Renal Disease

Single-cell sequencing dissects the transcriptional identity of activated fibroblasts and identifies novel persistent distal tubular injury patterns in kidney fibrosis – Scientific Reports

Time Stamp: January 3, 2024 12:00 AM
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Kidney fibrosis models exhibit proximal tubular loss, functional decline and novel cellular clusters

We established two clinically relevant kidney fibrosis models induced by UIR and UUO^35,36,37. At day 28 post-injury, both models exhibited key CKD features including renal parenchymal loss and blood flow decline³⁸ (Fig. 1a,b, Supplementary Fig. S1). Then, 10× Chromium scRNA-seq was carried out on the control, UIR and UUO kidney suspensions. Kidney cell populations were identified using unsupervised Uniform Manifold Approximation and Projection (UMAP) dimension reduction³⁹, and datasets were cleansed of potential doublets, ambient RNA and cells with high mitochondrial and hemoglobin components (Supplementary Figs. S2–S4). Controls exhibited podocyte (Nphs1/2), endothelial (Adgrl4) and epithelial clusters, including large PT S1 (Slc5a2/12), S2 (Cyp2e1/4b1, Slc22a6) and S3 (Acy3, Slc27a2) subpopulations and distal nephron tubular segments, represented by loop of Henle (Slc12a1), distal tubule (Slc12a3), collecting duct principal (Fxyd4) and intercalated (Atp6v1g3) cells (Figs. 1c, 2a, Supplementary Fig. S5, Supplementary Table S1)⁴⁰. UMAPs also revealed small myeloid clusters, including conventional dendritic (cDCs) 1 (Xcir1, Clec9a) and 2 (Clec10a)⁴¹, macrophages (C1qa) and neutrophils (S100a9), along with lymphoid B (Cd79a) and T/NK (Cd3g, Cd8a, Nkg7) cells⁴².

Both models caused remarkable cellular landscape changes, including dramatic PT decline (Fig. 1c, Supplementary Figs. S6 and S7a). We previously showed that AKI induced PT loss and dedifferentiation are restored to the mature gene expression as AKI resolves²⁹. Instead, substantial S1–S3 PT decline persisted in our long-term models, indicating AKI-to-CKD progression. However, the surviving UIR and UUO Day 28 PTs returned to normal gene expression, except the UUO S3 segment which exhibited persistent low solute-linked carrier encoding gene levels and pro-inflammatory signature³³ (Lyz2, C1qa/b, Ifi30, S100a8/9) (Supplementary Figs. S8 and S9). Moreover, scRNA-seq revealed a separate epithelial cluster, located between loop of Henle and distal tubule on the UMAP, which was present in the control and markedly expanded in the fibrotic kidneys (Fig. 1c, Supplementary Figs. S6 and S7a). We labeled them as “failed repair TECs” (frTECs) due to the simultaneous expression of epithelial (Slc14a2, Cdh1, Calb1), stromal (Cp) and injury related (Cryab, Dcdc2a, Sema5a) genes (Fig. 2a, Supplementary Figs. S5, S8 and S9).

Both models also exhibited evident inflammatory expansion, represented by increased myeloid and lymphoid infiltration along with proliferating macrophages (Figs. 1c, 2a, Supplementary Figs. S7b and S10a,b). While macrophages were the predominant immune fraction in the control, we observed Tcell/NK increase in the fibrotic kidneys. Moreover, scRNA-seq identified three separate fibroblast clusters, named “Fibro 1, 2 and 3” (Figs. 1c, 2b). We noted that Fibro 1 and 2 represented major stromal fractions of the fibrotic kidneys, while Fibro 3 cells were predominant in the controls (Supplementary Fig. S10). Validations on an independent control and injured mice cohorts verified substantial fibrotic remodeling, including statistically significant elevation of classical fibrosis markers Vim and αSma, inflammatory expansion and PT loss at the RNA and protein levels (Supplementary Figs. S11 and S12). Thus, we successfully established two models of kidney fibrosis exhibiting key CKD landmarks⁴³.

Prolonged kidney remodeling elicits pronounced epithelial-to-stromal crosstalk

Since our previous report showed enhanced cell-to-cell interactions in AKI²⁹, we dissected the nature of intercellular crosstalk in advanced kidney injuries^44,45 (Supplementary Table S2). Normal kidneys exhibited epithelial-to-epithelial, epithelial-to-stromal and epithelial-to-immune interactions via calmodulin, cadherin, G-protein coupled receptor, beta-2-microglobulin and urokinase pathways (Supplementary Figs. S13–S15). Both injury models caused enhanced communications (38,538 ligand-receptor pairs in UIR and 44,809 in UUO vs 24,984 pairs in control), including epithelial-to-stromal crosstalk (Supplementary Figs. S16–S19). scRNA-seq predicted that both injuries caused most notable increases in interactions between loop of Henle, collecting duct intercalated and PT S1 and 3 with Fibro 1 clusters, while the smallest number of receptor-ligand encoding gene pairs was found between collecting duct principal and stromal populations (Supplementary Fig. S20, Supplementary Table S3). TECs in the fibrotic kidneys interacted with stromal cells via collagen (Col4a1-Cd47/Itgav/Itgb1), osteopontin (Spp1-Cd44/Itgav/Itgb1), metalloproteinase 2 (Timp2-Itgb1), vascular cell adhesion molecule 1 (Vcam1-Itgb1/b7/a4) pathways. We also observed that Col18a1, which encodes the alpha chain of type XVIII collagen implicated in ureteric tree development⁴⁶ and which we previously reported in AKI²⁹, is elevated in the fibrotic kidney TECs, while it’s receptor encoding genes (Gpc1/4, Itga5/b1) are expressed in Fibro 1, 2 and 3 clusters, highlighting a putative interaction pathway present in both AKI and CKD models. scRNA-seq also showed that both models caused enhanced stromal-to-stromal and stromal-to-epithelial crosstalk (Supplementary Figs. S21 and S22). Fibroblasts interacted with each other and TECs via fibronectin, fibroblast growth factor 9/12, collagen, cadherin 1, transforming growth factor β (Tgfβ) and Wnt4 pathways. Of note, we revealed pronounced tubular-immune and stromal-immune communications, involving B cells, which is consistent with the recent report⁴².

Advanced kidney remodeling exhibits three separate secretory, contractile and migratory fibroblast clusters with distinctive pathway enrichments

Using two independent scRNA-seq platforms, we repeatedly identified three distinctive fibroblast clusters in control and both fibrosis models (Fig. 1c, Supplementary Fig. S23), thus we further examined their unique transcriptional identities. Pathway responsive gene analysis (PROGENy)⁴⁷ identified distinctions between Fibro 1, 2 and 3 clusters (Fig. 2c, Supplementary Fig. S24). Fibro 1 displayed elevation of TNFα, NFκB and JAK-STAT related genes while downregulating WNT and TGFβ. Fibro 2 was enriched for MAPK, EGFR and PI3K on the baseline and acquired WNT and TGFβ elevation in the injured kidneys. Fibro 3 exhibited WNT and TGFβ pathways enrichment on the baseline, which increased in both fibrosis models. Of note, cell division and death related p53 pathway was slightly upregulated in UIR and UUO Fibro 1 cluster and downregulated in Fibro 3 in both injured and control kidneys.

Comparative analysis showed that while all three fibroblast clusters expressed an established renal fibrosis marker Pdgfbβ²³, each population elicited unique molecular identity. Specifically, genes enriched in Fibro 1, including Col1a1, Fn1, Postn and Dcn, were related to ECM production, cartilage development and ossification, which led us to labelling them as “secretory fibroblasts” (Fig. 3a, Supplementary Table S4). RT-qPCR on an independent murine control, UIR and UUO Day 28 cohorts verified scRNA-seq predicted upregulation of ECM related genes, such as Col1a1 and Fn1, in a statistically significant manner (Fig. 3b). Cortical and medullary ECM deposition and fibrotic remodeling was also validated in both models by Picrosirius red staining (Supplementary Fig. S25a). Furthermore, immunofluorescence (IF) demonstrated remarkable elevation of Col1a1 cortical and medullary expression caused by both injuries in another validation experiment on an additional independently generated set of identical control, UIR and UUO animals (Fig. 3c). Thus, multiple rounds of independent validation reproducibly revealed the cortical and medullary spatial pattern of ECM deposition and fibrotic remodeling in both models.

While “Fibro 1” populations exhibited pronounced ECM production and connective tissue development related signature, “Fibro 2” clusters were marked by muscle development, differentiation and contraction related genes, including historical myofibroblast marker Acta2; thus, we identified them as “contractile” (Figs. 3a, 4a–d). Since Fibro 3 signature was enriched for hemopoiesis and immune system related biological processes along with neuron projection and dendrite development, we called them “migratory”. Fibro 3 was also the predominant fibroblast type in the normal kidney and exhibited the smallest fold increase in UIR and UUO, while Fibro 1 cluster was the most abundant in both injuries, exhibiting remarkable increase in the fibrotic kidneys (Fig. 2b, Supplementary Fig. S10c). The existence of fibroblasts with distinctive molecular identities was validated by high resolution IF for Myh11, uniquely labelling Fibro 2 cells according to our scRNA-seq findings (Fig. 4c,e, Supplementary Table S4). We observed the population of Myh11-positive stromal cells the interstitial spaces of control and injured kidneys (Fig. 4d). These cells exhibited low to absent Col1a1 expression, coherent with scRNA-seq data showing very low Col1a1 levels in “Fibro 2” (Fig. 3a). In the meantime, the surrounding Col1a1-enriched fibroblasts did not exhibit any Myh11 expression (Fig. 4d). These findings corroborate the scRNA-seq predicted molecular identities of healthy and fibrotic kidney stroma. In addition to the distinctions, three clusters had shared genes, related to TGFβ response, circulatory process and angiogenesis, cell migration and wound response⁴⁸ (Fig. 4f, Supplementary Fig. S25b). However, many markers, previously used to identify fibroblasts, including Vim, Lgals1, Tagln2 and Meis1/2, exhibited non-specific expression among off-target kidney populations (Fig. 3a).

Long-term kidney injury induced frTECs share transcriptional identity with embryonic and adult distal nephron tubular segments

Next, we questioned the transcriptional identity of tubular clusters in advanced kidney remodeling, particularly frTECs, which exhibited minimal presence in the normal kidney and expanded presence in the fibrotic kidneys (Figs. 1c and 5a). Gene ontology (GO)⁴⁹ analysis identified that frTECs were enriched for kidney and epithelium development (Pax2/8, Cited2, Sox4, Cd24a, Cdh6, Npnt)^50,51,52 (Fig. 5b, Supplementary Table S5), which indicates that these cells might be reverting to the dedifferentiated state as a result of injury⁵³. Moreover, frTECs exhibited signs of mesenchymal transcriptional signature²⁵, elevating genes related to locomotion, cell adhesion, muscle structure development and wounding. Since scRNA-seq demonstrated that frTECs are located between the distal segments of nephron tubule, including loop of Henle, distal tubule and collecting duct, and the fibroblast clusters on the UMAP (Fig. 1c), we asked what the cellular origins of this population might be. Bioinformatic comparison of our datasets to the previously generated E18 WT kidney scRNA-seq data (GSE214024) revealed that adult frTECs mainly align with embryonic loop of Henle, distal tubule and collecting duct (Fig. 5c, Supplementary Table S6). Moreover, comparison between adult tubular clusters produced substantial marker gene overlap between frTECs and distal nephron tubular segments, with only one gene common with S1–S3 PTs (Fig. 5d, Supplementary Table S7). Genes shared between frTECs, distal tubule, loop of Henle and collecting duct included kidney development (Cd24a, Sox4, Pkhd1)^51,52,54, cell adhesion (Epcam, Lgals3, Ezr, Dag1) and apoptotic process (Clu, Cldn7, S100a1) related genes. Of note, frTECs did not exhibit increased Ccl2, which was previously shown to be upregulated in the PTs of sepsis-induced AKI⁵⁵. scRNA-seq also revealed that control frTECs particularly elevated Calb1, which was also enriched in control distal tubules (Fig. 2a).

Prolonged UIR and UUO exhibit persistent distal spatial patterns of tubular injury, while the remaining proximal tubules restore normal gene expression

We noted that frTECs and distal nephron tubular segments exhibited overlapping epithelial injury related molecular identity (Fig. 6a, Supplementary Table S7). This signature included the clinically recognized tubular injury marker Lcn2⁵⁶ and other established markers of failed tubular repair, such as Spp1⁵⁷ and Krt7/8/18/19⁵⁸. This spatial tubular injury pattern was validated with IF on an independent control and fibrotic murine cohorts, which demonstrated increased Krt8 levels in E-cadherin (Ecad)-positive distal nephron tubular segments, including Uromodulin (Umod)-expressing loop of Henle of both UIR and UUO kidneys (Fig. 6b, Supplementary Fig. 26). While both scRNA-seq and IF showed moderate Krt8 expression only in parietal cells of the control collecting ducts, both models elicited dramatic overlap between Krt8 upregulation and Umod/Ecad-positive tubular segments. On the contrary, the remaining PTs restored normal mature gene expression signatures by Day 28 (Fig. 6a, Supplementary Figs. S5, S8 and S9). scRNA-seq revealed no elevation of Havcr1, an established marker of PT injury⁵⁹, in any tubular clusters (Fig. 6a). Moreover, the surviving PTs were spared by other tubular injury markers, including Krt8, which was not present in the remaining Lotus tetragonolobus lectin (LTL)-positive PTs while labelling cortical and medullary Umod-expressing loop of Henle and Dolichos biflorus agglutinin (DBA)-positive distal tubule/collecting duct (Supplementary Fig. S27).

We noted that frTECs expressed several markers previously used to label maladaptively repaired PTs, including Dcdc2a, Sema5a and Vcam1 (Figs. 2a, 6a)^33,60. Quantitative whole-kidney IF analysis revealed that both UIR and UUO result in dramatic Lrp2-positive PT decline and Vcam1 elevation at Day 28 (Fig. 7a). We observed that while normal kidneys express Vcam1 in the interstitial spaces, both injuries resulted in elevated intratubular Vcam1, including colocalization with Krt8 (Fig. 7b, Supplementary Fig. S28). To further dissect the tubular injury patterns in kidney fibrosis, we performed quantitative IF analysis of Krt8 and Vcam1 expression in the proximal (Lrp2-positive) and distal (Ecad-positive) nephron tubular segments (Fig. 7b). As we (Fig. 2a) and others⁶¹ show, Ecad exhibits negligent proximal tubular expression while labelling loop of Henle, distal tubule and both intercalated and parietal cells of the collecting duct in control and fibrotic kidneys. Our quantitative analysis revealed that Ecad-expressing tubular segments exhibited remarkably higher Krt8 and Vcam1 levels compared to Lrp2-positive proximal tubules in both injuries. Overall, our data shows that distal segments of the nephron tubule endure persistent and unresolved injury in advanced kidney fibrotic remodeling.

Kidney fibrosis causes renal developmental program reactivation in the stromal and distal nephron tubular populations

Among the molecular changes underlying pathologic long-term kidney remodeling, we noted robust reactivation of genes normally expressed during nephrogenesis, including Hox genes (Fig. 8a, Supplementary Fig. S29)⁶². Hoxd11 was elevated in Fibro 1 clusters of fibrotic kidneys, while some other isoforms including Hoxb6 were upregulated in distal segments of the UIR and UUO nephron tubule, which was validated with RNAscope (Fig. 8b). Our data also showed elevation of renal developmental genes Cd24a and Sox4 in both UIR and UUO. We recently reported that AKI elicited increased Sox4 and Cd24a expression, which returned to baseline as the injury resolved²⁹. However, scRNA-seq revealed persistent upregulation of these nephrogenic genes in both models of AKI-to-CKD transition, which was validated in additional UIR and UUO cohorts at the RNA and protein levels (Fig. 8c,d, Supplementary Fig. S30). Of note, both renal developmental genes were elevated in the distal nephron tubular segments, frTECs and fibroblasts, sparing the PTs. IF corroborated scRNA-seq findings, identifying that fibrosis caused Sox4 elevation in the loop of Henle, sparing the remaining LTL-positive PTs (Fig. 8e). Collectively, we revealed that AKI-to-CKD transition causes persistent nephrogenic signaling reactivation in multiple populations, including distal segments of the nephron tubule.

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Source: https://www.nature.com/articles/s41598-023-50195-0

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Single-cell sequencing dissects the transcriptional identity of activated fibroblasts and identifies novel persistent distal tubular injury patterns in kidney fibrosis – Scientific Reports

Kidney fibrosis models exhibit proximal tubular loss, functional decline and novel cellular clusters

Prolonged kidney remodeling elicits pronounced epithelial-to-stromal crosstalk

Advanced kidney remodeling exhibits three separate secretory, contractile and migratory fibroblast clusters with distinctive pathway enrichments

Long-term kidney injury induced frTECs share transcriptional identity with embryonic and adult distal nephron tubular segments

Prolonged UIR and UUO exhibit persistent distal spatial patterns of tubular injury, while the remaining proximal tubules restore normal gene expression

Kidney fibrosis causes renal developmental program reactivation in the stromal and distal nephron tubular populations

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