A Multi-layered Quantitative In Vivo Expression Atlas of the Podocyte Unravels Kidney Disease Candidate Genes

Abstract

SummaryDamage to and loss of glomerular podocytes has been identified as the culprit lesion in progressive kidney diseases. Here, we combine mass spectrometry-based proteomics with mRNA sequencing, bioinformatics, and hypothesis-driven studies to provide a comprehensive and quantitative map of mammalian podocytes that identifies unanticipated signaling pathways. Comparison of the in vivo datasets with proteomics data from podocyte cell cultures showed a limited value of available cell culture models. Moreover, in vivo stable isotope labeling by amino acids uncovered surprisingly rapid synthesis of mitochondrial proteins under steady-state conditions that was perturbed under autophagy-deficient, disease-susceptible conditions. Integration of acquired omics dimensions suggested FARP1 as a candidate essential for podocyte function, which could be substantiated by genetic analysis in humans and knockdown experiments in zebrafish. This work exemplifies how the integration of multi-omics datasets can identify a framework of cell-type-specific features relevant for organ health and disease.

Novel 3D analysis using optical tissue clearing documents the evolution of murine rapidly progressive glomerulonephritis

“…15 In this context, podocyte loss represents the major culprit for the development for glomerular diseases. 16 Podocytes were identified using histone-mediated nuclear expression of enhanced green fluorescent protein (eGFP) (Figure 2a). Total numbers of eGFP þ podocytes were quantified in intact using a combination of in vivo metabolic labeling and 3D morphometry ( Figure 2b).…”

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

“…Then proteins were reduced and alkylated as described above. Proteins were digested using a protease in-solution digestion protocol with a modified SP3 protocol (Hohne et al, 2018;Hughes et al, 2019) (50000 cells), or in solution digestion (Rinschen et al, 2018a). For all digestion steps, trypsin was used.…”

“…Further, only podocytes are isolated from one mouse, the other glomerular cells being indistinguishable through the expression of tdTomato. Additionally, the substantial expression of a foreign fluorescent protein within glomerular cells (up to 6%) (Rinschen et al, 2018a) could induce inevitable biochemical artefacts, depending on the biological system evaluated (for example protein degradation systems). These limitations are particularly important in diseases with altered glomerular composition such as podocyte loss or mesangial expansion.…”

Big science and big data in nephrology

“…76 First applications of deep proteomics include detailed characterization of human and mouse podocytes 77 that led to the discovery of FARP1, a previously unrecognized podocytespecific protein with a potential role in proteinuric kidney disease. 77 An ongoing area of research focuses on posttranslational protein modifications in glomerular tissue. In particular, large-scale phosphoproteomes, 78 integrated with genomic information, can be used to predict clinically relevant phosphorylation sites.…”