PT  - JOURNAL ARTICLE
AU  - Favian A. Hatje
AU  - Markus M. Rinschen
AU  - Uta Wedekind
AU  - Wiebke Sachs
AU  - Julia Reichelt
AU  - Tobias B. Huber
AU  - Sinah Skuza
AU  - Marlies Sachs
AU  - Stephanie Zielinski
AU  - Catherine Meyer-Schwesinger
TI  - Tripartite separation of glomerular cell-types and proteomes from reporter-free mice
AID  - 10.1101/2020.08.22.262584
DP  - 2020 Jan 01
TA  - bioRxiv
PG  - 2020.08.22.262584
4099  - http://biorxiv.org/content/early/2020/08/22/2020.08.22.262584.short
4100  - http://biorxiv.org/content/early/2020/08/22/2020.08.22.262584.full
AB  - Purpose The kidney glomerulus comprises a syncytium of podocytes, mesangial and endothelial cells, which jointly determine glomerular filtration barrier function, and thereby kidney and cardiovascular health. The understanding of this intricate functional unit and its intracellular communication beyond the transcriptome requires bulk isolation of these cell-types from glomeruli for subsequent biochemical investigations. Therefore, we developed a globally applicable tripartite isolation method for murine mesangial and endothelial cells and podocytes (timMEP).Methods Glomerular cells were separated via a novel FACS-sort depending on a cell-specific antibody labeling in wildtype mice or based on a combination of transgenic fluorescent protein expression and antibody labeling in mT/mG mice. The purity of isolated cell-types was validated by qPCR and immunoblot. The proteome of podocytes, mesangial and endothelial cells was determined and compared between species, ages and gender of wildtype and mT/mG mice. The method was also applied to the podocyte-targeting immunologic injury model of THSD7A-associated membranous glomerulonephritis.Results TimMEP enabled protein-biochemical analyses of podocytes, mesangial and endothelial cells derived from a single reporter free mouse. Proteomic analyses allowed the first characterization of podocyte, endothelial and mesangial proteomes of individual mice. Marker proteins for mesangial and endothelial proteins were determined, and protein-based interaction and intraglomerular cell communication networks were elucidated. Interestingly, analyses revealed significant cell-type specific proteome differences between mouse strains, artefacts induced by reporters, and alterations depending on gender and age. Within the glomerulus, timMEP resolved a fine-tuned initial stress response exclusively in podocytes after exposure to anti-THSD7A antibodies, which was not detectable using conventional analyses in whole glomeruli.Conclusion Globally applicable timMEP abolishes the need for costly, time- and animal-consuming breeding of mice to glomerular cell-type reporters. TimMEP enables glomerular cell-type resolved investigations at the transcriptional and protein biochemical level in health and disease, while avoiding reporter-based artefacts, paving the way towards the comprehensive and systematic characterization of glomerular cell-type biology.Key messagesA tripartite isolation method for mesangial, endothelial and podocyte cell-types from reporter-free mice.Generation of bulk cell-type samples and primary co-cultures for biochemical and protein-based analyses.Strain and transgene-dependent expression of proteins among glomerular cell-types, including protein profiles, intra-glomerular communication machineries, and reporter-dependent artefacts.Disease specific time-resolved resolution of glomerular cell-type’s response to injury.Competing Interest StatementThe authors have declared no competing interest.