Polyploid tubular cells initiate a TGF-β1 controlled loop that sustains polyploidization and fibrosis after acute kidney injury

Abstract

Polyploidization of tubular cells (TC) is triggered by acute kidney injury (AKI) to allow survival in the early phase after AKI, but in the long run promotes fibrosis and AKI-chronic kidney disease (CKD) transition. The molecular mechanism governing the link between polyploid TC and kidney fibrosis remains to be clarified. In this study, we demonstrate that immediately after AKI, expression of cell cycle markers mostly identifies a population of DNA-damaged polyploid TC. Using transgenic mouse models and single-cell RNA sequencing we show that, unlike diploid TC, polyploid TC accumulate DNA damage and survive, eventually resting in the G1 phase of the cell cycle. In vivo and in vitro single-cell RNA sequencing along with sorting of polyploid TC shows that these cells acquire a profibrotic phenotype culminating in transforming growth factor (TGF)-β1 expression and that TGF-β1 directly promotes polyploidization. This demonstrates that TC polyploidization is a self-sustained mechanism. Interactome analysis by single-cell RNA sequencing revealed that TGF-β1 signaling fosters a reciprocal activation loop among polyploid TC, macrophages, and fibroblasts to sustain kidney fibrosis and promote CKD progression. Collectively, this study contributes to the ongoing revision of the paradigm of kidney tubule response to AKI, supporting the existence of a tubulointerstitial cross talk mediated by TGF-β1 signaling produced by polyploid TC following DNA damage.NEW & NOTEWORTHY Polyploidization in tubular epithelial cells has been neglected until recently. Here, we showed that polyploidization is a self-sustained mechanism that plays an important role during chronic kidney disease development, proving the existence of a cross talk between infiltrating cells and polyploid tubular cells. This study contributes to the ongoing revision of kidney adaptation to injury, posing polyploid tubular cells at the center of the process.

Keywords: CKD; TGF-β1; fibrosis; polyploidy; tubular epithelial cells.

Graphical abstract

Figure 1.

Polyploid tubular cells (TC) with DNA damage accumulate after injury in vivo. A: Uniform Manifold Approximation and Projection (UMAP) of cluster distribution and of cell cycle distribution of mouse proximal tubular cells (PTC) at day 2 and 30 after unilateral ischemia reperfusion injury (uni-IRI). B: barplot showing experimental time distribution in cluster 8 and cluster 9. UMAP distribution of cell cycle activation (Pcna) (C), and cell cycle progression (Aurkb) genes (D). E: matrixplot showing expression of genes involved in cell cycle progression and inhibition. F–H: UMAP distribution of DNA damage markers H2afx, Topbp1, and Rad50. I: representative FACS analysis and gating strategy of mVenus+ TC stained for γH2AX in healthy and 2 days after uni-IRI (n = 5). J: percentage of γH2AX+/mVenus+ TC diploid (i.e., actively proliferating) and polyploid (i.e., undergoing endoreplication). K: representative FACS analysis and gating strategy of mCherry+ TC stained for γH2AX in healthy and 2 days after uni-IRI (n = 5). L: percentage of γH2AX+/mCherry+ TC diploid and polyploid showing accumulation over time in the polyploid population. Statistical significance was calculated by two-sided Mann–Whitney test; numbers on graphs represent exact P values. Bar plots: line = mean, whisker = outlier (coef. 1.5). n = number of mice.

Figure 2.

Polyploid tubular cells (TC) with DNA damage are profibrotic and actively produce transforming growth factor-β1 (TGF-β1) in vitro. A: barplot showing cell cycle phases in mouse polyploid clusters (8 and 9) divided in day 2 (t2) and 30 (t30) after unilateral ischemia reperfusion injury (uni-IRI). B: matrixplot of mouse polyploid clusters (8 and 9) divided in day 2 (t2) and 30 (t30) after uni-IRI, showing TGF-β1 and its receptors. C: Uniform Manifold Approximation and Projection (UMAP) showing human proximal tubular epithelial cell (hPTC) clusters. UMAP distribution of cell cycle activation (PCNA) (D), and cell cycle progression (AURKB) genes (E). F: UMAP distribution of DNA damage marker H2AFX. G–I: FACS analysis of mCherry-hPTC (cells in the G1 phase) showing the gating strategy for sorting. J: cell cycle distribution of diploid and polyploid mCherry-hPTC. A representative experiment out of 4 is shown. K: TGF-β1 gene expression in sorted polyploid mCherry-hPTC over diploid mCherry-hPTC (n = 4). Statistical significance was calculated by two-sided Mann–Whitney test; numbers on graph represent exact P value. Bar plots: line = mean, whisker = outlier (coef. 1.5). n = number of experiments.

Figure 3.

Polyploidization is a self-sustained mechanism stimulated by transforming growth factor-β1 (TGF-β1). A: Uniform Manifold Approximation and Projection (UMAP) showing sample distribution. B: first 20 characteristic genes of vehicle-treated human proximal tubular epithelial cell (hPTC) and TGF-β1-treated hPTC. C: gene set enrichment analysis showing activation of TGF-β pathway. D: gene set enrichment analysis showing activation of AKT pathway, one of the regulator of polyploidy in PTC. E: representative brightfield picture of mCherry-hPTC treated with vehicle (top) or TGF-β1 (bottom) for 48 h. Bar = 400 µm. F: representative picture of mCherry-hPTC treated with vehicle (top) or TGF-β1 (bottom) for 48 h. DAPI counterstains nuclei. Bar = 150 µm. G: cell cycle distribution of vehicle-treated mCherry-hPTC. H: cell cycle distribution of TGF-β1-treated mCherry-hPTC. I: total percentage of mCherry-hPTC in vehicle and TGF-β1 conditions (n = 6). J: percentage of polyploid mCherry-hPTC in vehicle-treated or TGF-β1-treated culture (n = 6). K: cell cycle distribution of TGF-β1-treated mCherry-hPTC. L: cell cycle distribution of TGF-β1 and Fresolimumab-treated mCherry-hPTC. M: total percentage of mCherry-hPTC in vehicle and TGF-β1 conditions (n = 5). N: cell cycle distribution of TGF-β1-treated mCherry-hPTC. O: cell cycle distribution of TGF-β1 and Verteporfin-treated mCherry-hPTC. P: total percentage of mCherry-hPTC in TGF-β1 and TGF-β1 with Verteporfin conditions (n = 4). Q–T: real-time PCR analysis of CTGF, CCL2, VIMENTIN, and SMAD3, following TGF-β1 stimulation and verteporfin treatment (n = 4). Statistical significance was calculated by two-sided Mann–Whitney test; numbers on graphs represent exact P values. Bar plots: line = mean, whisker = outlier (coef. 1.5). n = number of experiments; CTGF, connective tissue growth factor; CCL2, C-C motif chemokine ligand 2.

Figure 4.

Polyploid tubular cells (TC) interact with macrophages and fibroblasts to sustain tubulointerstitial fibrosis. A: dotplot showing transforming growth factor (Tgf)-β1, Tgf-β2, Tgf-βr1, and Tgf-βr2 distribution in all the mouse populations retrieved from kidneys 2 and 30 days after unilateral ischemia reperfusion injury (uni-IRI). B: barplots reporting the number of interactions occurring 2 days after uni-IRI between cluster 9, macrophages, and fibroblasts as source and all the cell types. C: barplots reporting the number of interactions occurring 30 days after uni-IRI between fibroblasts and macrophages as source and all the cell types. D: barplots reporting the number of interactions occurring 30 days after uni-IRI between the cluster 8 and 9 cells as target, and all the cell types. E and F: heatmap reporting the number of interactions between cell types, as source and target, at day 2 and 30 after uni-IRI. G: Circos plot of ligand-receptor interactions among polyploid cluster 9, 8, macrophages, and fibroblasts in kidneys at day 30 after uni-IRI. The populations producing the putative ligand (TGFB1-AR, COL4A1-Integrin a1b1, COL5A2-Integrin a1b1, COL6A3-Integrin a1b1, and FN1-Integrin aVb1) are shown. PTC, proximal tubular cells.

Figure 5.

Tubular cells (TC) interact with monocytes and fibroblasts activating proinflammatory and profibrotic pathways. A: schematic representation of experimental plan for coculture with monocytes. Real-time PCR analysis of hypoxia-inducible factor (HIF)1α (B), MAF(C), IL-6 (D), and transforming growth factor (TGF)-β1 (E) in monocytes following coculture with nonstimulated human proximal tubular epithelial cells (hPTC) and after TGF-β1 and H₂O₂ treatment (n = 4). F: schematic representation of experimental plan for coculture with fibroblasts. G and H: real-time PCR analysis of p21 and CCL2 in fibroblasts following coculture with nonstimulated hPTC and after TGF-β1 and H₂O₂ treatment (n = 4). Statistical significance was calculated by two-sided Mann–Whitney test; numbers on graphs represent exact P values. Bar plots: line = mean, whisker = outlier (coef. 1.5). Empty: transwell with monocytes or fibroblasts without hPTC; t0: monocytes after purification. n = number of experiments; MAF, musculoaponeurotic fibrosarcoma.

Figure 6.

Schematic representation of polyploid tubular cells (TC) response to acute kidney injury (AKI). In response to AKI, TC undergo polyploidization. Polyploid cells are characterized by DNA damage, cell cycle markers and p21 expression 2 days after AKI. In the long-term, polyploid TC start to secrete transforming growth factor (TGF)-β1, which triggers a feedback loop to generate further polyploid TC and activate macrophages and fibroblasts. CKD, chronic kidney disease; γH2AX, H2A Histone family member X; p21, CDKN1A; TGF-βR1, transforming growth factor β receptor 1.