Hatje, Favian A., Wedekind, Uta, Sachs, Wiebke, Loreth, Desiree 
ORCID: 0000-0003-0827-149X, Reichelt, Julia, Demir, Fatih ORCID: 0000-0002-5744-0205, Kosub, Christopher ORCID: 0000-0002-7425-4535, Heintz, Lukas ORCID: 0000-0003-1889-0470, Tomas, Nicola M., Huber, Tobias B., Skuza, Sinah, Sachs, Marlies, Zielinski, Stephanie, Rinschen, Markus M. and Meyer-Schwesinger, Catherine
(2021).
Tripartite Separation of Glomerular Cell Types and Proteomes from Reporter-Free Mice.
J. Am. Soc. Nephrol., 32 (9).
S. 2176 - 2195.
WASHINGTON:
AMER SOC NEPHROLOGY.
ISSN 1533-3450
Abstract
Background The glomerulus comprises podocytes, mesangial cells, 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 tripartite isolation method for murine mesangial and endothelial cells and podocytes (timMEP). Methods We separated glomerular cell types from wild-type or mT/mG mice via a novel FACS approach, and validated their purity. Cell type proteomes were compared between strains, ages, and sex. We applied timMEP to the podocyte-targeting, immunologic, THSD7A-associated, model of membranous nephropathy. Results timMEP enabled protein-biochemical analyses of podocytes, mesangial cells, and endothelial cells derived from reporter-free mice, and allowed for the characterization of podocyte, endothelial, and mesangial proteomes of individual mice. We identified marker proteins for mesangial and endothelial proteins, and outlined protein-based, potential communication networks and phosphorylation patterns. 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, that could not be detected by bulk glomerular analyses. The 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 toward the comprehensive and systematic characterization of glomerular cell biology.
| Item Type: | Journal Article | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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| URN: | urn:nbn:de:hbz:38-581559 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| DOI: | 10.1681/ASN.2020091346 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Journal or Publication Title: | J. Am. Soc. Nephrol. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Volume: | 32 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Number: | 9 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Page Range: | S. 2176 - 2195 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Date: | 2021 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Publisher: | AMER SOC NEPHROLOGY | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Place of Publication: | WASHINGTON | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| ISSN: | 1533-3450 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Language: | English | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Faculty: | Unspecified | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Divisions: | Unspecified | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Subjects: | no entry | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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| URI: | http://kups.ub.uni-koeln.de/id/eprint/58155 |
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