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

Abstract

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.

The classical pathway triggers pathogenic complement activation in membranous nephropathy

“…Recently, the slit diaphragm proteins nephrin and neph1 39 , as well as the actin-associated cytoplasmic protein synaptopodin 26 , have been suggested to be targets of complement-mediated injury in MN. In our THSD7A-immunized mice, nephrin and neph1 were substantially downregulated compared to controls (Fig.…”

“…6f ). In summary, these analyses suggest that extensive complement activation exacerbates podocyte injury through podocyte loss and compromising of the integral slit diaphragm proteins nephrin and neph1 40 .…”

Integrating basic science with translational research: the 13th International Podocyte Conference 2021

“…This approach of functional proteomics does not only measure protein levels, but also aims to characterize the functional role of proteins, their interaction partners, and posttranslational modifications under physiological and stress conditions. 88 , 89 , 90 , 91 , 92 , 93 , 94 , 95 Several variants in mitochondrial genes, such as PDSS2 , CoQ6 , and ADCK4 , 9 are described to be causative for SRNS, emphasizing the important role of mitochondria for podocyte biology. Paul Brinkkoetter investigated their function in podocytes and in particular their role as key signaling hub in the cell.…”

ScoMorphoFISH: A deep learning enabled toolbox for single‐cell single‐mRNA quantification and correlative (ultra‐)morphometry

“…To circumvent this, tissue has been either manually dissected (e.g. glomeruli from tubulointerstitium), 6 cell types were enriched by flow cytometry 7 or single‐cell‐RNA sequencing has been performed. 8 Unfortunately, the contextual and/or morphological information is lost in all approaches due to mechanical dissociation.…”