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Updated: Dec 29, 2025
A Modified Precipitation Method to Isolate Urinary Exosomes
Published on: January 16, 2015
Analysis of the polycystin complex (PCC) in human urinary exosome-like vesicles (ELVs)
Wendy A Lea1, Kerri McGreal1, Madhulika Sharma1
1The Jared Grantham Kidney Institute, University of Kansas Medical Center, Kansas City, KS 66160, USA.
Insights
Urinary exosome-like vesicles (ELVs) contain polycystin proteins involved in polycystic kidney disease. These proteins undergo extensive post-translational processing within ELVs, suggesting their utility in disease research.
Area of Science:
- Nephrology
- Molecular Biology
- Proteomics
Background:
- Polycystin-1 (PC1), polycystin-2 (PC2), and fibrocystin are key proteins in kidney function, encoded by PKD1, PKD2, and PKHD1 genes.
- These proteins assemble into the polycystin complex (PCC) within urinary exosome-like vesicles (ELVs).
- ELVs are nanoscale vesicles released into urine by kidney tubule cells.
Purpose of the Study:
- To investigate the post-translational modifications of PC1, PC2, and fibrocystin within human urinary ELVs.
- To characterize the cleavage events and complex formation of these proteins in ELVs.
Main Methods:
- Mass spectrometry (MS/MS) analysis of ELVs from individuals with PKD1 mutations and controls.
- Native gel electrophoresis to analyze the size and composition of the polycystin complex (PCC).
Main Results:
- Identified multiple novel cleavage events in PC1, PC2, and fibrocystin beyond known sites.
- The C-terminal region of PC1 shows three distinct cleavage events in vivo.
- Evidence suggests cleavage of fibrocystin and PC2 C-terminal tails prior to or within ELVs.
- The PCC is a large complex (>2 MDa), with cleaved ectodomains of PC1 and fibrocystin resolving at 300 kDa.
Conclusions:
- The three major human cystogenic proteins are present and extensively proteolytically processed in urinary ELVs.
- Urinary ELVs represent a valuable resource for studying the polycystin complex and its interacting proteins.
- Understanding these proteolytic events is crucial for elucidating the pathobiology of polycystic kidney diseases.
Abstract:
The polycystin-1 (PC1), polycystin-2 (PC2) and fibrocystin proteins, the respective products of the PKD1, PKD2 and PKHD1 genes, are abundant in urinary exosome-like vesicles (ELVs) where they form the polycystin complex (PCC). ELVs are 100 nm diameter membrane vesicles shed into the urine by the cells lining the nephron. Using MS/MS analysis of ELVs from individuals with PKD1 mutations and controls, we show that in addition to the well-described GPS/GAIN cleavage event in PC1 at 3048 aa and the proprotein convertase cleavage (PPC) event in fibrocystin at 3616 aa, there are multiple other cleavage events in these proteins. The C-terminal 11 transmembrane portion of PC1 undergoes three cleavage events in vivo. The absence of peptides from the C-terminal cytoplasmic tail of fibrocystin implies a cleavage event close to its single TM domain prior to loading onto the ELVs. There is also evidence that the C-terminal tail of PC2 is also cleaved in ELVs. Native gel analysis of the PCC shows that the entire complex is > 2 MDa in size and that N-terminal GPS/GAIN cleaved PC1 and PPC cleaved fibrocystin ectodomains can be released under non-reducing conditions and resolve at 300 kDa. This paper shows that the three major human cystogene proteins are detectable in human urinary ELVs and that all three undergo post-translational proteolytic processing. Human urinary ELVs may be a useful source of material in the search for proteins that interact with the PCC.
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