Single-Cell Transcriptome Profiling of the Kidney Glomerulus Identifies Key Cell Types and Reactions to Injury

Abstract

Background: The glomerulus is a specialized capillary bed that is involved in urine production and BP control. Glomerular injury is a major cause of CKD, which is epidemic and without therapeutic options. Single-cell transcriptomics has radically improved our ability to characterize complex organs, such as the kidney. Cells of the glomerulus, however, have been largely underrepresented in previous single-cell kidney studies due to their paucity and intractability.

Methods: Single-cell RNA sequencing comprehensively characterized the types of cells in the glomerulus from healthy mice and from four different disease models (nephrotoxic serum nephritis, diabetes, doxorubicin toxicity, and CD2AP deficiency).

Results: All cell types in the glomerulus were identified using unsupervised clustering analysis. Novel marker genes and gene signatures of mesangial cells, vascular smooth muscle cells of the afferent and efferent arterioles, parietal epithelial cells, and three types of endothelial cells were identified. Analysis of the disease models revealed cell type-specific and injury type-specific responses in the glomerulus, including acute activation of the Hippo pathway in podocytes after nephrotoxic immune injury. Conditional deletion of YAP or TAZ resulted in more severe and prolonged proteinuria in response to injury, as well as worse glomerulosclerosis.

Conclusions: Generation of comprehensive high-resolution, single-cell transcriptomic profiles of the glomerulus from healthy and injured mice provides resources to identify novel disease-related genes and pathways.

Keywords: anti-GBM disease; chronic kidney disease; diabetic glomerulopathy; glomerular disease; transcriptional profiling.

Graphical abstract

Figure 1.

Unbiased clustering of scRNA-seq data reveals the major cell types of the glomerulus. (A) T-distributed neighbor embedding (t-SNE) representation of 5287 glomerular cells from a healthy C57BL/6J mouse. Labels indicate clusters identified by unsupervised clustering analysis. (B) Violin plots of marker gene expression in each cluster shown in (A). (C) Violin plots showing expression of glomerular capillary endothelial cell (Ehd3) or arteriolar endothelial cell (Fbln2, Mgp, Trpv4, Bmx) marker genes in clusters 2 and 4 (D). PCA plot of normal glomerular cells. Cluster labels are identical to (A). Proximity of clusters 3 and 5 indicate high degree of similarity between the two clusters. (E) Violin plots showing expression levels of genes specific to mesangial cells or SMCs/JG cells. The numbers in parentheses indicate cluster number. (F) Immunofluorescence staining of kidney sections shows mesangial-specific expression of the identified marker genes. PDGFRβ, which stains mesangial cells in the glomerulus (dotted circle) and stromal cells outside the glomerulus, was used as reference. Scale bars, 20 μm. (G) Expression levels of the indicated genes are shown in a t-SNE plot of SMCs/JG cells. The two subclusters corresponding to AA SMCs and EA SMCs are outlined. (H) Immunofluorescence staining of kidney sections shows specific staining of calponin 1 in the vascular SMCs of the AA. Scale bar, 10 μm. Normalized expression levels are shown in the violin plots. DAPI, 4′,6-diamidino-2-phenylindole; α-SMA, α-smooth muscle actin.

Figure 2.

scRNA-seq analysis of nephritic mice reveal cell type–specific responses to injury. (A) T-distributed neighbor embedding (t-SNE) plot of control and nephritic mice at day 1. (B) Violin plots show the downregulation of podocyte-specific genes after nephrotoxic injury. Normalized expression levels are shown. (C) Heatmaps showing the top 100 most variable genes in each cell type. Each column represents a cell, each row represents a gene. (D) PCA plot of podocytes, mesangial cells, and endothelial cells from control and nephritic mice. (E) t-SNE plots of immune cells show increased immune infiltration after nephrotoxic injury. Cell types shown in the right panel were determined by expression of marker genes shown in Supplemental Figure 5L.

Figure 3.

The Hippo pathway is required for podocyte response to injury. (A) Immunoblots showing increased protein levels of TAZ and YAP in glomerular lysates 2 days after nephrotoxic injury. Blots are representative of two independent experiments. Vinculin was used as loading control. (B) YAP/TAZ target genes are induced in podocytes after nephrotoxic injury. Normalized expression levels are shown as violin plots. (C) Deletion of TAZ (Wwtr1^−/−) or YAP (Yap1^−/−) exacerbates proteinuria. Albumin-creatinine ratios (ACR) were measured from spot urine collected from mice at the indicated time points after nephrotoxic serum injection. (D) Kidney sections were stained with PAS and Masson trichrome stain 5 weeks after injection with nephrotoxic serum or saline and scored blindly for GN and fibrosis. Scoring criteria are described in the methods. Results were analyzed by multiple t tests using the two-stage linear step-up method (*q<0.05, **q<0.01, ****q<0.0001). WT, wild type.

Figure 4.

Glomerular cells display distinct responses to different types of injury. (A) PCA plots of podocytes, mesangial cells, and endothelial cells from control and diabetic mice. (B and C) Violin plots show increased expression of extracellular matrix and matrix-modifying genes in (B) podocytes and (C) mesangial cells from diabetic mice. (D) T-distributed neighbor embedding (t-SNE) plot of immune cells shows increased numbers of neutrophils and monocytes/macrophages in doxorubicin-treated mice. Immune cell types were determined by expression of marker genes shown in Supplemental Figure 8I. (E) Violin plot shows increased expression of Cxcl1 in mesangial cells from doxorubicin-treated mice. (F) PCA plots of podocytes, mesangial cells, and endothelial cells from wild-type and Cd2ap^−/− mice. (G) Graphs showing the fraction of mesangial cells expressing Ren1 after injury caused by nephrotoxic serum, diabetes, and doxorubicin.