RAPID COMMUNICATION www.jasn.org A Single-Cell Transcriptome Atlas of the Mouse Glomerulus Nikos Karaiskos,1 Mahdieh Rahmatollahi,2 Anastasiya Boltengagen,1 Haiyue Liu,1 Martin Hoehne ,2 Markus Rinschen,2,3 Bernhard Schermer,2,4,5 Thomas Benzing,2,4,5 Nikolaus Rajewsky,1 Christine Kocks ,1 Martin Kann,2 and Roman-Ulrich Müller 2,4,5 Due to the number of contributing authors, the affiliations are listed at the end of this article. ABSTRACT Background Three different cell types constitute the glomerular filter: mesangial depending on cell location relative to the cells, endothelial cells, and podocytes. However, to what extent cellular heteroge- glomerular vascular pole.3 Because BP ad- neity exists within healthy glomerular cell populations remains unknown. aptation and mechanoadaptation of glo- merular cells are key determinants of kidney Methods We used nanodroplet-based highly parallel transcriptional profiling to function and dysregulated in kidney disease, characterize the cellular content of purified wild-type mouse glomeruli. we tested whether glomerular cell type sub- Results Unsupervised clustering of nearly 13,000 single-cell transcriptomes identi- sets can be identified by single-cell RNA fied the three known glomerular cell types. We provide a comprehensive online sequencing in wild-type glomeruli. This atlas of gene expression in glomerular cells that can be queried and visualized using technique allows for high-throughput tran- an interactive and freely available database. Novel marker genes for all glomerular scriptome profiling of individual cells and is cell types were identified and supported by immunohistochemistry images particularly suitable for identifying novel obtained from the Human Protein Atlas. Subclustering of endothelial cells celltypesaswellassubsetsandnovelmarker revealed a subset of endothelium that expressed marker genes related to endo- genes of known cell populations.4–6 thelial proliferation. By comparison, the podocyte population appeared more ho- mogeneous but contained three smaller, previously unknown subpopulations. METHODS Conclusions Our study comprehensively characterized gene expression in individ- ual glomerular cells and sets the stage for the dissection of glomerular function at Glomerular isolation and preparation of the single-cell level in health and disease. single-cell suspensions were carried out J Am Soc Nephrol 29: ccc–ccc, 2018. doi: https://doi.org/10.1681/ASN.2018030238 Significance Statement The glomerular filtration unit consists of three Glomeruli are the key functional units of Received March 6, 2018. Accepted April 28, 2018. fi tightly intertwined cell types: the endothelium, the kidney ltration apparatus. Within N.K.,M.Rahmatollahi,M.K.,andR.-U.M.contrib- the mesangium, and podocytes. Despite es- each glomerulus, a capillary tuft is struc- uted equally to this work. tablished physiologic cues acting on these cells within a single glomerulus, cellular heteroge- turally maintained by mesangial cells and Published online ahead of print. Publication date neity in the healthy glomerulus remains poorly provides a three-layered filtration barrier available at www.jasn.org. characterized. To address this problem, the consisting of endothelial cells, the glo- Correspondence: Dr. Christine Kocks, Systems Bi- authors performed large-scale transcriptional merular basement membrane, and po- ology of Gene Regulatory Elements, Berlin Institute profiling of 13,000 mouse glomerular cells. docytes.1,2 Although these three cell for Medical Systems Biology, Max Delbrück Center They provide a comprehensive atlas of gene types within the glomerular tuft have for Molecular Medicine in the Helmholtz Associa- expression for the known glomerular cell types tion, Robert-Roessle-Str. 10, 13125 Berlin, Ger- and describe potential subpopulations for en- long been established, it is as yet un- many, or Dr. Roman-Ulrich Müller, Department II of dotheliumandpodocytes.Aninteractive,freely Internal Medicine and Center for Molecular Medi- known whether individual cells within accessible web tool allows for querying and the glomerulus respond to cues to which cine Cologne, University of Cologne, Kerpener Strasse 62, 50937 Cologne, Germany. Email: visualizing the data. The study highlights the they are physiologically exposed. Such [email protected] or roman-ulrich. power of single-cell RNA sequencing to study cues include changing pressure gradi- [email protected] gene expression in the kidney and sets the ents along the capillaries and mechanical stage for future investigations of glomerular Copyright © 2018 by the American Society of dysfunction in disease. strain on mesangial cells, which may differ Nephrology J Am Soc Nephrol 29: ccc–ccc, 2018 ISSN : 1046-6673/2907-ccc 1 RAPID COMMUNICATION www.jasn.org as described7 on 8-week-old wild-type concentration used).4,8,12 To obtain by sequencing method, pairwise corre- CD1 male mice. Flow-sorted cells were high-quality single-cell data, we used a lations by cell type supported our cell dehydrated in methanol,8 stored and previously developed algorithm to score type assignments. shipped at 270°C,andrehydratedfor cell type–specific marker genes12 (Sup- We continued by characterizing glo- highly parallel single-cell transcriptome plemental Table 2) and removed 1768 merularcelltypesinmoredetailandaimed profiling by Drop-seq.4,8 This method probable doublets. The final dataset con- to identify novel cell-specificmarkersby predominantly detects 39 ends of polya- tained 12,954 cells, with a median of 630 assessing highly variable genes between denylated mRNA as well as long noncod- genes and 950 unique molecular identi- clusters.4 Established cell-specificmarker ing RNA molecules. Single-cell data were fiers per cell at a sequencing depth of genes for endothelium, mesangium, and processed, and genes were quantified approximately 9400 aligned reads per podocytes as well as genes described as with Drop-seq tools v. 1.124 and further cell (Supplemental Figure 1B, Supple- relevant to the respective cell type in the analyzed with “dropbead”8 and Seurat.5 mental Table 1). literature (Supplemental Methods has de- Marker gene identification was carried As shown in Figure 1B, unsupervised tails) were comprehensively reproduced out with Seurat function “FindAllMark- clustering of the remaining 12,954 single as specifically expressed, validating our ers”5 and visual inspection of violin plots cells identified five major cell clusters. unsupervised clustering (Figure 2A, Sup- as well as images from the Human Pro- On the basis of marker genes, three of plemental Figure 2).7,14,15 Importantly, tein Atlas.9 Immunofluorescence stain- these clusters corresponded to known expression of several previously reported ing was carried out on glomeruli of glomerular cell populations: podocytes key podocyte genes did not seem to be Nphs2-Cre/mTmG reporter mice10 using (80%), mesangial cells (2%), and endo- exclusive to podocytes, a finding bear- affinity-purified rabbit antibodies. Images thelial cells (12%). The other two clus- ing important implications for future were obtained using confocal microscopy. ters corresponded to tubular cells (6%) studies on the function of such genes Animal experiments were approved by the and a small group of immune cells in kidneys. Examples for such genes in- Landesamt für Natur, Umwelt und Verbrau- (0.2%). Glomerular cell type clusters clude Podxl (for which previously de- cherschutz Nordrhein-Westfalen (LANUV showed specificexpressionofestab- scribed endothelial expression was NRW, AZ 2013.A 375). Statistical methods lished marker genes (Figure 1, C and D, confirmed16), Actn4,andItgb1 (Figure were used as indicated. Supplemental Table 3), and all replicates 2, Supplemental Figure 2). Conse- Raw and processed datasets are avail- contributed to the observed cell clusters quently, we aimed to identify novel able from the Gene Expression Omnibus (Supplemental Figure 1C). Hierarchical cell-specificmarkersforallthreeglo- repository (GSE111107). The interactive clustering of aggregated reads from all merular cell types (Figure 2B). A large online database is available at https:// replicates and cell types indicated high proportion of these markers was cor- shiny.mdc-berlin.de/mgsca/. correlations according to cell type and roborated on the protein level by im- independent of the replicate (Supple- munostaining images obtained from mental Figure 1D). To control for effects the Human Protein Atlas (Figure 2B).9 RESULTS of single-cell preparation, we compared Novel markers represent a wide variety single-cell RNAseq data with bulk of molecular functions, including the Figure 1A shows the study design. We polyA-RNAseq libraries prepared from transcription factor Meis2 identified as isolated glomeruli by magnetic bead per- glomeruli before and after dissociation specific to endothelial cells and disease fusion followed by magnetic separation into single cells (bulk1 and bulk2, re- genes, such as Pde3a (the gene mutated and rigorous washing (Supplemental spectively) (Figure 1A). Although the in autosomal dominant hypertension Figure 1A),11 generated single-cell sus- single-cell data showed good correla- with brachydactyly, which was identi- pensions by enzymatic digestion, and tions with both bulk mRNAseq datasets fied as specific to mesangial cells), as performed highly parallel single-cell (Supplemental Figure 1E), it became well as the E3-ubiquitin-ligase Wsb2,a RNA sequencing using the Drop-seq apparent that single-cell dissociation af- novel podocyte marker. Taken