A conditionally immortalized Gli1-positive kidney mesenchymal cell line models myofibroblast transition

Abstract

Glioma-associated oncogene homolog-1 (Gli1)-positive resident mesenchymal stem cell-like cells are the predominant source of kidney myofibroblasts in fibrosis, but investigating Gli1-positive myofibroblast progenitor activation is hampered by the difficulty of isolating and propagating primary cultures of these cells. Using a genetic strategy with positive and negative selection, we isolated Kidney-Gli1 (KGli1) cells that maintain expression of appropriate mesenchymal stem cell-like cell markers, respond to hedgehog pathway activation, and display robust myofibroblast differentiation upon treatment with transforming growth factor-β (TGF-β). Coculture of KGli1 cells with endothelium stabilizes capillary formation. Single-cell RNA sequencing (scRNA-seq) analysis during differentiation identified autocrine ligand-receptor pair upregulation and a strong focal adhesion pathway signal. This led us to test the serum response factor inhibitor CCG-203971 that potently inhibited TGF-β-induced pericyte-to-myofibroblast transition. scRNA-seq also identified the unexpected upregulation of nerve growth factor (NGF), which we confirmed in two mouse kidney fibrosis models. The Ngf receptor Ntrk1 is expressed in tubular epithelium in vivo, suggesting a novel interstitial-to-tubule paracrine signaling axis. Thus, KGli1 cells accurately model myofibroblast activation in vitro, and the development of this cell line provides a new tool to study resident mesenchymal stem cell-like progenitors in health and disease.

Keywords: chronic kidney disease; fibrosis; myofibroblast; pericyte; single-cell RNA sequencing; transcriptomics; transforming growth factor-β.

Fig. 1.

Gli1 labels myofibroblast progenitors across multiple organs. A: tdTomato-labeled Gli1+ cells from Gli1-CreERt²; R26^{tdTomato/DTR-LoxP}; H-2kb^SV40tsA58/WT mice were isolated by FACS from bone chip, heart, lung, aorta, brain, and liver. All cells either had endogenous expression of αSMA or could differentiate into myofibroblasts after treatment with TGF-β. Scale bars on organ samples and fluorescent images of cells (columns 1, 5, and 6) represent 20 µm, whereas scale bars on phase contrast (columns 2, 3, and 4) represent 100 µm. B: quantitative PCR analysis of the primary cell cultures show induction of αSMA in all organ-derived Gli1 cells except lung. Each cell type was tested for myofibroblast differentiation by treatment with 10 ng/ml TGF-β for 24 h. *P < 0.05, unpaired Student’s t-test. Gli1, glioma-associated oncogene homolog-1; TGF-β, transforming growth factor-β.

Fig. 2.

Creation and characterization of Kidney-Gli1 (KGli1) cell lines. A: Gli1 is expressed mainly in the corticomedullary region, adjacent to endomucin-positive endothelial cells. Quadruple transgenic mice (Gli1-CreERt²; R26^{tdTomato/DTR-LoxP}; H-2kb^SV40tsA58/WT) were given tamoxifen at 6 wk old to activate cre-recombinase. Mice were then euthanized, and kidneys were harvested, minced up, and plated out on cell culture plates. After treatment with diphtheria toxin, all non-Gli1 expressing cells (non-tdTomato cells) died off, leaving only Gli1 positive cells. Scale bars on tissue samples represent 20 µm, whereas scale bars on cells represent 100 µm. B: SV40 expression can be activated by culturing of cells at 33°C in the presence of IFNγ and turned off at 39°C. Low levels of SV40 remain when cultured at 37°C with IFNγ, with almost no expression seen at 37°C without IFNγ. C: Gli1 cells are positive for PDGFRβ, CD146, nestin, and NG2 and lack expression of calponin, similar to in vivo. DTX, diphtheria toxin; Gli1, glioma-associated oncogene homolog-1.

Fig. 3.

Kidney Gli1 cells are myofibroblast progenitors. A: kidney Gli1 cells differentiate into myofibroblasts in vitro by treatment with TGF-β. Myofibroblast expression is characterized by increases in αSMA and Ctgf. Increased αSMA is also seen on a proteins level. B: TGF-β induces αSMA protein in association with the actin cytoskeleton. C: kidney Gli1 cells respond to the smoothened agonist, SAG, in vitro after 24 h. A Gli protein inhibitor, GANT61, blocks TGF-β-induced myofibroblast induction, similar to in vivo. D: TGF-β blocks expression of PPARγ, whereas PPARγ agonist rosiglitazone (Rosi) suppresses TGF-β-induced differentiation. ADRP is a PPARγ target gene, which is still repressed by TGF-β. Scale bar, 20 µm. **P < 0.01, ***P < 0.001; statistically significant differences are denoted by letters, with P < 0.05. Gli1, glioma-associated oncogene homolog-1; TGF-β, transforming growth factor-β.

Fig. 4.

Kidney Gli1 cells stabilize vasculogenesis. A: two-dimensional cocultures of HUVEC and Gli1 cells form capillary-like structures on Matrigel. Gli1 cells migrate and associate closely with HUVEC cells, similar to in vivo. In addition, Gli1 cells help to stabilize and improve vasculature networks after 6 h of coculturing (B–E, see materials and methods Coculture Assays). *P < 0.05, **P < 0.01, unpaired Student’s t-test between untreated and treated. Scale bar, 200 µm. Gli1, glioma-associated oncogene homolog-1; HUVEC, human umbilical vein endothelial cells; px, pixels.

Fig. 5.

TGF-β-treated kidney-derived Gli1 cell single-cell RNA sequencing shows increased myofibroblast marker gene expression. A: tSNE plot of hour 0 (red), hour 6 (green), hour 12 (teal) and hour 24 (purple) shows clear separation of hour 0 to all three time points, with a directional differentiation pattern (arrow). B: violin plots of fibrosis-related genes showing an increase in fibrotic gene signature over time. C: heatmap of ligands and receptors upregulated in all TGF-β-treated samples. Receptor list includes receptors, pseudo-receptors, and computer predicted binding receptors. D: KEGG pathway analysis of all receptors and ligands identified from hour 6, hour 12, and hour 24 combined. Scale is represented as the −log₁₀ of the P value. *P < 0.05. ECM, extracellular matrix; Gli1, glioma-associated oncogene homolog-1; TGF-β, transforming growth factor-β; tSNE, t-distributed stochastic neighbor embedding.

Fig. 6.

Unsupervised clustering of cells identifies a critical myofibroblast expansion phase before terminal differentiation. A: tSNE plot of the unsupervised cluster analysis. Seven distinct clusters were identified and annotated based on their gene expression. Arrow indicates the directionality of differentiation through the various stages of myofibroblast differentiation. B: fibrotic gene expression of the various clusters. C: bioinformatic cell cycle scoring of the clusters identifies the myofibroblast expansion cluster as being almost completely in a proliferative stage. D: percentage of cells within the cluster that are in either G1/S or G2/M phase. E: breakdown of each cluster in pseudo-time. Differentiation goes from the start (Gli1 MSC) through to the end (late myofibroblasts) with one branch point corresponding to an undifferentiated stage. Gli1, glioma-associated oncogene homolog-1; Gli1 MSC, Gli1 mesenchymal stromal cells; tSNE, t-distributed stochastic neighbor embedding.

Fig. 7.

Focal adhesion pathway activation drives myofibroblast differentiation. A: heatmap of a select number of genes regulated by serum response factor (SRF) that are involved in focal adhesion from all time points. Expression levels are log fold change compared with hour 0. B: validation of a subset of SRF target genes by quantitative PCR in kidney Gli1 cells. C: dose response of CCG-203971 in kidney Gli1 cells treated with 1 ng/ml of TGF-β to determine therapeutic dosage. A 10 µM concentration of CCG-203971 (−log 2 M) was chosen and had the ability to block TGF-β-induced expression of αSMA. D: fluorescent staining of cells treated with either 1 ng/ml TGF-β alone or 1 ng/ml TGF-β and 10 µM CCG-203971 for 72 h. ***P < 0.001; statistically significant differences are denoted by letters, with P < 0.05. Gli1, glioma-associated oncogene homolog-1; TGF-β, transforming growth factor-β.

Fig. 8.

Ngf is upregulated in kidney myofibroblasts after TGF-β in vitro and after fibrotic injury in vivo. A: tSNE plot showing expression levels of Ngf RNA across all time points. Ngf showed the biggest increase at hour 12. Pseudo-temporal ordering shows Ngf expression turning on the most in the latter stages of differentiation. B: quantitative PCR confirmation of increased Ngf mRNA expression after treatment of KGli1 cells with TGF-β. C: rat kidney fibroblast cell line does not upregulate NGF in response to TGF-β. D: increased NGF mRNA expression after unilateral ischemia-reperfusion injury (IRI). E: increased Ngf expression during acute kidney injury after unilateral ureteral obstruction (UUO). F: Ngf protein expression is also increased after UUO. G: TrkA protein is expressed in tubular epithelium in human kidney. *P < 0.05, ***P < 0.001; statistically significant differences are denoted by letters, with P < 0.05. Gli1, glioma-associated oncogene homolog-1; KGli1, kidney-Gli1; NGF, nerve growth factor; NS, not significant; TGF-β, transforming growth factor-β; tSNE, t-distributed stochastic neighbor embedding. [Image from The Human Protein Atlas (version 18; https://www.proteinatlas.org) with permission under Creative Commons Attribution-ShareAlike 3.0 International License.]