BASIC RESEARCH www.jasn.org

Global Analysis Reveals the Complexity of the Human Glomerular

† † Rachel Lennon,* Adam Byron,* Jonathan D. Humphries,* Michael J. Randles,* † ‡ † | Alex Carisey,* Stephanie Murphy,* David Knight, Paul E. Brenchley, Roy Zent,§ and Martin J. Humphries*

*Wellcome Trust Centre for Cell-Matrix Research and ‡Biological Mass Spectrometry Core Facility, Faculty of Life Sciences, and †Faculty of Medical and Human Sciences, University of Manchester, Manchester, United Kingdom; §Division of Nephrology, Department of Medicine, Vanderbilt University Medical Center, Nashville, Tennessee; and |Department of Medicine, Veterans Affairs Hospital, Nashville, Tennessee

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 . This catalog includes all previously identified glomerular components plus many new and abundant components. Relative quantification showed a dominance of IV, collagen I, and isoforms in the glomerular ECM together with abundant collagen VI and TINAGL1. Protein net- work 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 impor- tance 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.

J Am Soc Nephrol 25: 939–951, 2014. doi: 10.1681/ASN.2013030233

The glomerulus is a sophisticated organelle com- In addition to operating as a signaling platform, prisinguniquecellularandextracellularmatrix(ECM) ECM provides a structural scaffold for adjacent cells components. Fenestrated capillary endothelial cells and has a tissue-specific molecular composition.3,4 and overlying podocytes are separated by a special- ized glomerular (GBM), and Received March 11, 2013. Accepted September 10, 2013. these three components together form the filtration Published online ahead of print. Publication date available at barrier. Mesangial cells and their associated ECM, www.jasn.org. the mesangial matrix, exist between adjacent capil- Present address: Adam Byron, Edinburgh Cancer Research lary loops and maintain the three-dimensional or- United Kingdom Centre, Institute of Genetics and Molecular ganizationof the capillary bundle.Inturn,theparietal Medicine, University of Edinburgh, Edinburgh, United Kingdom. epithelial cells and ECM of Bowman’s capsule enclose Correspondence: Dr. Rachel Lennon, Wellcome Trust Centre for this network of capillaries. Cells adhere to ECM pro- Cell-Matrix Research, Michael Smith Building, University of teins by adhesion receptors, and these interactions are Manchester, Manchester M13 9PT, United Kingdom. Email: Rachel. required to maintain intact barrier function of the [email protected] glomerulus.1,2 Copyright © 2014 by the American Society of Nephrology

J Am Soc Nephrol 25: 939–951, 2014 ISSN : 1046-6673/2505-939 939 BASIC RESEARCH www.jasn.org

Candidate-based investigations of glomerular ECM have fo- Therefore, to interrogate this complexity effectively, a systems- cused on the GBM and shown that it resembles the typical level understanding of glomerular ECM is required. To address basal lamina found in multicellular organisms, containing a the need for a global analysis of the extracellular environment core of (collagen IV, , and nidogens) within the glomerulus, we used mass spectrometry (MS)-based and (agrin, , and colla- proteomics of glomerular ECM fractions to define the human gen XVIII).5 Mesangial and parietal cell ECMs have been less glomerular ECM proteome. well investigated; nonetheless, they are also thought to contain similar core components in addition to other glycoproteins, including fibronectin.6,7 Thus, the glomerulus consists of a RESULTS combination of condensed ECM within the GBM and Bow- man’s capsule and loose ECM supporting the mesangial cells. Isolation of Glomerular ECM The ECM compartments in the glomerulus are thought to Using purified isolates of human glomeruli (Figure 1A), we be distinct and exhibit different functional roles. The GBM is developed a fractionation approach to collect glomerular integral to the capillary wall and therefore, functionally linked ECM proteins (Figure 1B). Before homogenization and solu- to glomerular filtration.5 Mutations of tissue-restricted iso- bilization, extracted glomeruli appeared acellular, suggestive forms of collagen IV (COL4A3, COL4A4,andCOL4A5)and of successful ECM enrichment (Figure 1A). Western blotting laminin (LAMB2), which are found in the GBM, cause signif- confirmed enrichment of ECM proteins (collagen IVand lam- icant barrier dysfunction and ultimately, renal failure.8,9 Less inin) and depletion of cytoplasmic and nuclear proteins is understood about the functions of mesangial and parietal (nephrin, actin, and lamin B1) in the ECM fraction (Figure cell ECMs, although expansion of the mesangial compartment 1C). Glomerular ECM fractions from three adult human kid- is a histologic pattern seen across the spectrum of glomerular neys were then extracted and analyzed by MS. disease.10 Compositional investigation of the distinct glomerular Ontology Enrichment Analysis of ECM Fractions ECM compartments is limited by the technical difficulties of All proteins identified by MS were allocated to categories separation. Early investigations of GBM constituents used the according to their (GO) assignment, and the relative insolubility of ECM proteins to facilitate separation enrichment of GO terms in the dataset was assessed using GO from cellular proteins in the glomerulus but did not separate enrichment analysis, which is described in Supplemental the GBM from mesangial and parietal cells ECMs.11,12 Re- Methods. The network of enriched GO terms clustered into cently, studies incorporating laser microdissection of glomer- three subnetworks, and the largest, most confidently identified ular sections have been coupled with proteomic analyses.13,14 subnetwork was for ECM-related GO terms (Figure 2A). A These studies report both cellular and ECM components and large majority of other proteins were included in two addi- typically require pooled material from glomerular sections to tional subnetworks representing mitochondrial and cytoskel- improve protein identification. The ability of laser microdis- etal GO terms, and the presence of these proteins in the ECM section to separate glomerular ECM compartments has not yet fractions is likely to be caused by the strength of their inter- been tested; however, this approach will be limited by the molecular interactions with ECM proteins. Spectral counting amount of protein that is possible to retrieve. To achieve was used to determine the enrichment of ECM in each of the good coverage of ECM proteins within a tissue, proteomic four fractions collected from isolated human glomeruli. There studies need to combine a reduction in sample complexity was 38% enrichment of extracellular proteins in the glomer- with maximal protein quantity. Currently, the inability to sep- ular ECM fractions (Figure 2B), and this result compares arate glomerular ECM compartments in sufficient quantity favorably with the 12%–30% enrichment of ECM proteins re- is a limitation that prohibits proteomic studies of these struc- ported in comparable proteomic studies.15 tures; however, for other tissues, proteomic analysis of ECM has been achieved by enrichment of ECM combined with sam- Relative Abundance of Glomerular ECM Proteins ple fractionation.15 To determine the relative abundance of glomerular ECM Although the composition of the ECM in other tissues has proteins, we used MS to analyze additional glomerular ECM been addressed using proteomic approaches,15 studies of glo- fractions from three human kidneys, and we quantified the merular ECM to date have used candidate-based technologies. proteins using a intensity approach,20 which is de- These studies have identified key molecular changes during scribed in Supplemental Methods. Using this analysis, the 10 development and disease and highlighted the compositional most abundant proteins were a combination of , lam- and organizational dynamics of glomerular ECM. Nonethe- inin 521, and heparan sulfate proteoglycans (Figure 2C, Sup- less, the extracellular environment within the glomerulus is plemental Table 1), which have all previously been reported in the setting for a complex series of interactions between both the glomerulus. In addition, we found collagen VI and the structural ECM proteins and ECM-associated proteins, such TINAGL1 among the 10 most abundant pro- as growth factors16–18 and proteases,19 which together pro- teins, and the detection of these two components was con- vide a specialized niche to support glomerular cell function. firmed by Western blotting (Figure 2D). Relative quantification

940 Journal of the American Society of Nephrology J Am Soc Nephrol 25: 939–951, 2014 www.jasn.org BASIC RESEARCH

further categorized as basement membrane proteins (Table 1), other structural ECM proteins (Table 2), or ECM-associated pro- teins (Supplemental Table 2). There are a number of published glomerular proteo- mic studies,21–25 and a comparison was made with protein identifications in our dataset (Figure 3). Of two published studies for which complete MS datasets were avail- able,22,25 neither study enriched for glo- merular ECM or analyzed more than one biologic replicate. We detected all 15 ECM proteins reported by Yoshida et al.22 (which represents 8% of ECM proteins detected in this study) and 76% of 91 ECM proteins reported by Cui et al.25 (which represents 39% of ECM proteins detected in this study) based on identification in one bio- logic replicate, which was reported in these published studies. Furthermore, we identi- fied12timesasmanyECMproteinsas Yoshida et al.22 and 2 times as many ECM proteins as Cui et al.25 Therefore, our study yielded the greatest number of ECM pro- teins from glomeruli to date, likely owing to the use of effective ECM enrichment be- fore state-of-the-art MS analysis.

Creation of a Protein Interaction Network To visualize the components of glomerular ECM as a network of interacting proteins, the identified proteins were mapped onto a Figure 1. Isolation of enriched glomerular ECM. (A) Human glomeruli were isolated by curated protein interaction database (Sup- differential sieving, yielding .95% purity. Before homogenization, glomeruli appeared plemental Methods) to generate an inter- acellular (right). (B) A proteomic workflow for the isolation of enriched glomerular ECM by action network (Figure 4A). Statistical fractionation (details in Concise Methods). (C) Coomassie staining and Western blotting analysis showed that the network was – (WB) of fractions 1 3 and the ECM fraction probing for the extracellular proteins with more clustered than expected by chance, pancollagen IV and panlaminin probes and the intracellular proteins nephrin, actin, and indicative of preferential interaction of pro- lamin B1. M, molecular mass marker. teins in a nonrandom network topology (Supplemental Table 3). Interestingly, base- ment membrane and structural ECM pro- was also performed using spectral counting, which was compared teins were involved in more interactions with other proteins in with the peptide intensity approach, and further showed that the network than ECM-associated proteins (Figure 4B). Topolog- collagen VI and TINAGL1 were among the most abundant pro- ical network analysis confirmed that basement membrane and teins detected in the glomerular ECM (Supplemental Figure 2). other structural ECM proteins formed a highly connected core subnetwork, whereas ECM-associated proteins were less clustered Defining the Glomerular ECM Proteome in the network (Supplemental Figures 3 and 4). These data sug- Using the GO classification of extracellular region proteins and gest that structural ECM proteins mediate multiple sets of pro- crossreferencing with the human matrisome project3 (de- tein–protein interactions and thus, have important roles in the scribed in Supplemental Methods), we identified 144 extracel- assembly and organization of glomerular ECM. lular proteins in the glomerular ECM, including all previously known glomerular ECM components. Only proteins identi- Localization of Glomerular ECM Proteins fied in at least two of three biologic replicate analyses were Validation of protein expression was performed by searching included in the dataset, and identified ECM proteins were the Human Protein Atlas (HPA) database26 for 144 glomerular

J Am Soc Nephrol 25: 939–951, 2014 Proteome of the Glomerular Matrix 941 BASIC RESEARCH www.jasn.org

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.

ECM proteins identified in this study (Figure 5). Glomerular a5andlaminina5. Protein expression was confirmed for 78 immunostaining was not available for 20 proteins, including ECM components (63% of proteins with available HPA data) the known glomerular ECM proteins collagen IV a3, a4, and and reported as negative for 46 ECM components, although

942 Journal of the American Society of Nephrology J Am Soc Nephrol 25: 939–951, 2014 www.jasn.org BASIC RESEARCH

Table 1. Basement membrane proteins in the glomerular ECM proteome Basement Membrane Proteins Gene Name Molecular Mass (kDa) Abundance (nSC) Classification Agrin AGRN 215 3.075 Glycoprotein Collagen a1(XV) chain COL15A1 142 0.052 Collagen Collagen a1(XVIII) chain COL18A1 154 5.230 Collagen Collagen a1(IV) chain COL4A1 161 12.515 Collagen Collagen a2(IV) chain COL4A2 168 17.709 Collagen Collagen a3(IV) chain COL4A3 162 8.155 Collagen Collagen a4(IV) chain COL4A4 164 8.961 Collagen Collagen a5(IV) chain COL4A5 161 3.778 Collagen Collagen a6(IV) chain COL4A6 164 0.589 Collagen Fibulin-1 FBLN1 77 0.029 Glycoprotein Fibrillin-1 FBN1 312 1.403 Glycoprotein Fibronectin FN1 263 1.893 Glycoprotein ECM protein FRAS1 FRAS1 443 0.039 Glycoprotein Hemicentin-1 HMCN1 613 0.007 Glycoprotein Perlecan HSPG2 467 7.012 Laminin subunit a2 LAMA2 343 0.043 Glycoprotein Laminin subunit a5 LAMA5 400 9.482 Glycoprotein Laminin subunit b1 LAMB1 198 0.977 Glycoprotein Laminin subunit b2 LAMB2 196 14.745 Glycoprotein Laminin subunit g1 LAMC1 178 8.818 Glycoprotein Nidogen-1 NID1 136 10.247 Glycoprotein Nidogen-2 NID2 151 1.249 Glycoprotein Tubulointerstitial nephritis antigen TINAG 55 12.873 Glycoprotein von Willebrand factor A domain–containing protein 1 VWA1 47 1.171 Glycoprotein The glomerular ECM proteome was further categorized according to GO annotation. Twenty-four basement membrane proteins were identified by MS. Relative protein abundance is shown as normalized spectral counts (nSCs).

Table 2. Other structural ECM proteins in the glomerular ECM proteome Other Structural ECM Proteins Gene Name Molecular Mass (kDa) Abundance (nSC) Classification Asporin ASPN 44 1.199 Proteoglycan BGN 42 2.268 Proteoglycan Collagen a1(XII) chain COL12A1 333 0.073 Collagen Collagen a1(I) chain COL1A1 139 0.253 Collagen Collagen a2(I) chain COL1A2 129 0.594 Collagen Collagen a1(III) chain COL3A1 139 0.143 Collagen Collagen a1(VI) chain COL6A1 109 12.685 Collagen Collagen a2(VI) chain COL6A2 109 5.889 Collagen Collagen a3(VI) chain COL6A3 344 11.145 Collagen DCN 40 0.067 Proteoglycan Dermatopontin DPT 24 0.397 Glycoprotein EMILIN-1 EMILIN1 107 0.699 Glycoprotein Fibrinogen a-chain FGA 95 0.954 Glycoprotein Fibrinogen b-chain FGB 56 1.965 Glycoprotein g-A of fibrinogen g-chain FGG 49 2.002 Glycoprotein MGP 12 0.557 Glycoprotein Nephronectin NPNT 65 3.707 Glycoprotein Periostin, osteoblast-specific factor POSTN 90 0.191 Glycoprotein RPE-spondin RPESP 30 1.364 Glycoprotein TGF-b–induced protein ig-h3 TGFBI 75 0.369 Glycoprotein Tubulointerstitial nephritis antigen-like TINAGL1 52 20.010 Glycoprotein VTN 54 9.015 Glycoprotein von Willebrand factor A domain–containing protein 5B2 VWA5B2 133 0.024 Glycoprotein von Willebrand factor A domain–containing protein 8 VWA8 211 0.076 Glycoprotein The glomerular ECM proteome was categorized according to GO annotation. Twenty-four proteins were identified as having a structural role but without basement membrane association, and they were termed other structural ECM proteins. Relative protein abundance is shown as normalized spectral counts (nSCs).

J Am Soc Nephrol 25: 939–951, 2014 Proteome of the Glomerular Matrix 943 BASIC RESEARCH www.jasn.org

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.25 and Yoshida et al.22). 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 re- ported one biologic replicate. Large node size indicates proteins detected in at least two biologic replicates in this study. there were notable false negatives, including collagen IV a2, more than one ECM compartment (Figure 5B), suggesting a collagen XVIII, and laminin b1. Immunohistochemistry relies common core of protein components between the glomerular on antibody specificity, and therefore, combining expression ECM compartments. data from antibody-based investigations with MS data has the potential to significantly increase the number of protein identi- Colocalization of Novel Glomerular ECM Proteins fications. Indeed, our proteomic dataset increased the number of To confirm the expression of new glomerular ECM proteins, ECM proteins detected in the glomerulus by 59% compared which were either abundant in our MS analysis or not detected with HPA data alone. We also evaluated the HPA database to in the HPA database, we conducted colocalization studies. determine the pattern of immunostaining as GBM, mesangial Collagen VI and TINAGL1 were among the most abundant matrix, Bowman’s capsule, or a combination of compartments proteins detected in this study (Supplemental Figure 2), and (Supplemental Methods). Most components were present in they were validated by Western blotting (Figure 2D). Collagen

944 Journal of the American Society of Nephrology J Am Soc Nephrol 25: 939–951, 2014 www.jasn.org BASIC RESEARCH

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 inter- actions. 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.

VI was also present in the highly connected subnetwork of known glomerular ECM proteins in addition to many poten- ECM proteins (Supplemental Figures 3 and 4). Immunohisto- tially novel components. Moreover, using immunohistochem- chemical and correlation intensity analysis of human renal cortex istry, we have confirmed the MS identification of TINAGL1, revealed colocalization of TINAGL1 with collagen IV a1, which collagen VI, nephronectin, and vitronectin within specific has a mesangial pattern of immunostaining, whereas collagen VI glomerular compartments. These findings show that the com- overlapped with both collagen IV a1andcollagenIVa3/lam- position of glomerular ECM is far more complex than previously inin, which both have a GBM pattern of immunostaining (Figure appreciated and imply that many more ECM components may 6, A and C). MS analysis also detected nephronectin and vitro- contribute to glomerular development and disease processes. nectin (Table 2). Correlation intensity analysis showed that neph- This unbiased, global approach to define the glomerular ronectin localized in the GBM and mesangial matrix, whereas ECM proteome shows that this specialized ECM has a core of vitronectin localized in the mesangial matrix alone (Figure 6, B highly connected extracellular components. In adult human and D). This objective and quantitative method of colocalization glomeruli, 144 ECM proteins, including all previously de- allows protein expression to be correlated with reliable markers scribed components, were identified. In addition, we found predominating in distinct ECM compartments. With antibodies many more structural and regulatory ECM proteins, revealing of suitable specificity, this approach could be extended to map the complexity of the glomerular ECM. This proteome is the full proteome into glomerular ECM compartments. comparable in size with ECM profiles recently reported for vasculature-, lung-, and bowel-derived ECMs.3,27,28 In our analysis, several novel glomerular proteins were highly abun- DISCUSSION dant, including collagen VI and TINAGL1. Collagen VI is a structural component that forms microfibrils, and it is impor- We used an ECM enrichment strategy coupled with an tant for muscle function29,30; however, its role in the glomer- unbiased proteomics approach to confirm the presence of all ulus has not been investigated. Our study shows that collagen

J Am Soc Nephrol 25: 939–951, 2014 Proteome of the Glomerular Matrix 945 BASIC RESEARCH www.jasn.org

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; 2, 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.

VI is present in a highly connected core network of proteins in angiogenesis.33 However, its function within the glomerulus is the glomerular ECM, and it is localized within both the GBM undefined. Both of these highly abundant proteins may have and mesangial matrix. TINAGL1 was also abundantly ex- roles in barrier function or glomerular disease. pressed in glomerular ECM, where it predominantly localized To assess the relative abundance of ECM proteins, we used to the mesangial matrix. TINAGL1 (also known as TIN-Ag_RP, two distinct methods of quantification (peptide intensity lipocalin-7, oxidised LDL-responsive gene 2 and androgen reg- analysis20 and spectral counting), and both approaches gave ulated gene 1) is a glycoprotein, and it is structurally related to very similar results. Peptide intensity analysis revealed collagen TINAG, a tubular basement membrane component, which is IV and laminin isoforms to be the most abundant proteins in the antigenic target in autoimmune anti–tubular basement the glomerular ECM, and both analyses revealed abundant membrane disease.31 TINAGL1 has a proteolytically inactive collagen VI and TINAGL1. There were some differences between cathepsin domain,32 and it has been shown to have a role in the methods for the quantification of collagen isoforms, but the

946 Journal of the American Society of Nephrology J Am Soc Nephrol 25: 939–951, 2014 www.jasn.org BASIC RESEARCH

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 a3, and (B) collagen IV a1. (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 a3 and (D) colocalization of collagen VI, TINAGL1, nephronectin, and vitronectin with collagen IV a1. stoichiometry of different proteins is difficult to determine by discovery of unknown ECM proteins. Therefore, the findings any global proteomic methodology, including antibody-based presented in this study provide relative patterns of glomerular techniques, which rely on probe immunoaffinity. To perform ECM protein enrichment and pave the way for additional in- absolute protein quantification by MS, labeled peptide standards depth studies of the ECM components identified. for each protein could be used; however, it would be a significant Compared with other large-scale glomerular proteomic studies, undertaking for a large protein dataset and would not permit the which have identified up to a total of 1800 proteins,22,25 this

J Am Soc Nephrol 25: 939–951, 2014 Proteome of the Glomerular Matrix 947 BASIC RESEARCH www.jasn.org investigation had the greatest yield of ECM proteins, thus high- were chosen to reduce the influence of age and sex. Normal renal lighting the importance of sample fractionation for improving functionwas determined by prenephrectomy serum creatinine values. protein identification. However, the large-scale requirement At 4°C, renal cortex (2.5 g) was finely diced and pressed onto a 250-mm for the analysis prohibited the separation of glomerular ECM sieve (Endecotts, London, UK) using a 5-ml syringe plunger. Glomer- components, and therefore, protein localization was deter- uli were rinsed through sieves (250 and 150 mm) with cold PBS and mined by immunohistochemistry. Although laser microdissec- separated from tubular fragments by collection on both 150- and 100-mm tion studies have the ability to precisely define the anatomic sieves. Retained glomeruli were retrieved into 10 ml PBS and washed structure for analysis, the total number of proteins identified in another three times with PBS and interval centrifugation. Glomerular the most recent of these studies, using a variety of tissue sam- purity was consistently .95% as determined by counting whole glo- ples, ranged from 114 to 340, which is fewer proteins than the meruli and nonglomerular fragments using phase-contrast light large-scale glomerular studies and significantly fewer ECM pro- microscopy. teins compared with this study.13,34,35 Future developments in technologies, which may couple tissue imaging with improved Isolation of Enriched Glomerular ECM sensitivity of molecular analysis, may allow direct compositional This method was adapted from previously published methods38 and analysis of ECM within distinct tissue compartments.36,37 used to reduce the complexity of protein samples for MS analysis by The profile of glomerular ECM that we have described is removing cellular components and enriching for ECM proteins. All likely to represent a snapshot of a highly dynamic extracellular steps were carried out at 4°C to minimize proteolysis. Pure glomer- environment that changes under the influence of cellular, ular isolates from three human kidneys were incubated for 30 min- physical, and chemical environmental cues. Nonetheless, the utes in extraction buffer (10 mM Tris, 150 mM NaCl, 1% [vol/vol] datasets provide a valuable resource for additional investigation Triton X-100, 25 mM EDTA, 25 mg/ml leupeptin, 25 mg/ml aprotinin, of the composition and complexity of glomerular ECM. Our and 0.5 mM 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochlo- methodology can now be applied to the comparison of ride) to solubilize cellular proteins, and samples were then centri- glomerular ECMs in vivo in the context of development or fuged at 14,0003g for 10 minutes to yield fraction 1. The remaining disease. Systems-level analysis will be integral to the down- pellet was incubated for 30 minutes in alkaline detergent buffer (20 stream interrogation of data to identify informative, predic- mM NH4OH and 0.5% [vol/vol] Triton X-100 in PBS) to further tive networks of ECM composition and function. Combined solubilize cellular proteins and disrupt cell–ECM interactions. Sam- with the methodologies and datasets described herein, such ples were then centrifuged at 14,0003g for 10 minutes to yield frac- analyses will enable the construction of a dynamic glomerular tion 2. The remaining pellet was incubated for 30 minutes in a ECM interactome and help to build understanding of how deoxyribonuclease buffer (10 mg/ml deoxyribonuclease I [Roche, this network may alter during glomerular development and Burgess Hill, UK] in PBS) to degrade DNA. The sample was centri- disease. fuged at 14,0003g for 10 minutes to yield fraction 3, and the final pellet was resuspended in reducing sample buffer (50 mM TriszHCl, pH 6.8, 10% [wt/vol] glycerol, 4% [wt/vol] SDS, 0.004% [wt/vol] CONCISE METHODS bromophenol blue, and 8% [vol/vol] b-mercaptoethanol) to yield the ECM fraction. Samples were heat denatured at 70°C for 20 Antibodies minutes. Monoclonal antibodies used were against actin (clone AC-40; Sigma- Aldrich, Poole, UK), nephronectin (ab64419; Abcam, Cambridge, Western Blotting UK), TINAGL1 (ab69036; Abcam), vitronectin (ab11591; Abcam), After SDS-PAGE, resolved proteins were transferred to nitrocellulose and collagen IV chain–specific antibodies (provided by B. Hudson, membrane (Whatman, Maidstone, UK). Membranes were blocked Vanderbilt University Medical Center, Nashville, TN). Polyclonal anti- with casein blocking buffer (Sigma-Aldrich) and probed with primary bodies used were against pancollagen IV (ab6586; Abcam), panlaminin antibodies diluted in blocking buffer containing 0.05% (vol/vol) (ab11575; Abcam), pancollagen VI (ab6588; Abcam), lamin B1 Tween 20. Membranes were washed with Tris-buffered saline (10 mM (ab16048; Abcam), and nephrin (ab58968; Abcam). Secondary antibod- TriszHCl, pH 7.4, and 150 mM NaCl) containing 0.05% (vol/vol) ies against rabbit IgG conjugated to tetramethylrhodamineisothiocyanate Tween 20 and incubated with species-specific fluorescent dye– and mouse or rat IgG conjugated to FITC (Jackson ImmunoResearch conjugated secondary antibodies diluted in blocking buffer contain- Laboratories, Inc., West Grove, PA) were used for immunofluorescence; ing 0.05% (vol/vol) Tween 20. Membranes were washed in the dark secondary antibodies conjugated to Alexa Fluor 680 (Life Technologies, and then scanned using the Odyssey infrared imaging system (LI- Paisley, UK) or IRDye 800 (Rockland Immunochemicals, Glibertsville, COR Biosciences, Cambridge, UK) to visualize bound antibodies. PA) were used for Western blotting. MS Data Acquisition Isolation of Human Glomeruli Protein samples were resolved by SDS-PAGE and visualized by Normal renal cortex from human donor kidneys technically unsuit- Coomassie staining. Gel lanes were sliced and subjected to in-gel trypsin able for transplantation was used with full ethical approval (reference digestion asdescribed previously39 Liquid chromatography–tandem MS 06/Q1406/38). Three adult men donors between 37 and 63 years of age analysis was performed using a nanoACQUITY UltraPerformance

948 Journal of the American Society of Nephrology J Am Soc Nephrol 25: 939–951, 2014 www.jasn.org BASIC RESEARCH liquid chromatography system (Waters, Elstree, UK) coupled online through MetaVue software (Molecular Devices, Wokingham, UK). to an LTQVelos mass spectrometer (Thermo Fisher Scientific, Waltham, Images were collected using a CoolSnap HQ camera (Photometrics, MA) or offline to an Orbitrap Elite analyzer (Thermo Fisher Scientific) Tucson, AZ) and separate 49,6-diamidino-2-phenylindole, FITC, and for experiments incorporating analysis with Progenesis (Supple- Cy3 filters (U-MWU2, 41001, and 41007a, respectively; Chroma, mental Methods). were concentrated and desalted on a Olching, Germany) to minimize bleed through between the differ-

Symmetry C18 preparative column (20-mm length, 180-mminner ent channels. Images were processed and analyzed using Fiji/ImageJ diameter, 5-mm particle size, 100-Å pore size; Waters). Peptides were software (version 1.46r; National Institutes of Health, Bethesda, separated on a bridged ethyl hybrid C18 analytical column (250-mm MD). Raw images were subjected to signal rescaling using linear length, 75-mm inner diameter, 1.7-mm particle size, 130-Å pore size; transformation for display in the figures. For calculation of the Waters) using a 45-minute linear gradient from 1% to 25% (vol/vol) Pearson’scorrelationcoefficient, a region of interest was drawn, acetonitrile in 0.1% (vol/vol) formic acid at a flow rate of 200 nl/min. and a threshold was set to restrict analysis to a single glomerulus. Peptides were selected for fragmentation automatically by data- The coefficient was measured using the Intensity Correlation Analysis dependent analysis. plugin for Fiji/ImageJ,44 and the subsequent plotting steps were per- formed using MATLAB (version R2012a; MathWorks, Natick, MA). MS Data Analyses Tandem mass spectra were extracted using extract_msn (Thermo Statistical Analyses 6 Fisher Scientific) executed in Mascot Daemon (version 2.2.2; Matrix All measurements are shown as mean SEM. Box plots indicate 25th 3 Science, London, UK). Peak list files were searched against a modified and 75th percentiles (lower and upper bounds, respectively), 1.5 in- version of the International Protein Index Human Database (version terquartile range (whiskers), and median (black line). Numbers of pro- – – 3.70; release date, March 4, 2010) containing 10 additional contam- tein protein interactions were compared using Kruskal Wallis one-way inant and reagent sequences of nonhuman origin using Mascot ANOVA tests with post hoc Bonferroni correction. GO enrichment anal- fi ’ – (version 2.2.03; Matrix Science).40 Carbamidomethylation of cysteine yses were compared using modi ed Fisher sexacttestswithBenjamini , fi was set as a fixed modification; oxidation of and hydrox- Hochberg correction. P values 0.05 were deemed signi cant. ylation of proline and lysine were allowed as variable modifications. Only tryptic peptides were considered, with up to one missed cleav- age permitted. Monoisotopic precursor mass values were used, and ACKNOWLEDGMENTS only doubly and triply charged precursor ions were considered. Mass tolerances for precursor and fragment ions were 0.4 and 0.5 Da, Mass spectrometry was performed in the Biological Mass Spec- respectively. MS datasets were validated using rigorous statistical trometry Core Facility, Faculty of Life Sciences, University of Man- algorithms at both the peptide and protein levels41,42 implemented chester, and we thank Stacey Warwood for advice and technical in Scaffold (version 3.00.06; Proteome Software, Portland, OR). Pro- support. We thank Julian Selley for bioinformatic support. Micro- tein identifications were accepted on assignment of at least two scopy was performed in the Bioimaging Core Facility, Faculty of Life unique validated peptides with $90% probability, resulting in Sciences, University of Manchester. The collagen IV chain-specific $99% probability at the protein level. These acceptance criteria re- antibodies were kindly provided by the Billy Hudson research group, sulted in an estimated protein false discovery rate of 0.1% for all Department of Medicine, Vanderbilt Medical Center. datasets. This work was supported by Wellcome Trust Intermediate Fel- lowship Award Reference 090006 (to R.L.) and Wellcome Trust Grant MS Quantification and Proteomic Analyses 092015(to M.J.H.). R.Z. is supported byVeteransAffairs Merit Awards MS quantification and proteomic data analyses were performed as 1I01BX002196-01, DK075594, DK069221, and DK083187, and an previously described39,43 with the modifications described in Supple- American Heart Association Established Investigator Award. The mass mental Methods. The mass spectrometry proteomics data have been spectrometer and microscopes used in this study were purchased with deposited to the ProteomeXchange Consortium with the dataset grantsfromtheBiotechnologyandBiologicalSciencesResearchCouncil, identifier PXD000456 (http://www.ebi.ac.uk/pride). the Wellcome Trust, and the University of Manchester Strategic Fund.

Immunohistochemistry and Image Analysis DISCLOSURES Formalin-fixed, paraffin-embedded tissue blocks were sectioned at 5 mm. Sections were dewaxed and treated with recombinant protein- None. ase K (Roche Diagnostics, Indianapolis, IN) for 15 minutes. Sections were blocked with 5% (vol/vol) donkey serum (Sigma-Aldrich) and REFERENCES 1.5% (vol/vol) BSA (Sigma-Aldrich) for 30 minutes and primary antibodies overnight at 4°C. Sections were washed three times with PBS, incubated with secondary antibodies, mounted with polyvinyl 1. Pozzi A, Jarad G, Moeckel GW, Coffa S, Zhang X, Gewin L, Eremina V, Hudson BG, Borza DB, Harris RC, Holzman LB, Phillips CL, Fassler R, alcohol mounting medium (Fluka 10981; Sigma-Aldrich), and im- Quaggin SE, Miner JH, Zent R: Beta1 expression by podocytes aged using a BX51 upright microscope (Olympus, Southend on Sea, is required to maintain glomerular structural integrity. Dev Biol 316: UK) equipped with a 203 UPlan Fln 0.50 objective and controlled 288–301, 2008

J Am Soc Nephrol 25: 939–951, 2014 Proteome of the Glomerular Matrix 949 BASIC RESEARCH www.jasn.org

2. Has C, Spartà G, Kiritsi D, Weibel L, Moeller A, Vega-Warner V, Waters membrane. Composition and organization of type IV collagen chains, A, He Y, Anikster Y, Esser P, Straub BK, Hausser I, Bockenhauer D, Dekel and the linkage of alpha3(IV) and alpha5(IV) chains. JBiolChem272: B, Hildebrandt F, Bruckner-Tuderman L, Laube GF: Integrin a3muta- 17023–17032, 1997 tions with kidney, lung, and skin disease. NEnglJMed366: 1508– 20. Silva JC, Gorenstein MV, Li GZ, Vissers JP, Geromanos SJ: Absolute 1514, 2012 quantification of proteins by LCMSE: A virtue of parallel MS acquisition. 3. Naba A, Clauser KR, Hoersch S, Liu H, Carr SA, Hynes RO: The mat- MolCellProteomics5: 144–156, 2006 risome: In silico definition and in vivo characterization by proteomics of 21. Xu BJ, Shyr Y, Liang X, Ma LJ, Donnert EM, Roberts JD, Zhang X, Kon V, normal and tumor extracellular matrices. Mol Cell Proteomics 11: Brown NJ, Caprioli RM, Fogo AB: Proteomic patterns and prediction of M111.014647, 2012 glomerulosclerosis and its mechanisms. JAmSocNephrol16: 2967– 4. Yurchenco PD: Basement membranes: Cell scaffoldings and signaling 2975, 2005 platforms. Cold Spring Harb Perspect Biol 3: pii: a004911, 2011 22. Yoshida Y, Miyazaki K, Kamiie J, Sato M, Okuizumi S, Kenmochi A, 5. Miner JH: The glomerular basement membrane. Exp Cell Res 318: 973– Kamijo K, Nabetani T, Tsugita A, Xu B, Zhang Y, Yaoita E, Osawa T, 978, 2012 Yamamoto T: Two-dimensional electrophoretic profiling of normal 6. Miner JH: Renal basement membrane components. Kidney Int 56: human kidney glomerulus proteome and construction of an extensible 2016–2024, 1999 markup language (XML)-based database. Proteomics 5: 1083–1096, 7. Courtoy PJ, Kanwar YS, Hynes RO, Farquhar MG: Fibronectin locali- 2005 zation in the rat glomerulus. J Cell Biol 87: 691–696, 1980 23. Miyamoto M, Yoshida Y, Taguchi I, Nagasaka Y, Tasaki M, Zhang Y, Xu 8. Barker DF, Hostikka SL, Zhou J, Chow LT, Oliphant AR, Gerken SC, B, Nameta M, Sezaki H, Cuellar LM, Osawa T, Morishita H, Sekiyama S, Gregory MC, Skolnick MH, Atkin CL, Tryggvason K: Identification of Yaoita E, Kimura K, Yamamoto T: In-depth proteomic profiling of the mutations in the COL4A5 collagen gene in . Science normal human kidney glomerulus using two-dimensional protein pre- 248: 1224–1227, 1990 fractionation in combination with liquid chromatography-tandem mass 9. Zenker M, Aigner T, Wendler O, Tralau T, Müntefering H, Fenski R, Pitz spectrometry. J Proteome Res 6: 3680–3690, 2007 S, Schumacher V, Royer-Pokora B, Wühl E, Cochat P, Bouvier R, Kraus C, 24. Zhang Y, Yoshida Y, Xu B, Magdeldin S, Fujinaka H, Liu Z, Miyamoto M, Mark K, Madlon H, Dötsch J, Rascher W, Maruniak-Chudek I, Lennert T, Yaoita E, Yamamoto T: Comparison of human glomerulus proteomic Neumann LM, Reis A: Human laminin beta2 deficiency causes con- profiles obtained from low quantities of samples by different mass genital nephrosis with mesangial sclerosis and distinct eye abnormali- spectrometry with the comprehensive database. Proteome Sci 9: 47, ties. Hum Mol Genet 13: 2625–2632, 2004 2011 10. Fogo AB: Mesangial matrix modulation and glomerulosclerosis. Exp 25. Cui Z, Yoshida Y, Xu B, Zhang Y, Nameta M, Magdeldin S, Makiguchi T, Nephrol 7: 147–159, 1999 Ikoma T, Fujinaka H, Yaoita E, Yamamoto T: Profiling and annotation of 11. Spiro RG: Studies on the renal glomerular basement membrane. human kidney glomerulus proteome. Proteome Sci 11: 13, 2013 Preparation and chemical composition. J Biol Chem 242: 1915–1922, 26. Uhlen M, Oksvold P, Fagerberg L, Lundberg E, Jonasson K, Forsberg 1967 M, Zwahlen M, Kampf C, Wester K, Hober S, Wernerus H, Björling L, 12. van den Heuvel LP, van den Born J, van de Velden TJ, Veerkamp JH, Ponten F: Towards a knowledge-based Human Protein Atlas. Nat Bi- Monnens LA, Schroder CH, Berden JH: Isolation and partial charac- otechnol 28: 1248–1250, 2010 terization of heparan sulphate proteoglycan from the human glomer- 27. Didangelos A, Yin X, Mandal K, Baumert M, Jahangiri M, Mayr M: ular basement membrane. Biochem J 264: 457–465, 1989 Proteomics characterization of extracellular space components in the 13. Shapiro JP, Biswas S, Merchant AS, Satoskar A, Taslim C, Lin S, Rovin human aorta. Mol Cell Proteomics 9: 2048–2062, 2010 BH, Sen CK, Roy S, Freitas MA: A quantitative proteomic workflow for 28. Angel PM, Nusinow D, Brown CB, Violette K, Barnett JV, Zhang B, characterization of frozen clinical biopsies: Laser capture microdissec- Baldwin HS, Caprioli RM: Networked-based characterization of extra- tion coupled with label-free mass spectrometry. J Proteomics 77: 433– cellular matrix proteins from adult mouse pulmonary and aortic valves. 440, 2012 JProteomeRes10: 812–823, 2011 14. Nakatani S, Wei M, Ishimura E, Kakehashi A, Mori K, Nishizawa Y, Inaba 29. Bönnemann CG: The collagen VI-related myopathies Ullrich congenital M, Wanibuchi H: Proteome analysis of laser microdissected glomeruli muscular dystrophy and . Handb Clin Neurol 101: from formalin-fixed paraffin-embedded kidneys of autopsies of diabetic 81–96, 2011 patients: Nephronectin is associated with the development of diabetic 30. Bonaldo P, Braghetta P, Zanetti M, Piccolo S, Volpin D, Bressan GM: glomerulosclerosis. Nephrol Dial Transplant 27: 1889–1897, 2012 Collagen VI deficiency induces early onset myopathy in the mouse: An 15. Byron A, Humphries JD, Humphries MJ: Defining the extracellular animal model for Bethlem myopathy. Hum Mol Genet 7: 2135–2140, matrix using proteomics [published online ahead of print February 19, 1998 2013]. Int J Exp Pathol 10.1111/iep.12011 31. Miyazato H, Yoshioka K, Hino S, Aya N, Matsuo S, Suzuki N, Suzuki Y, 16. Eremina V, Sood M, Haigh J, Nagy A, Lajoie G, Ferrara N, Gerber HP, Sinohara H, Maki S: The target antigen of anti-tubular basement Kikkawa Y, Miner JH, Quaggin SE: Glomerular-specificalterationsof membrane antibody-mediated interstitial nephritis. Autoimmunity 18: VEGF-A expression lead to distinct congenital and acquired renal dis- 259–265, 1994 eases. JClinInvest111: 707–716, 2003 32. Wex T, Lipyansky A, Brömme NC, Wex H, Guan XQ, Brömme D: TIN- 17. Welsh GI, Hale LJ, Eremina V, Jeansson M, Maezawa Y, Lennon R, Pons ag-RP, a novel catalytically inactive cathepsin B-related protein with DA, Owen RJ, Satchell SC, Miles MJ, Caunt CJ, McArdle CA, EGF domains, is predominantly expressed in vascular smooth muscle Pavenstädt H, Tavaré JM, Herzenberg AM, Kahn CR, Mathieson PW, cells. 40: 1350–1357, 2001 Quaggin SE, Saleem MA, Coward RJ: signaling to the glomerular 33. Brown LJ, Alawoki M, Crawford ME, Reida T, Sears A, Torma T, Albig podocyte is critical for normal kidney function. Cell Metab 12: 329–340, AR: Lipocalin-7 is a matricellular regulator of angiogenesis. PLoS One 5: 2010 e13905, 2010 18. Yokoi H, Mukoyama M, Mori K, Kasahara M, Suganami T, Sawai K, 34. Kley RA, Maerkens A, Leber Y, Theis V, Schreiner A, van der Ven PF, Yoshioka T, Saito Y, Ogawa Y, Kuwabara T, Sugawara A, Nakao K: Uszkoreit J, Stephan C, Eulitz S, Euler N, Kirschner J, Müller K, Meyer Overexpression of growth factor in podocytes worsens HE, Tegenthoff M, Fürst DO, Vorgerd M, Müller T, Marcus K: A com- diabetic nephropathy in mice. Kidney Int 73: 446–455, 2008 bined laser microdissection and mass spectrometry approach reveals 19. Kahsai TZ, Enders GC, Gunwar S, Brunmark C, Wieslander J, Kalluri R, new disease relevant proteins accumulating in aggregates of filamin- Zhou J, Noelken ME, Hudson BG: Seminiferous tubule basement opathy patients. Mol Cell Proteomics 12: 215–227, 2013

950 Journal of the American Society of Nephrology J Am Soc Nephrol 25: 939–951, 2014 www.jasn.org BASIC RESEARCH

35. Mu Y, Chen Y, Zhang G, Zhan X, Li Y, Liu T, Li G, Li M, Xiao Z, Gong X, 41. Keller A, Nesvizhskii AI, Kolker E, Aebersold R: Empirical statistical Chen Z: Identification of stromal differentially expressed proteins in the model to estimate the accuracy of peptide identifications made by MS/ colon carcinoma by quantitative proteomics. Electrophoresis 34: 1679– MS and database search. Anal Chem 74: 5383–5392, 2002 1692, 2013 42. Nesvizhskii AI, Keller A, Kolker E, Aebersold R: A statistical model for 36. Seeley EH, Caprioli RM: 3D imaging by mass spectrometry: A new identifying proteins by tandem mass spectrometry. Anal Chem 75: frontier. Anal Chem 84: 2105–2110, 2012 4646–4658, 2003 37. Angel PM, Caprioli RM: Matrix-assisted laser desorption ionization 43. Rashid ST, Humphries JD, Byron A, Dhar A, Askari JA, Selley JN, Knight D, imaging mass spectrometry: In situ molecular mapping. Biochemistry Goldin RD, Thursz M, Humphries MJ: Proteomic analysis of extracellular 52: 3818–3828, 2013 matrix from the hepatic stellate cell line LX-2 identifies CYR61 and Wnt-5a 38. Todorovic V, Desai BV, Eigenheer RA, Yin T, Amargo EV, Mrksich M, as novel constituents of fibrotic liver. JProteomeRes11: 4052–4064, 2012 Green KJ, Patterson MJ: Detection of differentially expressed basal cell 44. Li Q, Lau A, Morris TJ, Guo L, Fordyce CB, Stanley EF: A syntaxin 1, proteins by mass spectrometry. Mol Cell Proteomics 9: 351–361, 2010 Galpha(o), and N-type calcium channel complex at a presynaptic nerve 39. Humphries JD, Byron A, Bass MD, Craig SE, Pinney JW, Knight D, terminal: Analysis by quantitative immunocolocalization. JNeurosci24: Humphries MJ: Proteomic analysis of integrin-associated complexes 4070–4081, 2004 identifies RCC2 as a dual regulator of Rac1 and Arf6. Sci Signal 2: ra51, 2009 40. Perkins DN, Pappin DJ, Creasy DM, Cottrell JS: Probability-based protein identification by searching sequence databases using mass This article contains supplemental material online at http://jasn.asnjournals. spectrometry data. Electrophoresis 20: 3551–3567, 1999 org/lookup/suppl/doi:10.1681/ASN.2013030233/-/DCSupplemental.

J Am Soc Nephrol 25: 939–951, 2014 Proteome of the Glomerular Matrix 951