Proteomic Analysis Identifies Distinct Glomerular Extracellular Matrix in Collapsing Focal Segmental Glomerulosclerosis
Authors: Merchant Michael L. , Barati Michelle T. , Caster Dawn J. , Hata Jessica L. , Hobeika Liliane , Coventry Susan , Brier Michael E. , Wilkey Daniel W. , Li Ming , Rood Ilse M. , Deegens Jeroen K. , Wetzels Jack F. , Larsen Christopher P. , Troost Jonathan P. , Hodgin Jeffrey B. , Mariani Laura H. , Kretzler Matthias , Klein Jon B. , McLeish Kenneth R. 
Published: June 2020

Significance Statement
Extracellular matrix (ECM) replaces glomerular capillaries in FSGS. To evaluate differences in ECM remodeling between collapsing FSGS (cFSGS) and FSGS not otherwise specified (FSGS-NOS), we performed a proteomic analysis of glomerular ECM composition using human biopsies. Abundance of 58 ECM proteins differed: 41 were more abundant in cFSGS and 17 in FSGS-NOS. Increased abundance and coexpression of cathepsin B, cathepsin C, and annexin A3 characterized cells infiltrating glomerular tufts in cFSGS. These cells expressed markers of activated parietal epithelial cells, but not markers of podocytes. This work demonstrates multiple mechanisms of how dysregulated ECM remodeling underlies focal sclerosis. The work supports the important role of parietal epithelial cells in disease histopathology and identifies them as a possible therapeutic target, particularly for cFSGS.

Background
The mechanisms leading to extracellular matrix (ECM) replacement of areas of glomerular capillaries in histologic variants of FSGS are unknown. This study used proteomics to test the hypothesis that glomerular ECM composition in collapsing FSGS (cFSGS) differs from that of other variants.

Methods
ECM proteins in glomeruli from biopsy specimens of patients with FSGS not otherwise specified (FSGS-NOS) or cFSGS and from normal controls were distinguished and quantified using mass spectrometry, verified and localized using immunohistochemistry (IHC) and confocal microscopy, and assessed for gene expression. The analysis also quantified urinary excretion of ECM proteins and peptides.

Results
Of 58 ECM proteins that differed in abundance between cFSGS and FSGS-NOS, 41 were more abundant in cFSGS and 17 in FSGS-NOS. IHC showed that glomerular tuft staining for cathepsin B, cathepsin C, and annexin A3 in cFSGS was significantly greater than in other FSGS variants, in minimal change disease, or in membranous nephropathy. Annexin A3 colocalized with cathepsin B and C, claudin-1, phosphorylated ERK1/2, and CD44, but not with synaptopodin, in parietal epithelial cells (PECs) infiltrating cFSGS glomeruli. Transcripts for cathepsins B and C were increased in FSGS glomeruli compared with normal controls, and urinary excretion of both cathepsins was significantly greater in cFSGS compared with FSGS-NOS. Urinary excretion of ECM-derived peptides was enhanced in cFSGS, although in silico analysis did not identify enhanced excretion of peptides derived from cathepsin B or C.

Conclusions
ECM differences suggest that glomerular sclerosis in cFSGS differs from that in other FSGS variants. Infiltration of activated PECs may disrupt ECM remodeling in cFSGS. These cells and their cathepsins may be therapeutic targets.