BASIC RESEARCH www.jasn.org Glomerular Cell Cross-Talk Influences Composition and Assembly of Extracellular Matrix † Adam Byron,* Michael J. Randles,* Jonathan D. Humphries,* Aleksandr Mironov,* † ‡ ‡ ‡ Hellyeh Hamidi,* Shelley Harris, Peter W. Mathieson, Moin A. Saleem, Simon C. Satchell, | † Roy Zent,§ Martin J. Humphries,* and Rachel Lennon* *Wellcome Trust Centre for Cell-Matrix Research, Faculty of Life Sciences and †Faculty of Medical and Human Sciences, University of Manchester, Manchester, United Kingdom; ‡Academic Renal Unit, Faculty of Medicine and Dentistry, University of Bristol, Bristol, United Kingdom; §Department of Medicine, Vanderbilt University Medical Center, Nashville, Tennessee; and |Department of Medicine, Veterans Affairs Hospital, Nashville, Tennessee ABSTRACT The glomerular basement membrane (GBM) is a specialized extracellular matrix (ECM) compartment within the glomerulus that contains tissue-restricted isoforms of collagen IV and laminin. It is integral to the capillary wall and therefore, functionally linked to glomerular filtration. Although the composition of the GBM has been investigated with global and candidate-based approaches, the relative contributions of glomerular cell types to the production of ECM are not well understood. To characterize specific cellular contributions to the GBM, we used mass spectrometry–based proteomics to analyze ECM isolated from podocytes and glomerular endothelial cells in vitro. These analyses identified cell type–specificdifferences in ECM composition, indicating distinct contributions to glomerular ECM assembly. Coculture of podo- cytes and endothelial cells resulted in an altered composition and organization of ECM compared with monoculture ECMs, and electron microscopy revealed basement membrane–like ECM deposition be- tween cocultured cells, suggesting the involvement of cell–cell cross-talk in the production of glomerular ECM. Notably, compared with monoculture ECM proteomes, the coculture ECM proteome better resembled a tissue-derived glomerular ECM dataset, indicating its relevance to GBM in vivo.Protein network analyses revealed a common core of 35 highly connected structural ECM proteins that may be important for glomerular ECM assembly. Overall, these findings show the complexity of the glomerular ECM and suggest that both ECM composition and organization are context-dependent. J Am Soc Nephrol 25: 953–966, 2014. doi: 10.1681/ASN.2013070795 The glomerular basement membrane (GBM) is in- Forexample, collagen IVnetworksare predominantly tegral to the glomerular capillary wall, and it directly comprised of a1anda2 chains; however, at the contributes to barrier function.1,2 The ultrastructural appearance of the GBM shows that it is unusually Received July 28, 2013. Accepted December 16, 2013. thick (300–350 nm) as a result of two basement mem- branes fusing during glomerular development.3 This A.B. and M.J.R. contributed equally to this work. specialized extracellular matrix (ECM) supports two Published online ahead of print. Publication date available at distinct cell types: glomerular endothelial cells www.jasn.org. (GEnCs) on the inner capillary wall and podocytes, Present address: Dr. Adam Byron, Edinburgh Cancer Research United Kingdom Centre, Institute of Genetics and Molecular which form a mesh-like covering of the outer surface. Medicine, University of Edinburgh, Edinburgh, United Kingdom. However, there is limited understanding about the Correspondence: Dr. Rachel Lennon, Wellcome Trust Centre for relative contribution of each cell type to the regulation Cell-Matrix Research, Michael Smith Building, University of of ECM synthesis and organization in the GBM. Manchester, Manchester M13 9PT, United Kingdom. Email: Some unique ECM isoforms, essential for normal [email protected] barrier function, characterize the mature glomerulus. Copyright © 2014 by the American Society of Nephrology J Am Soc Nephrol 25: 953–966, 2014 ISSN : 1046-6673/2505-953 953 BASIC RESEARCH www.jasn.org capillary loop stage of glomerular development, there is an iso- 1B, iii). In contrast, podocytes had wider cell–cell junctions form switch to the expression of the collagen IV a3a4a5 net- (2963.4 nm) (Figure 1B, v), more lysosomes (Figure 1B, vi), work in the GBM.4,5 This network is thought to provide the and greater cell width (6.160.49 mm) (Figure 1, B and C). tensile strength necessary to withstand filtration forces.5,6 Sim- ilar changes in laminin isoforms have been reported, switching Glomerular Cells In Vitro Exhibit Differential ECM from a combination of laminin 111 and 511 to a predominance Protein Synthesis of laminin 521 in the mature GBM.7 The importance of glomer- Tocollect ECM proteins for proteomic analysis, a fractionation ular ECM composition for normal barrier function is high- workflow was adapted from previous studies (Figure 2A).17,18 lighted by human genetic mutations, which result in the absence Cell-derived ECMs were extracted from both GEnC and po- of the collagen IV a3a4a5 network in Alport syndrome8 and the docyte monocultures and analyzed by MS. We identified 127 absenceoflaminin521inPiersonsyndrome.9 However, there extracellular proteins in GEnC samples (Supplemental Table is a much wider spectrum of inherited and acquired glomerular 1) and 142 extracellular proteins in podocyte samples (Sup- disease with associated abnormalities in the GBM, which is not plemental Table 2). There was considerable (55%) overlap explained by defects in collagen IV or laminin.10,11 Therefore, between proteins detected in GEnC and podocyte ECMs, building understanding of ECM composition in an unbiased suggesting a common core of ECM proteins, but cell type– manner could help to identify additional genetic defects and specific proteins were also identified (Figure 2B). Further- appreciate mechanisms of ECM regulation in the glomerulus. more, 44% of proteins in the recently described in vivo These questions can be initially addressed with studies in glomerular ECM proteome15 were identified in cell-derived vitro, because cell-based investigations allow the dissection of ECMs (Figure 2B). Statistical analysis of all identified proteins molecular pathways in a manner not feasible in animal studies. in the cell-derived ECM samples revealed differential ECM Conditionally immortalized human glomerular cells have protein synthesis between GEnCs and podocytes (Figure 2C, been derived to facilitate these investigations12,13; however, Supplemental Table 3). Although gene ontology (GO) enrich- their ability to synthesize and organize ECM has not been fully ment analysis highlighted significantly enriched GO terms as- investigated. In vivo, these cells are highly specialized, and ex sociated with ECM proteins for both GEnC and podocyte vivo, they may lose critical paracrine and environmental cues samples (Supplemental Table 4), there was also evidence of required to maintain a differentiated state.14 To test this hy- cell type–specific differences between the ECMs. Specifically, pothesis and inform mechanisms of ECM regulation in the there was significant enrichment of vascular development glomerulus, global investigation of cell-derived ECM processes in GEnC ECM, whereas cell adhesion processes represents a powerful, unbiased analytical approach. were enriched in podocyte ECM (Figure 2D, Supplemental Using an ECM enrichment strategy coupled to mass spec- Table 4). These data indicate a functional distinction between trometry (MS)–based proteomics, we recently described a GEnC and podocyte ECMs. Moreover, the expected associa- glomerular ECM proteome derived from isolated human glo- tion of GEnC with endothelial cellular functions suggests that meruli.15 Toextend this global analysis, we analyzed cell-derived this analysis has the capability to identify proteins with tissue- ECM from GEnCs and podocytes to determine their relative con- specificfunctions. tribution to ECM production and regulation in the glomerulus. In contrast to the compositional differences highlighted by the GO analysis, previously identified glomerular ECM com- ponents were detected to a similar extent in ECMs from both RESULTS cell types (Figure 2B), suggesting that the ECMs are qualita- tively closely related. To examine putative interactions be- Human Glomerular Cells Synthesize and Organize ECM tween the identified ECM proteins, interaction networks In Vitro were generated from a curated protein interaction database Toinvestigate ECM production by glomerular cells on either side (Supplemental Material). ECM protein interaction networks of the GBM, we prepared monocultures of human GEnCs and had similar topologies, with highly connected subnetworks of podocytes in vitro. Both cell types synthesized and deposited basement membrane and other structural ECM proteins (Fig- ECM (Figure 1A), and this process did not seem disrupted by ure 3, Supplemental Figures 1 and 2). Highly connected the removal of cells before ECM extraction (Figure 1A, right proteins tend to be involved in important biologic functions. panels). Ultrastructural analysis of both glomerular cell types Interestingly, basement membrane proteins had a higher revealed a thin basal layer of ECM (Figure 1B). In addition, number of protein–protein interactions than ECM-associated GEnCs and podocytes exhibited distinct morphology (Figure proteins, suggesting that these molecules play key roles in glo- 1, B and C). GEnCs had more caveolae, which were visible as merular ECM organization (Figure 3, B and D). These findings small (50- to 100-nm diameter)