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Subfractionation, characterization, and in-depth proteomic analysis of glomerular membrane vesicles in human urine.

Hogan MC, Johnson KL, Zenka RM, Charlesworth MC, Madden BJ, Mahoney DW, Oberg AL, Huang BQ, Leontovich AA, Nesbitt LL, Bakeberg JL, McCormick DJ, Bergen HR, Ward CJ - Kidney Int. (2013)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Division of Nephrology and Hypertension, Department of Internal Medicine, Mayo Clinic, Rochester, Minnesota, USA.

ABSTRACT
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.

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GMVs from individuals with glomerular diseases: 6A Crude exosomes from one normal (C) and three glomerular disease cases #1–3. THP resolves at 85–100kDa & albumin at 60–67kDa. Albumin mainly remains in solution under these conditions with only moderate enhancement in the nephrotic individuals. 6B Transilluminated ultracentrifuge tubes with gradients obtained from cases with glomerular disease: .#1 and 2 resolved well whereas the #3 resolved less well defined bands, although we did not have any difficulty fractionating bands of interest in #3. 6C: Pellets from D2O gradients SDS PAGE Coomassie brilliant blue stained prior to gel sectioning (#3 has a small contaminating THP band at 85kDa). 6D: Representative TEM of ELVs from individual #1, fractions A, B and C.
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Figure 6: GMVs from individuals with glomerular diseases: 6A Crude exosomes from one normal (C) and three glomerular disease cases #1–3. THP resolves at 85–100kDa & albumin at 60–67kDa. Albumin mainly remains in solution under these conditions with only moderate enhancement in the nephrotic individuals. 6B Transilluminated ultracentrifuge tubes with gradients obtained from cases with glomerular disease: .#1 and 2 resolved well whereas the #3 resolved less well defined bands, although we did not have any difficulty fractionating bands of interest in #3. 6C: Pellets from D2O gradients SDS PAGE Coomassie brilliant blue stained prior to gel sectioning (#3 has a small contaminating THP band at 85kDa). 6D: Representative TEM of ELVs from individual #1, fractions A, B and C.

Mentions: Using identical conditions we isolated GMV subfractions from three individuals with glomerular disease (2 membranous nephropathy cases and another individual with IgG3 membranoproliferative glomerulonephritis (figure 6). The initial ultracentrifugation spin precipitated THP as usual (figure 6A)but with only small amounts of albumin retained in the pellet (figure 6C; compare control with cases) implying that it is retained in solution under these conditions with negligible albumin in the respective pellets following the D2O sucrose gradient step (figure 6C) step. Using the same gel slice technique we identified a total of 5657 proteins using the Q-Exactive™ mass spectrometer (Supplemental table 4). GMV morphology was identical to the healthy control samples but importantly we detected the all the nephrotic syndrome proteins and several others not detected in the healthy controls (e.g. nephrin, TRPC6, INF2, IQGAP1; and the idiopathic membranous nephropathy target autoantigen PLA2R; Supplemental table 4).


Subfractionation, characterization, and in-depth proteomic analysis of glomerular membrane vesicles in human urine.

Hogan MC, Johnson KL, Zenka RM, Charlesworth MC, Madden BJ, Mahoney DW, Oberg AL, Huang BQ, Leontovich AA, Nesbitt LL, Bakeberg JL, McCormick DJ, Bergen HR, Ward CJ - Kidney Int. (2013)

GMVs from individuals with glomerular diseases: 6A Crude exosomes from one normal (C) and three glomerular disease cases #1–3. THP resolves at 85–100kDa & albumin at 60–67kDa. Albumin mainly remains in solution under these conditions with only moderate enhancement in the nephrotic individuals. 6B Transilluminated ultracentrifuge tubes with gradients obtained from cases with glomerular disease: .#1 and 2 resolved well whereas the #3 resolved less well defined bands, although we did not have any difficulty fractionating bands of interest in #3. 6C: Pellets from D2O gradients SDS PAGE Coomassie brilliant blue stained prior to gel sectioning (#3 has a small contaminating THP band at 85kDa). 6D: Representative TEM of ELVs from individual #1, fractions A, B and C.
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Related In: Results  -  Collection

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Figure 6: GMVs from individuals with glomerular diseases: 6A Crude exosomes from one normal (C) and three glomerular disease cases #1–3. THP resolves at 85–100kDa & albumin at 60–67kDa. Albumin mainly remains in solution under these conditions with only moderate enhancement in the nephrotic individuals. 6B Transilluminated ultracentrifuge tubes with gradients obtained from cases with glomerular disease: .#1 and 2 resolved well whereas the #3 resolved less well defined bands, although we did not have any difficulty fractionating bands of interest in #3. 6C: Pellets from D2O gradients SDS PAGE Coomassie brilliant blue stained prior to gel sectioning (#3 has a small contaminating THP band at 85kDa). 6D: Representative TEM of ELVs from individual #1, fractions A, B and C.
Mentions: Using identical conditions we isolated GMV subfractions from three individuals with glomerular disease (2 membranous nephropathy cases and another individual with IgG3 membranoproliferative glomerulonephritis (figure 6). The initial ultracentrifugation spin precipitated THP as usual (figure 6A)but with only small amounts of albumin retained in the pellet (figure 6C; compare control with cases) implying that it is retained in solution under these conditions with negligible albumin in the respective pellets following the D2O sucrose gradient step (figure 6C) step. Using the same gel slice technique we identified a total of 5657 proteins using the Q-Exactive™ mass spectrometer (Supplemental table 4). GMV morphology was identical to the healthy control samples but importantly we detected the all the nephrotic syndrome proteins and several others not detected in the healthy controls (e.g. nephrin, TRPC6, INF2, IQGAP1; and the idiopathic membranous nephropathy target autoantigen PLA2R; Supplemental table 4).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Division of Nephrology and Hypertension, Department of Internal Medicine, Mayo Clinic, Rochester, Minnesota, USA.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus