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

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Tripartite Separation of Glomerular Cell-Types and Proteomes From Reporter-Free Mice. / Hatje, Favian; Wedekind, Uta; Sachs, Wiebke; Loreth, Desiree; Reichelt, Julia; Demir, Fatih; Kosub, Christopher; Heintz, Lukas; Tomas, Nicola; Huber, Tobias; Skuza, Sinah; Sachs, Marlies; Zielinski, Stephanie; Rinschen, Markus; Meyer-Schwesinger, Catherine.

in: J AM SOC NEPHROL, Jahrgang 32, Nr. 9, 09.2021, S. 2175-2193.

Publikationen: SCORING: Beitrag in Fachzeitschrift/Zeitung › SCORING: Zeitschriftenaufsatz › Forschung › Begutachtung

Harvard

Hatje, F, Wedekind, U, Sachs, W, Loreth, D, Reichelt, J, Demir, F, Kosub, C, Heintz, L, Tomas, N, Huber, T, Skuza, S, Sachs, M, Zielinski, S, Rinschen, M & Meyer-Schwesinger, C 2021, 'Tripartite Separation of Glomerular Cell-Types and Proteomes From Reporter-Free Mice', J AM SOC NEPHROL, Jg. 32, Nr. 9, S. 2175-2193. https://doi.org/10.1681/ASN.2020091346

APA

Hatje, F., Wedekind, U., Sachs, W., Loreth, D., Reichelt, J., Demir, F., Kosub, C., Heintz, L., Tomas, N., Huber, T., Skuza, S., Sachs, M., Zielinski, S., Rinschen, M., & Meyer-Schwesinger, C. (2021). Tripartite Separation of Glomerular Cell-Types and Proteomes From Reporter-Free Mice. J AM SOC NEPHROL, 32(9), 2175-2193. https://doi.org/10.1681/ASN.2020091346

Vancouver

Hatje F, Wedekind U, Sachs W, Loreth D, Reichelt J, Demir F et al. Tripartite Separation of Glomerular Cell-Types and Proteomes From Reporter-Free Mice. J AM SOC NEPHROL. 2021 Sep;32(9):2175-2193. https://doi.org/10.1681/ASN.2020091346

Bibtex

@article{22bf99e0cc8843d49ad0d72c73e7487d,
title = "Tripartite Separation of Glomerular Cell-Types and Proteomes From Reporter-Free Mice",
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.",
author = "Favian Hatje and Uta Wedekind and Wiebke Sachs and Desiree Loreth and Julia Reichelt and Fatih Demir and Christopher Kosub and Lukas Heintz and Nicola Tomas and Tobias Huber and Sinah Skuza and Marlies Sachs and Stephanie Zielinski and Markus Rinschen and Catherine Meyer-Schwesinger",
year = "2021",
month = sep,
doi = "10.1681/ASN.2020091346",
language = "English",
volume = "32",
pages = "2175--2193",
journal = "J AM SOC NEPHROL",
issn = "1046-6673",
publisher = "American Society of Nephrology",
number = "9",

}

RIS

TY - JOUR

T1 - Tripartite Separation of Glomerular Cell-Types and Proteomes From Reporter-Free Mice

AU - Hatje, Favian

AU - Wedekind, Uta

AU - Sachs, Wiebke

AU - Loreth, Desiree

AU - Reichelt, Julia

AU - Demir, Fatih

AU - Kosub, Christopher

AU - Heintz, Lukas

AU - Tomas, Nicola

AU - Huber, Tobias

AU - Skuza, Sinah

AU - Sachs, Marlies

AU - Zielinski, Stephanie

AU - Rinschen, Markus

AU - Meyer-Schwesinger, Catherine

PY - 2021/9

Y1 - 2021/9

N2 - 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.

AB - 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.

U2 - 10.1681/ASN.2020091346

DO - 10.1681/ASN.2020091346

M3 - SCORING: Journal article

C2 - 34074698

VL - 32

SP - 2175

EP - 2193

JO - J AM SOC NEPHROL

JF - J AM SOC NEPHROL

SN - 1046-6673

IS - 9

ER -