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Tripartite Separation of Glomerular Cell-Types and Proteomes From Reporter-Free Mice

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DOI

  • Favian Hatje, Univ Med Ctr Hamburg Eppendorf, University of Hamburg, University Medical Center Hamburg-Eppendorf
  • ,
  • Uta Wedekind, Univ Med Ctr Hamburg Eppendorf, University of Hamburg, University Medical Center Hamburg-Eppendorf
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  • Wiebke Sachs, Univ Med Ctr Hamburg Eppendorf, University of Hamburg, University Medical Center Hamburg-Eppendorf
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  • Desiree Loreth, Univ Med Ctr Hamburg Eppendorf, University of Hamburg, University Medical Center Hamburg-Eppendorf
  • ,
  • Julia Reichelt, Univ Med Ctr Hamburg Eppendorf, University of Hamburg, University Medical Center Hamburg-Eppendorf
  • ,
  • Fatih Demir
  • Christopher Kosub, Univ Med Ctr Hamburg Eppendorf, University of Hamburg, University Medical Center Hamburg-Eppendorf
  • ,
  • Lukas Heintz, Univ Med Ctr Hamburg Eppendorf, University of Hamburg, University Medical Center Hamburg-Eppendorf
  • ,
  • Nicola Tomas, Univ Med Ctr Hamburg Eppendorf, University of Hamburg, University Medical Center Hamburg-Eppendorf
  • ,
  • Tobias Huber, Univ Med Ctr Hamburg Eppendorf, University of Hamburg, University Medical Center Hamburg-Eppendorf
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  • Sinah Skuza, Univ Med Ctr Hamburg Eppendorf, University of Hamburg, University Medical Center Hamburg-Eppendorf
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  • Marlies Sachs, Univ Med Ctr Hamburg Eppendorf, University of Hamburg, University Medical Center Hamburg-Eppendorf
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  • Stephanie Zielinski, Univ Med Ctr Hamburg Eppendorf, University of Hamburg, University Medical Center Hamburg-Eppendorf
  • ,
  • Markus Rinschen
  • Catherine Meyer-Schwesinger, Univ Med Ctr Hamburg Eppendorf, University of Hamburg, University Medical Center Hamburg-Eppendorf

Background: The glomerulus comprises podocytes, mesangial, and endothelial cells, which jointly determine glomerular filtration. Understanding this intricate functional unit beyond the transcriptome requires bulk isolation of these cell-types for biochemical investigations. We developed a globally applicable t ripartite i solation method for m urine m esangial and e ndothelial cells and p odocytes (timMEP). Methods: Glomerular cell-types were separated via a novel FACS-sort approach from wildtype or mT/mG mice and the purity validated. Cell-type proteomes were compared between strains, ages, and sex. TimMEP was applied to the podocyte-targeting immunologic THSD7A-associated membranous nephropathy model. Results: TimMEP enabled protein-biochemical analyses of podocytes, mesangial, and endothelial cells derived from reporter-free mice and allowed the characterization of podocyte, endothelial, and mesangial proteomes of individual mice. Marker proteins for mesangial and endothelial proteins were identified and protein-based potential communication networks and phosphorylation patterns outlined. The analysis detected cell-type specific proteome differences between mouse strains and alterations depending on sex, age, and transgene. After exposure to anti-THSD7A antibodies, timMEP resolved a fine-tuned initial stress response chiefly in podocytes, which bulk glomerular analyses could not detect. Combination of proteomics with super-resolution imaging revealed a specific loss of slit-diaphragm but not of other foot process proteins, unraveling a protein-based mechanism of podocyte injury in this animal model. Conclusion: TimMEP enables glomerular cell-type resolved investigations at the transcriptional and protein-biochemical level in health and disease, while avoiding reporter-based artifacts, paving the way towards the comprehensive and systematic characterization of glomerular cell-type biology.

OriginalsprogEngelsk
TidsskriftJournal of the American Society of Nephrology : JASN
ISSN1046-6673
DOI
StatusE-pub ahead of print - 1 jun. 2021

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