Abstract: SA-PO457
Urinary CRK1 Positive Vesicles Yield Novel Insight into Signaling Through Extracellular Vesicles in the Kidney
Session Information
- Development and Regenerative Medicine
November 09, 2019 | Location: Exhibit Hall, Walter E. Washington Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Development, Stem Cells, and Regenerative Medicine
- 500 Development, Stem Cells, and Regenerative Medicine
Authors
- Homeyer, Inka Christina, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
- Puelles, Victor G., University Medical Center Hamburg-Eppendorf, Hamburg, Germany
- Obser, Anja, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
- Oberüber, Valerie Thalia, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
- Shafikhani, Sasha H., Rush Medical College, Chicago, Illinois, United States
- Huber, Tobias B., University Medical Center Hamburg-Eppendorf, Hamburg, Germany
- Braun, Fabian, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
Background
While specific functions of extracellular vesicles (EVs) have been discovered in many fields of biology and medicine, very little is known about their role in kidney health and disease. Recently, a new subgroup of EVs was identified in human and murine cell culture as well as a model of glomerulonephritis. These vesicles are shed upon apoptosis and trigger proliferation in neighboring cells, hence named apoptotic compensatory proliferative signaling vesicles (ACPSVs). As these vesicles could be separated from kidney tissue, we aimed to determine whether a fraction is shed into the urine and further analyze their biological properties.
Methods
We established a protocol of differential centrifugation and filtration to separate ACPSVs from urine samples of healthy control subjects. Multiplex immunofluorescence microscopy, Western Blot and FACS were used to validate the presence of CRKI+ EVs, determine their cellular origin and assess the baseline characteristics of these urinary vesicles.
Results
The employed protocol lead to a robust isolation of spherical vesicles ranging between 800nm and 1,8µm in diameter with a subfraction containing the ACPSV marker protein CRKI. Further protein analysis revealed the presence of marker proteins for all segments of the nephron colocalizing with both CRKI positive and negative EVs. FACS analysis and multiplex immunofluorescence enabled us to determine and quantify the specific vesicle fractions originating from i.e podocytes, PECs and collecting duct cells.
Conclusion
Our study represents the first analysis of urinary CRKI containing vesicles of the ACPSV size range. Taken into account the presence of podocyte marker proteins in the separated vesicle fraction, we hypothesize, that these are not only shed upon apoptosis, hence would not call them urinary ACPSVs. Ongoing investigations aim to validate the potential to initiate proliferation on different renal cell types and to determine differences in their function and content in the state of renal diseases. As these vesicles can be easily isolated in a high purity, they also represent a valuable source for biomarker research in various nephropathies.