Single-Cell Transcriptomics Reveal Disrupted Kidney Filter Cell-Cell Interactions After Early And Selective Podocyte Injury Ⅱ
Jul 01, 2024
Results
Single-Cell Profiling of >29,000 Glomerular-Enriched Kidney Cells
Current single-cell protocols for whole kidney identify <2.5% glomerular cells,9 and although a purified glomerular preparation using magnetic beads enriches for this population,22 it fails to capture other cell types of the kidney that may be of interest. A sieving method was used to simultaneously enrich glomeruli and capture additional kidney cell types to extend these findings and develop a detailed understanding of cell-cell interactions within the glomerulus in the context of the entire cellular landscape of the kidney, before and after podocyte injury.
To identify the early transcriptional effects of podocyte ablation in a cell typeespecific manner, scRNAseq was performed with kidney tissue from WT and iCTCFpod / mice collected by serial sieving after 1 week of doxycycline treatment (Figure 1A). Kidney tissue from four WT animals (four biological replicates) and four iCTCFpod / animals yielded a total of 14,783 WT and 14,727 iCTCFpod / cells profiled after filtering (Supplemental Figure S1, A and B; Supplemental Tables S1 and S2). Data were normalized to remove effects due to the number of UMIs and percentage of mitochondrial reads. After integration of WT and iCTCFpod / samples, a low resolution of clustering was used to detect nine clusters (Figure 1B). Biological replicates of WT and iCTCFpod / samples were distributed among all clusters (Supplemental Figure S2, A-C). All cell types of the glomerulus, as well as additional kidney cell types, were identified using established and data-derived markers (Figure 1C; Supplemental Table S3; Supplemental Figure S3A). Glomerular cells, representing a total 82.1% of all recovered cells [36.9% podocytes, 42.1% glomerular endothelial cells (GECs), 2.7% mesangial cells, and 0.36% PECs] were isolated and reclustered (Figure 1D). Four clusters of podocytes, five clusters of GECs, and one cluster each of mesangial cells and PECs, expressing canonical cell-type markers, were identified (Figure 1E; Supplemental Figure S3B). Biological replicates of WT and iCTCFpod / samples were distributed throughout all clusters (Supplemental Figure S2, D-F; Supplemental Table S4). As anticipated, given that podocyte-specific CTCF deletion leads to histologically detectable podocyte loss at 2 weeks,13 iCTCFpod / samples contained a lower percentage of podocytes than WT samples (Figure 1F; Supplemental Table S5). iCTCFpod / samples had 13.1% fewer podocytes, 11.9% more GECs, and 1.5% more mesangial cells than WT samples (Supplemental Table S5). PECs contributed <1% to either of the iCTCFpod / or WT samples. This finding suggests that disruption of transcriptional programs critical for podocyte survival precedes histologically detectable podocyte injury and loss and highlights the power of scRNAseq in discerning subtle changes that can be missed by histologic analysis.
not significantly differentially expressed. Theyaxis has been limited to 100.Dashed line indicates default parameters: cutoffP Z 0.25. C:Visualization of theoverrepresentation analysis of genes uniquely differentially expressed in podocyte 1 presented as a network or enrichment map. Overlapping gene sets clustertogether.
Podocyte-Specific Inducible CTCF Deletion Leads to Gene Expression Changes in All Glomerular Cell Types
Differential expression analysis was performed in each of 11 clusters comparing iCTCFpod / and WT cells (Supplemental Table S6). Genes were considered differentially expressed if found in at least 10% of cells in a given cluster, with a minimum absolute log fold change of 0.1 and an adjusted P < 0.05. Ctcf was differentially expressed in each of the four podocyte clusters, with mean log fold changes of 0.390, 0.189, 0.331, and 0.303, respectively. The numbers of cells in each cluster were downsampled and the differential expression analysis repeated to compare the number of differentially expressed genes among the 11 clusters. The podocyte clusters had the most differentially expressed genes, followed by GEC-1 and GEC-2 (Figure 1G). The remaining three clusters of GECs, along with the mesangial cells and PECs, had far fewer differentially expressed genes (Figure 1G), suggesting that GEC-1 and GEC-2, among all glomerular clusters, were most affected by the sequelae of CTCF deletionedriven podocyte injury.
Disease-Associated Gene Programs Identified in Specific Podocyte and Endothelial Cell Clusters
This study first sought to examine how the individual podocyte clusters respond to injury. A Venn diagram of the differentially expressed genes in each of the four podocyte clusters revealed that podocyte 1 had the most uniquely differentially expressed genes of the four podocyte clusters (Figure 2A; Supplemental Table S7). The observation that one of four podocyte clusters was more prominently affected is in agreement with prior histologic data indicating that not all podocytes are affected with equal severity in the face of injury and highlights the power of scRNAseq to molecularly characterize the heterogeneity of cell states within the same cell type.23 An overrepresentation analysis was performed to identify gene programs enriched as a consequence of CTCF loss in the podocyte 1 cluster (Supplemental Table S8). The top enriched terms were visualized with an enrichment map to cluster mutually overlapping gene sets (Figure 2B). A prominent group of enriched terms was mitochondrial functions, including ATP synthesis, mitochondrial organization, electron transport chain, and oxidative phosphorylation (Figure 2B). These data extend recent work pointing to mitochondrial dysfunction as a sign of podocyte injury.24 In addition, human genetics have pointed to the importance of mitochondrial functions in podocytes, including several mutations in the CoQ biosynthesis pathway (PDSS1, PDSS2, COQ2, COQ6, and ADCK4) that cause nephrotic syndrome, mainly in children.25,26 It has therefore been postulated that podocyte mitochondrial dysfunction may represent a prominent cell state associated with all diseases that stem from podocyte loss.24 The data provide support for this notion at single-cell resolution, suggesting that mitochondrial dysfunction may represent the earliest injury state in a specific population of podocytes, leading to podocyte loss. One of the main groups of enriched terms from the overrepresentation analysis (Figure 2B) was cytoskeletal organization as well as a related single enriched term for cell-matrix adhesion. The actin cytoskeleton plays an essential role in maintaining the podocytes' unique and complex structure, and adhesion to the GBM is essential for podocyte function.27 Mature focal adhesions contain hundreds of proteins that link the actin cytoskeleton, receptor matrix binding, intracellular signal transduction, and actin polymerization. One of the most down-regulated genes in podocyte 1 was Rhpn1 (Supplemental Figure S4A), an essential component for establishing podocyte cytoskeleton dynamics and maintaining podocyte foot process architecture.28 The Arp2/3 complex component Arpc3, a driver of actin polymerization, was upregulated in the podocyte 1 cluster (Figure 2C; Supplemental Figure S4B). A gene for an additional component of the complex, Actr2, was also up-regulated in both podocyte 1 and podocyte 4. In addition, Wasl, whose protein product, N-WASP, activates the Arp2/3 complex and is required for the maintenance of podocyte foot processes in vivo29, was also up-regulated in podocyte 1, podocyte 3, and podocyte 4. The cytoskeletal regulator Arhgdia, whose deletion is associated with nephrotic syndrome in mice,30 was down-regulated in podocyte 1 (Figure 2C). The expression levels of two Rho GTPases that are well established regulators of the actin cytoskeleton and cell adhesion dynamics31 were also altered: RhoA was up-regulated and RhoC was down-regulated, as was a central downstream effector of Rac1, Pak1, consistent with a prior study.23 Furthermore, Cd2ap, a critical podocyte actin cytoskeleton component,27,32 was up-regulated in podocyte 1. Together, these changes suggest that one of the earliest podocyte responses to CTCF deletionedriven injury is to alter critical components of the actin cytoskeleton in a struggle to maintain attachment to the GBM and thus survive the injury. In addition, scRNAseq identified specific mediators of this response matched to a specific population of podocytes, pointing to putative targets for early therapeutic intervention.
The GBM is a meshwork of extracellular matrix proteins situated between podocytes and GECs that provides structural support for the glomerular capillaries, harbors ligands for receptors on the surface of the adjacent GECs, podocytes, and mesangial cells, and contributes to glomerular filter selectivity.33 One of the critical components of the GBM is collagen type IV ɑ5 (Col4a5); mutations in this gene cause Alport syndrome and FSGS in humans.33,34 Variants in Col4a3, another structural component of the GBM, were recently identified by a comprehensive genome-wide association study in DKD.35 Therefore, changes to GBM components as a consequence of podocyte injury are broadly relevant to many kidney diseases. Whether expression of genes encoding structural components of the GBM were disrupted in the model was queried. Col4a5 was up-regulated in iCTCFpod / podocytes (with a log fold change of 0.130) (Supplemental Figure S4C; Supplemental Table S6). Ligand receptor analysis suggested that Col4a5 upregulation in podocyte 1 leads to increased interactions with cells of all five GEC clusters and mesangial cells through several integrins (Figure 3A). The expression of Col4a5 in Nphs2-expressing podocytes was significantly increased (P < 0.0001; Welch-corrected two-tailed t-test) (Figure 3B). The observed Col4a5 up-regulation, with spatial resolution, was validated by HCR, a method that generates single-molecule fluorescence via in situ hybridization36 (Figure 3C; Supplemental Figure S5). This result confirmed that individual gene data derived from the single-cell transcriptomic experiment could be independently validated, with spatial resolution, bolstering the validity of the conclusions drawn by computational analyses. Furthermore, these data highlight that the key response to injury by a specific population of podocytes is to up-regulate collagen production, which may account for the prominent or thick GBM observed in progressive diseases, such as DKD.35
GEC-1 and GEC-2 had nearly as many differentially expressed genes as podocyte clusters 2 to 4, whereas the remaining GEC clusters, mesangial cells, and PECs had fewer differentially expressed genes. Therefore, these two endothelial clusters were most affected by podocyte injury. GEC-1 expressed >600 uniquely differentially expressed genes (Supplemental Figure S6A; Supplemental Table S9). Overrepresentation analysis indicated that the uniquely differentially expressed genes in GEC-1 were enriched in gene programs for cell migration and adhesion (Supplemental Figure S6, B and C; Supplemental Table S10). These analyses revealed the earliest transcriptional changes that occur in GECs as a response to podocyte injury.
Modeling Intercellular Communication Reveals
Key Interactions between Cell Types in Response to Early Podocyte Injury
Having identified disrupted gene programs in several distinct cell clusters, the study next sought to understand how glomerular cell-cell crosstalk was influenced by podocyte injury. The study first probed the list of differentially expressed genes in podocyte, GEC, and mesangial cell clusters. The expression of two key autocrine prosurvival ligands, Vegfa and Pdgfb, as well as the expression of the PDGFB receptor, Pdgfrb, were disrupted (Supplemental Table S6). Vegfa expression was decreased in all podocyte clusters (mean log fold change between 0.034 and 0.149). In contrast, Pdgfb expression was increased in all GEC clusters (mean log fold change between 0.031 and 0.140), and the receptor Pdgfrb was up-regulated in mesangial cells (mean log fold change of 0.155). This analysis suggested that podocyte injury leads to decreased expression of the prosurvival ligand Vegfa, negatively affecting GECs. To compensate, GECs may up-regulate the prosurvival ligand Pdgfb, triggering mesangial cells to upregulate the receptor Pdgfrb.
NicheNet, a novel algorithm that infers how ligandreceptor interactions derived from expression data may affect specific targets by integrating preexisting knowledge of signaling and regulatory networks, was used to more deeply investigate ligand-receptor interactions and their putative target genes.20 NicheNet was applied to model interactions between podocytes and GECs as well as mesangial cells and GECs that could potentially induce differentially expressed genes (target genes) in GECs in the setting of podocyte injury (Figure 4A). For this analysis, all clusters of each cell type were combined into a single cluster. The top predicted ligands expressed by mesangial cells and/or podocytes were pleiotrophin (Ptn), angiopoietin 2 (Angpt2), Col4a1, vascular cell adhesion molecule 1, bone morphogenetic protein 4 (Bmp4), Ephrin B1, connective tissue growth factor, and semaphoring 3e (Sema3e) (Figure 4B). The expression pattern of GEC receptors through which these ligands are known to act were mapped next to the ligand activity analysis. In addition, the predicted target genes that were differentially expressed in iCTCFpod / GECs compared with WT controls were also mapped. The results in Figure 4B are summarized as circular plots in Figure 4, C and D.
Several ligands, receptors, and target genes were notable from this analysis. Pleiotrophin, a ligand highly expressed in mesangial cells (Figure 4B), is a secreted growth factor that can bind and inhibit protein tyrosine phosphatase receptor type B, stimulating endothelial cell migration via increased Tek (Tie2)/Angpt137 and Kdr (Vegfr2)/Vegfa38 signaling. Accordingly, Tek was significantly up-regulated in GECs (Figure 4B). Angpt2, highly expressed in mesangial cells, is an antagonistic ligand of Tek, inhibiting the binding of Angpt1.39 Because Angpt1 signaling is known to promote podocyte survival and a disruption of the Angpt1/Angpt2 ratio contributes to the development of DKD,40 this ligandreceptor analysis appeared to point to maladaptive Tek signaling in GECs as one of the earliest consequences of podocyte injury.
This study also found prominent changes in type IV collagen expression in several glomerular cells (Figure 4B).Normally produced by healthy podocytes, collagen IV heterotrimers transition from ɑ1ɑ1ɑ2 to ɑ3ɑ4ɑ5 during glomerular development, which is necessary for proper GBM formation and function.33,34 Up-regulation of Col4a1, along with expansion of the mesangial matrix, is frequently observed in DKD.41,42 In the current analysis, Col4a1 was expressed by both mesangial cells and GECs (mean expression of ligand heatmap) (Figure 4B) and was specifically up-regulated in iCTCFpod / GECs compared with WT controls. These data suggest that GECs up-regulate Col4a1 in the setting of podocyte injury, which may lead to a stiffer, fibrotic GBM that contributes to the development of segmental sclerosis.33
Bmp4 was a highly expressed mesangial ligand identified by NicheNet analysis (Figure 4, B-D). BMPs play key roles in kidney development and disease.43 Bmp4 is up-regulated in the setting of diabetic nephropathy in rats,42 and treatment of mice with diabetes with an anti-BMP4 antibody prevents the up-regulation of Col4a1 and mesangial matrix expansion.44 Of particular interest, mesangial Bmp4 was found to trigger GEC Smad6 expression (Figure 4B). Expression of Smad6 is induced by BMPs and in a negativefeedback loop, Smad6 specifically inhibits BMPs, including BMP4.45 Smad6 was down-regulated in iCTCFpod / GECs compared with WT controls, suggesting increased BMP signaling in endothelial cells in the setting of podocyte injury.
One of the prioritized ligands identified in podocytes was Sema3e (Figure 4, B-D). Class 3 semaphorins are secreted proteins that function in a variety of biological processes, including angiogenesis, lymphangiogenesis, and disease.46 Sema3a is up-regulated in human DKD,47 and Sema3g was recently identified as a podocyte-specific gene that protects podocytes from inflammation in vivo. 48 The role of Sema3e in the podocyte remains unclear. The current analysis revealed a previously unrecognized receptor-ligand pair whereby podocyte Sema3e interacts with plexinD1 on GECs to trigger several downstream gene targets in the setting of podocyte injury (Figure 4, C and D).
Among several newly identified GEC target genes, Hes1 and Cx3cl1 were also found in the adhesion gene programs enriched in GEC-1 (Figure 4, B-D; Supplemental Figure S6). Hes1 is a target of Notch signaling, which plays an important role in the developing kidney, but reactivation can lead to fibrosis.49 Cx3cl1 is a chemokine mainly produced by glomerular endothelium that acts as a chemoattractant and adhesion molecule for its receptor, Cx3cr1, which is ubiquitously expressed on mononuclear and circulatory lymphatic leukocytes.50 Cx3cl1 has been implicated in a variety of kidney diseases, including DKD, IgA nephropathy, and glomerulonephritis.50 Hes1 and Cx3cl1 were both up-regulated in iCTCFpod / GECs compared with WT controls, suggesting that GECs respond to podocyte injury by up-regulating proinflammatory and profibrosis programs.
Finally, because many of the ligands and targets identi- fied in the NicheNet analysis are implicated in DKD, in both rodent models as well as humans, the data set was compared with a single nuclei transcriptomic data set of early human DKD.10 A specific comparison was made of differentially expressed genes between human and mouse cell clusters. Of the 138 differentially expressed genes in human DKD GECs, 25 genes were also identified in mouse iCTCFpod / endothelial cells, including Col4a1 (Supplemental Figure S7; Supplemental Table S11). Together, these results suggest that endothelial cell programs frequently disrupted in human DKD can be partially recapitulated in the mouse model of early glomerular injury via selective podocyte ablation. In addition, these results suggest that, in addition to podocytes, there may be potential endothelialspecific targets and therapeutic strategies to halt or slow the progression of complex and highly prevalent kidney diseases, such as DKD.
