urinary vesicles. (A) Electron micrograph of negatively stained urinary vesicles. (B) Histogram of vesicle diameter.

Fig. 3.

1D gel electrophoresis of urinary vesicles. (A) Coomassie-blue-stained gel of urinary vesicles. Shown is one of the two gels analyzed in the present study. The number of proteins identified in each molecular mass range is indicated. (B) Immunoblot of the same sample with anti-THP antibody; the large bulge is due to a large amount of THP.

Fig. 4.

Distribution of identified proteins by subcellular origin. Protein classification is based on analysis by using a harvester search. Protein classes are color-coded to correspond to Table 3.

Fig. 5.

Instances of identification of ubiquitin. Ubiquitin was identified by LC-MS/MS (A) and immunoblot by using anti-ubiquitin antibody (B) on 1D gels over a molecular mass range from ≈10 kDa to ≈400 kDa, presumably representing a variety of ubiquitinated proteins.

Fig. 6.

Immunoblots of proteins in human urine exosomes (Urine) and kidney regions (Cortex, Outer Medulla, and Inner Medulla). Immunoblots were probed with antibodies to the following proteins: ACE, angiotensin-converting enzyme; ALIX, ALG-2 interacting protein X; APN; AQP1; AQP2; CA4, carbonic anhydrase type IV; CD9; CLIC1, chloride intracellular channel-1; αENaC, epithelial sodium channel α; βENaC, epithelial sodium channel β; γENaC, epithelial sodium channel γ; MUC1, mucin-1; NCC; NKCC2, Na-K-2Cl cotransporter type 2; PODXL, podocalyxin; RAB4; RAB5B; RAB11; SNX18, sorting nexin-18; sorcin; TSG101; and PC1, polycystin-1. In the PC1 immunoblot, an additional lane was added (PC1+), which was loaded with PC1 protein extracted from Madin-Darby canine kidney cells with stable expression of human PC1 protein (a kind gift of Feng Qian, The Johns Hopkins University, Baltimore).

Fig. 7.

Process of exosome formation and release into the urine. Ub, ubiquitin; AP, adaptor protein; ESCRT, endosomal sorting complex required for transport; ALIX, ALG-2 interacting protein X.