CLINICAL RESEARCH www.jasn.org Proteomic Analysis Identifies Distinct Glomerular Extracellular Matrix in Collapsing Focal Segmental Glomerulosclerosis Michael L. Merchant,1 Michelle T. Barati,1 Dawn J. Caster ,1 Jessica L. Hata,2 Liliane Hobeika,3 Susan Coventry,2 Michael E. Brier,1 Daniel W. Wilkey,1 Ming Li,1 Ilse M. Rood,4 Jeroen K. Deegens,4 Jack F. Wetzels,4 Christopher P. Larsen,5 Jonathan P. Troost,6 Jeffrey B. Hodgin,7 Laura H. Mariani,8 Matthias Kretzler ,8 Jon B. Klein,1,9 and Kenneth R. McLeish1 Due to the number of contributing authors, the affiliations are listed at the end of this article. ABSTRACT 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 spectrom- etry, 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, ca- thepsin 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, phos- phorylated 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 ca- thepsins may be therapeutic targets. JASN 31: ccc–ccc, 2020. doi: https://doi.org/10.1681/ASN.2019070696 Received July 14, 2019. Accepted April 13, 2020. FSGS is the most common glomerular disorder lead- Published online ahead of print. Publication date available at 1,2 ing to ESKD in North America. FSGS consists of a www.jasn.org. spectrum of histopathology with multiple causes, Correspondence: Dr. Kenneth R. McLeish, Division of Nephrol- various presentations, and different outcomes and ogy and Hypertension, Department of Medicine, University of responses to therapy. The Columbia classification de- Louisville, Baxter I Research Building, Rm 102 South, 570 S. fines five patterns of histopathologic changes Preston Street, Louisville, KY 40202, or Dr. Michael L. Merchant, Division of Nephrology and Hypertension, Department of Med- in FSGS: collapsing variant (collapse of the glo- icine, University of Louisville, Baxter I Research Building, Rm 202 merulartuftwithepithelialcellhyperplasia),tip South, 570 S. Preston Street, Louisville, KY 40202. E-mail: k. variant (lesions at the urinary pole), perihilar var- [email protected] or [email protected] iant (lesions at the vascular pole), cellular variant Copyright © 2020 by the American Society of Nephrology JASN 31: ccc–ccc, 2020 ISSN : 1046-6673/3108-ccc 1 CLINICAL RESEARCH www.jasn.org (endocapillary hypercellularity), and FSGS not otherwise Significance Statement specified (FSGS-NOS) for lesions that do not meet the cri- teria for the other four classes.3 The collapsing FSGS Extracellular matrix (ECM) replaces glomerular capillaries in FSGS. (cFSGS) variant defines a group of patients with more se- To evaluate differences in ECM remodeling between collapsing fi vere nephrotic syndrome, lower eGFR at presentation, and a FSGS (cFSGS) and FSGS not otherwise speci ed (FSGS-NOS), we performed a proteomic analysis of glomerular ECM composition 4–7 higher likelihood of progression to ESKD. Additionally, using human biopsies. Abundance of 58 ECM proteins differed: 41 cFSGS is associated with proliferation of parietal epithelial were more abundant in cFSGS and 17 in FSGS-NOS. Increased cells (PECs) and altered podocyte differentiation.8–10 The abundance and coexpression of cathepsin B, cathepsin C, and an- unique histologic and clinical characteristics suggest the nexin A3 characterized cells infiltrating glomerular tufts in cFSGS. pathophysiology of cFSGS differs from other FSGS variants. These cells expressed markers of activated parietal epithelial cells, fi but not markers of podocytes. This work demonstrates multiple A histopathologic nding common to all variants of FSGS mechanisms of how dysregulated ECM remodeling underlies focal is focal and segmental deposition of new extracellular matrix sclerosis. The work supports the important role of parietal epithelial (ECM) that obliterates glomerular capillaries. ECM is a com- cells in disease histopathology and identifies them as a possible plex molecular structure that provides a physical scaffold for therapeutic target, particularly for cFSGS. all tissues and regulates cell and tissue physiology. ECM com- fi position is tissue speci c and undergoes continuous 13 Halt Protease and Phosphatase Inhibitor [78442; Thermo 11–15 16 remodeling. Proteomic studies by our group and by Fisher Scientific, Waltham, MA]) and stored at 280°C. Lennon et al.17 identified about 250 proteins that comprise the normal glomerular ECM. Although alterations in glomer- Urine Collection ular ECM components occur in FSGS,9,18–21 a comprehensive Single void urine samples were centrifuged at 1200 3 g for analysis of ECM composition and differences among FSGS 15 minutes at 4°C to remove cellular debris. Supernatants were variants has not been performed. Thus, the contribution aliquoted into 15 ml vials and stored at 280°C until used for of abnormal ECM composition to development and pro- sample preparation. Urine was diluted with an equal volume gression of FSGS remains unclear. Based on the unique char- of 0.5% trifluoroacetic acid and then transferred to a Sartorius acteristics of cFSGS, we postulated that glomerular ECM Vivaspin-2 concentrator (Vivaproducts, Littleton, MA) composition in cFSGS differs from the other variants, and with a 5000 Da mol wt cutoff Hydrosart membrane. The those differences may contribute to the unique characteris- Vivaspin-2wasspunat32203 g for 15 minutes at 4°C in an tics of cFSGS. To address this hypothesis, proteomic and Eppendorf 5810R centrifuge. Peptides were concentrated transcriptomic analysis of glomeruli isolated from human and desalted using 1 ml Oasis HLB (Waters Corporation, biopsies was performed. Milford, MA) solid phase extraction cartridges as previously described.22 The ultrafiltrate was rinsed three times using 10 mM HEPES/0.5 mM EDTA, pH 7.6, and recovered by METHODS pipetting. The peptide and protein sample concentrations were determined by determined using a mBCA kit (Pierce, Study Approval Rockford, IL). The University of Louisville Human Studies Committee ap- proved sample collection and use of de-identified samples ECM Enrichment and Protein Extraction provided by coauthors’ institutions. All study subjects pro- Protein extraction was performed using a two-step procedure vided informed consent before sample collection, except previously reported.16 Isolated glomerular sections were de- where samples were deemed exempt. All studies were conduc- cellularized with 25 mM ammonium hydroxide/0.5% Triton ted in compliance with the Declaration of Helsinki. X-100 plus HALT Protease and Phosphatase Inhibitor Cock- tail (Thermo Fisher Scientific) followed by protein extraction Tissue Collection of residual ECM-enriched pellet using an acid-labile surfac- De-identified, formalin-fixed, paraffin-embedded renal tissue tant (0.1% ProteaseMAX Surfactant in 0.05 M ammonium was provided by collaborating pathologists after confirmation bicarbonate) with heating for 5 minutes at 95°C. The isolated of the diagnosis based on the Columbia classification criteria.3 proteins were digested overnight at 37°C with 20 ng Kidney tissue was cut into 10-mm sections on polyethylene sequencing-grade modified trypsin in 100 ml of 1 M urea terephthalate membrane frame slides and stained with Mayer with 0.1 M Tris hydrochloride, pH 8.5. The digests were fil- hematoxylin following Leica LMD6500 Laser Microdissection tered using the YM-10 (10,000 Da mol wt cutoff) device. The System staining protocol. Laser capture microdissection of filtered and digested samples were desalted and concentrated glomeruli was performed such that the Bowman’scapsule using a reversed phase C18 PROTO Ultra MicroSpin desalting was excluded from samples (Supplemental Figure 1). Between column (Nest Group, Southborough, MA). The recovered 38 and 124 glomerular sections from 12 to 26 glomeruli for peptides were lyophilized and resuspended in 2% acetoni- each subject were collected into tubes containing 20 ml trile/0.1% formic acid before quantification using a Nano- of storage buffer (10 mM HEPES pH 7.0, 0.67 mM EDTA, Drop 2000 (Thermo Fisher Scientific) at 205 nm. 2 JASN JASN 31: ccc–ccc,2020 www.jasn.org