Global analysis reveals the complexity of the human glomerular extracellular matrix

Abstract

The glomerulus contains unique cellular and extracellular matrix (ECM) components, which are required for intact barrier function. Studies of the cellular components have helped to build understanding of glomerular disease; however, the full composition and regulation of glomerular ECM remains poorly understood. We used mass spectrometry-based proteomics of enriched ECM extracts for a global analysis of human glomerular ECM in vivo and identified a tissue-specific proteome of 144 structural and regulatory ECM proteins. This catalog includes all previously identified glomerular components plus many new and abundant components. Relative protein quantification showed a dominance of collagen IV, collagen I, and laminin isoforms in the glomerular ECM together with abundant collagen VI and TINAGL1. Protein network analysis enabled the creation of a glomerular ECM interactome, which revealed a core of highly connected structural components. More than one half of the glomerular ECM proteome was validated using colocalization studies and data from the Human Protein Atlas. This study yields the greatest number of ECM proteins relative to previous investigations of whole glomerular extracts, highlighting the importance of sample enrichment. It also shows that the composition of glomerular ECM is far more complex than previously appreciated and suggests that many more ECM components may contribute to glomerular development and disease processes. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium with the dataset identifier PXD000456.

Figure 1.

Isolation of enriched glomerular ECM. (A) Human glomeruli were isolated by differential sieving, yielding >95% purity. Before homogenization, glomeruli appeared acellular (right). (B) A proteomic workflow for the isolation of enriched glomerular ECM by fractionation (details in Concise Methods). (C) Coomassie staining and Western blotting (WB) of fractions 1–3 and the ECM fraction probing for the extracellular proteins with pancollagen IV and panlaminin probes and the intracellular proteins nephrin, actin, and lamin B1. M, molecular mass marker.

Figure 2.

MS analysis of enriched glomerular ECM fractions. (A) GO enrichment analysis of the full MS dataset. Nodes (circles) represent enriched GO terms, and edges (gray lines) represent overlap of proteins between GO terms. Node color indicates the significance of GO term enrichment; node diameter is proportional to the number of proteins assigned to each GO term. Edge weight is proportional to the number of proteins shared between connected GO terms. The full list of GO terms is detailed in Supplemental Figure 1. (B) All four protein fractions were analyzed by MS, and spectral counting was used to determine the enrichment of ECM proteins (identified by GO analysis). The mean ECM enrichment was 38% from three biologic replicates. (C) Relative quantification for the 10 most abundant ECM proteins detected by MS. Relative protein abundance was calculated using peptide intensity as described in Supplemental Methods. Gene names are shown for clarity, and collagen (COL) IV and laminin isoforms are combined as one value. (D) Western blotting (WB) confirmed enrichment of TINAGL1 and collagen VI in glomerular ECM.

Figure 3.

Comparison of the glomerular ECM proteome with published glomerular proteomic datasets. The glomerular ECM proteome identified in this study was compared with other glomerular proteomic studies for which full datasets were available (studies by Cui et al. and Yoshida et al.). Numbers of proteins in each intersection set of the area proportional Euler diagram are in bold italics. ECM proteins were categorized as basement membrane, other structural ECM, or ECM-associated proteins, and they were colored and arranged accordingly. Nodes (circles) are labeled with gene names for clarity. ECM proteins detected in any of the three biologic replicates reported in this study were included in the comparison with other proteomic datasets; these published datasets each reported one biologic replicate. Large node size indicates proteins detected in at least two biologic replicates in this study.

Figure 4.

Interaction network analysis of human glomerular ECM. (A) Protein interaction network constructed from enriched glomerular ECM proteins identified by MS. Nodes (circles) represent proteins, and edges (gray lines) represent reported protein–protein interactions. ECM proteins were categorized as basement membrane, other structural ECM, or ECM-associated proteins, and they were colored and arranged accordingly. Nodes are labeled with gene names for clarity. (B) Distribution of degree (number of protein–protein interactions per protein) for basement membrane, other structural ECM, or ECM-associated proteins. Data points are shown as circles; outliers are shown as diamonds. **P<0.01. NS, P≥0.05.

Figure 5.

Localization of glomerular ECM proteins in the HPA database. (A) The HPA was searched for glomerular ECM proteins identified in at least two biologic replicates in this study. (Left) Glomerular immunostaining was reviewed (+, detected; −, not detected; N/A, data not available in the HPA), and (right) localization was determined as GBM, mesangial matrix (MM), Bowman’s capsule (BC), or a combination of these ECM compartments. (B) ECM proteins were categorized as basement membrane, other structural ECM, or ECM-associated proteins, and (right) they were colored and arranged accordingly. Proteins not detected or without data in glomeruli in the HPA are shown separately. Nodes (circles) are labeled with gene names for clarity. (Left) Node diameter (proteins localized in glomeruli in the HPA only) is proportional to the intensity of glomerular immunostaining in the HPA.

Figure 6.

Colocalization of novel and known glomerular ECM proteins. (A and B) Immunohistochemistry of human renal cortex was used to examine the colocalization of collagen VI, TINAGL1, nephronectin, and vitronectin with laminin, (A) collagen IV α3, and (B) collagen IV α1. (C and D) Bar charts show intensity correlation quotients calculated from immunohistochemistry images (n=6–10 images for each analysis), showing (C) colocalization of laminin, collagen VI, and nephronectin with collagen IV α3 and (D) colocalization of collagen VI, TINAGL1, nephronectin, and vitronectin with collagen IV α1.