Tripartite separation of glomerular cell-types and proteomes from reporter-free mice

Abstract

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.

α-Parvin Defines a Specific Integrin Adhesome to Maintain the Glomerular Filtration Barrier

“…; podocyte proteome C, Rinschen et al .) 14 , 48 – 50 . IAC components were determined as “expressed” with a cutoff expression of >50 transcript per million on mRNA level or with a log 2 fold-change cell-type/other glomerular cells between −0.5 and 0.5 on a proteome level.…”

“…In addition, recently published murine podocyte RNA sequencing and glomerular proteome datasets were used for external validation of the podocyte ITAM dataset (MES, GEC, and podocyte proteome A, Hatje et al; podocyte proteome-B, Schell et al; podocyte proteome C, Rinschen et al). 14,[48][49][50] IAC components were determined as "expressed" with a cutoff expression of .50 transcript per million on mRNA level or with a log 2 fold-change celltype/other glomerular cells between 20.5 and 0.5 on a proteome level. IAC components were determined as celltype-specific enriched with a statistical significant log 2 fold change cell-type/other glomerular cells .0.5 and .50 transcript per million on mRNA level, or a statistical significant log 2 fold change podocyte/nonpodocyte .0.5 on proteome level.…”

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.…”

The Intertwining of Autophagy and the Ubiquitin Proteasome System in Podocyte (Patho)Physiology

“…Recent data from our lab suggest that podocytes have an exceptional high dependence on a functioning proteasomal system in vivo, already at young age. Thereby, a new technique enabling a reporter-free bulk separation of glomerular cell-types from wildtype mice for protein biochemical investigations shows that podocytes express higher levels of proteasomal proteins in comparison to mesangial and glomerular endothelial cells [131] in 8 to 10-weekold mice. The strong podocyte dependence on the proteasome for regular proteostasis is further stressed by the finding that podocyte-specific deficiency of rpt3, a subunit of the 19S RP of the proteasome leads to early onset podocyte injury (albuminuria at 4 weeks of age) and loss, with atrophic kidneys at 14 weeks of age [132].…”