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 tripartite isolation method for murine mesangial and endothelial cells and podocytes (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.

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.

Kidney filtration is ensured in the glomerulus by the interaction of podocytes, endothelial and mesangial cells. In comparison to autophagy and mitochondrial function, the proteasome system represents a completely unexplored aspect of cellular metabolism especially in kidney cells, even though it is getting more and more clear that proteasome alterations are central to many glomerular metabolic and immune pathologies. Here we identify the proteasome to be critical in a conserved cell type specific modality. Combining genetic and inhibitor-based human, pig, mouse, and Drosophila models we demonstrate that the proteasome ensures filtration barrier integrity, with podocyte metabolism depending on the constitutive proteasome whereas glomerular endothelial cells depend on the immunoproteasome. Endothelial immunoproteasome deficiency as well as proteasome inhibition disrupt the filtration barrier in mice, resulting in pathologic immunoglobulin deposition under the slit diaphragm and glomerular basement membrane alterations. Mechanistically, a reduced endocytic activity was identified, which relates to altered membrane recycling and turnover of endocytic receptors for collagen 4 and immunoglobulins. Our findings expand the concept of the (immuno)proteasome as a control protease for protein degradation and antigen presentation to an orchestrator of endocytosis and will lead to new therapeutic principles in targeting disease-associated glomerular protein accumulations.