Lineage tracing reveals distinctive fates for mesothelial cells and submesothelial fibroblasts during peritoneal injury

Abstract

Fibrosis of the peritoneal cavity remains a serious, life-threatening problem in the treatment of kidney failure with peritoneal dialysis. The mechanism of fibrosis remains unclear partly because the fibrogenic cells have not been identified with certainty. Recent studies have proposed mesothelial cells to be an important source of myofibroblasts through the epithelial-mesenchymal transition; however, confirmatory studies in vivo are lacking. Here, we show by inducible genetic fate mapping that type I collagen-producing submesothelial fibroblasts are specific progenitors of α-smooth muscle actin-positive myofibroblasts that accumulate progressively in models of peritoneal fibrosis induced by sodium hypochlorite, hyperglycemic dialysis solutions, or TGF-β1. Similar genetic mapping of Wilms' tumor-1-positive mesothelial cells indicated that peritoneal membrane disruption is repaired and replaced by surviving mesothelial cells in peritoneal injury, and not by submesothelial fibroblasts. Although primary cultures of mesothelial cells or submesothelial fibroblasts each expressed α-smooth muscle actin under the influence of TGF-β1, only submesothelial fibroblasts expressed α-smooth muscle actin after induction of peritoneal fibrosis in mice. Furthermore, pharmacologic inhibition of the PDGF receptor, which is expressed by submesothelial fibroblasts but not mesothelial cells, attenuated the peritoneal fibrosis but not the remesothelialization induced by hypochlorite. Thus, our data identify distinctive fates for injured mesothelial cells and submesothelial fibroblasts during peritoneal injury and fibrosis.

Figure 1.

Col1a1-GFP identifies collagen-producing cells in normal and injured peritoneum. (A) Collagen-producing cells expressing enhanced GFP under the regulation of the collagen type I (α1) (Col1a1) promoter and enhancers are SM fibroblasts (arrows), not cytokeratin⁺ MCs (arrowheads), in normal peritoneum of Col1a1-GFP^Tg mice. (B and C) Col1a1-GFP⁺ SM fibroblasts (arrows) express PDGFRβ and vimentin in normal peritoneum. Cells (arrowheads) above Col1a1-GFP⁺ SM fibroblasts, suggesting MCs are PDGFRβ^– and weak vimentin⁺. (D) Cell numbers of Col1a1-GFP⁺ collagen–producing cells and thickness of the nidogen⁺ scar in Col1a1-GFP^Tg mice increase within 4 days after intraperitoneal injection of hypochlorite. Cytokeratin⁺ MCs after hypochlorite injury (arrowheads) also express Col1a1-GFP. (E) Myofibroblasts, characterized by αSMA⁺ and Col1a1-GFP⁺ coexpression, accumulate in the thickened laminin⁺ scar 7 days after hypochlorite injury. Col1a1-GFP⁺ cells on the peritoneal surface, suggesting injured MCs (arrowheads), do not express αSMA. (F) Cytokeratin⁺ MCs (arrowheads) express Col1a1-GFP, not αSMA, 7 days after hypochlorite injury. (G) Col1a1-GFP⁺ cells on the peritoneal surface, suggesting injured MCs (arrowheads), do not express PDGFRβ 7 days after hypochlorite injury. (H) Vimentin is expressed in Col1a1-GFP⁺ cells 7 days after hypochlorite injury. Arrowheads indicate Col1a1-GFP⁺ cells on the peritoneal surface, suggesting injured MCs. All images are taken at the original magnification from the peritoneal covering of the liver unless otherwise specified. Bar, 20 μm. Original magnification, ×630.

Figure 2.

WT1 expression in the adult peritoneum enables efficient labeling of MCs as well as a minor population of SM fibroblasts. (A) Experimental schema for cohort labeling in WT1^CreERT2/+;ROSA26^fstdTomato and WT1^CreERT2/+;ROSA26^fstdTomato;Col1a1-GFP^Tg mice from 10 weeks of age. Analysis is performed on the peritoneal covering of liver 2 weeks after cohort labeling. (B) Cytokeratin⁺;WT1-RFP⁺ (arrowheads) and cytokeratin⁺;WT1-RFP^– MCs (asterisks) are shown in normal peritoneum of WT1^CreERT2/+;ROSA26^fstdTomato mice. Cytokeratin^–;WT1-RFP⁺ cells (arrows) are occasionally seen. The graph shows the percentage of cytokeratin⁺ MCs labeled with WT1-RFP and the percentage of WT1-RFP⁺ cells without cytokeratin expression (mean±SEM, n=6). (C) Three-dimensional images with XZ stacks show cytokeratin⁺;WT1-RFP⁺ MCs (arrowheads) on the surface of normal peritoneum of WT1^CreERT2/+;ROSA26^fstdTomato mice. (D) Vimentin is expressed by both WT1-RFP⁺ cells (arrowheads, suggesting MCs) and WT1-RFP^– cells (arrows, suggesting SM fibroblasts) in normal peritoneum of WT1^CreERT2/+;ROSA26^fstdTomato mice. (E) WT1-RFP⁺;Col1a1-GFP^– MCs (arrowheads) and WT1-RFP^–;Col1a1-GFP⁺ SM fibroblasts (asterisks) separated by the laminin⁺ basal lamina are shown in normal peritoneum of WT1^CreERT2/+;ROSA26^fstdTomato;Col1a1-GFP^Tg mice. WT1-RFP⁺;Col1a1-GFP⁺ SM fibroblasts (arrow) are occasionally seen. The graph shows the percentage of Col1a1-GFP⁺ SM fibroblasts labeled with WT1-RFP and the percentage of WT1-RFP⁺ MCs without Col1a1-GFP expression (n=6). (F) Representative plot shows FACS analysis of peritoneal cells prepared from WT1^CreERT2/+;ROSA26^fstdTomato;Col1a1-GFP^Tg mice after intraperitoneal retention of trypsin-EDTA. The graph shows the percentage of Col1a1-GFP⁺ SM fibroblasts labeled with WT1-RFP and the percentage of WT1-RFP⁺ MCs without Col1a1-GFP expression (n=3). Bar, 20 μm. Original magnification, ×630.

Figure 3.

Injured peritoneum is remesothelialized by surviving MCs. (A) The experimental schema shows cohort labeling followed by hypochlorite injury in WT1^CreERT2/+;ROSA26^fstdTomato mice. Analysis is performed on the peritoneal covering of liver 10 days after injury. (B) Low-powered (upper panel) and high-powered (lower panel) images show cytokeratin⁺;WT1-RFP⁺ MCs (arrows) on the surface of the thickened nidogen⁺ scar. Arrowheads indicate the denuded peritoneum. Only rare WT1-RFP⁺ cells are noted within or beneath the nidogen⁺ scar. The graph beneath shows the percentage of cytokeratin⁺ MCs labeled with WT1-RFP at this time point (n=6). (C) Three-dimensional images with YZ and XZ stacks show WT1-RFP⁺ MCs (arrowheads) on the surface of the thickened nidogen⁺ scar. Only rare WT1-RFP⁺ cells (arrows) are noted within the nidogen⁺ scar. (D) Active proliferation of WT1-RFP⁺ MCs after injury is shown by Ki67 expression (asterisks). (E) Images show that most of WT1-RFP⁺ cells are found above (MCs, arrowheads) or below (SM fibroblasts, arrows) the thickened laminin⁺ scar after injury. Extremely rare WT1-RFP⁺;αSMA⁺ myofibroblasts (asterisks) are within the thickened laminin⁺ scar. The graph beneath shows the percentage of αSMA⁺ myofibroblasts coexpressing WT1-RFP (n=6). (F) Three-dimensional images with YZ and XZ stacks show that most of WT1-RFP⁺ cells are on the peritoneal surface (MCs, arrowheads) after injury. WT1-RFP⁺;αSMA⁺ myofibroblasts (asterisks) are extremely rare. Bar, 20 μm. Original magnification, ×200 in B upper panel; ×630 in B lower panel and C–F.

Figure 4.

Overexpression of TGF-β1 in the peritoneum induces peritoneal fibrosis in WT1^CreERT2/+;ROSA26^fstdTomato mice. (A) Experimental schema for cohort labeling in WT1^CreERT2/+;ROSA26^fstdTomato mice followed by injection of AdTGF-β1 transgenic expression. Analysis is performed on the peritoneal covering of liver 10 days after AdTGF-β1. (B) Low-powered (upper panel) and high-powered images (lower panel) show large areas of the peritoneal surface devoid of WT1-RFP⁺ MCs. Occasional WT1-RFP⁺ cells (SM fibroblasts, arrows) are found beneath the accumulated αSMA⁺ myofibroblasts. Rare WT1-RFP⁺;αSMA⁺ myofibroblasts (asterisks) are identified. (C) Surviving WT1-RFP⁺ MCs (arrowheads) on the surface of injured peritoneum do not express αSMA. Rare WT1-RFP⁺;αSMA⁺ myofibroblasts (asterisks), amounting to 14.6% of αSMA⁺ myofibroblasts, are seen exclusively within the thickened laminin⁺ scar. WT1-RFP⁺;αSMA^– SM fibroblast (arrows) is located beneath the scar. (D) Images show WT1-RFP⁺;cytokeratin⁺ MCs on the peritoneal surface after AdTGF-β1 (arrowheads). A WT1-RFP⁺;cytokeratin^– SM fibroblast (arrows) is seen. (E) MCs isolated and cultured from peritoneal WT1-RFP⁺;PDGFRβ-APC^– cells of WT1^CreERT2/+;ROSA26^fstdTomato mice after cohort labeling lack expression of αSMA in culture medium alone (CON), whereas activate expression of αSMA in the presence of TGF-β1 for 2 days. Scale bar, 20 μm. Original magnification, ×200 in B upper panel; ×630 in B lower panel and C–E.

Figure 5.

SM fibroblasts are the major precursors of collagen-producing cells during peritoneal fibrosis after hypochlorite injury. (A) Experimental schema for cohort labeling of Col1a2⁺ cells and hypochlorite peritoneal injury in Col1a2-CreERT^Tg;ROSA26^fstdTomato;Col1a1-GFP^Tg mice. Analysis is performed on the peritoneal covering of liver before and 7 days after hypochlorite injury. (B and C) Images show tamoxifen-induced cohort labeling of Col1a1-GFP⁺ SM fibroblasts with Col1a2-RFP before (B) and 7 days after hypochlorite injury (C). Arrowheads and arrows indicate Col1a1-GFP⁺ with and without coexpression of Col1a2-RFP, respectively. Numerous DAPI⁺ nuclei without labeling of RFP or GFP noted at the surface of normal peritoneum suggest MCs (asterisks) (B). (D) The graph shows the percentages of Col1a1-GFP⁺ cells that coexpress the fate reporter Col1a2-RFP and the proportion of Col1a2-RFP⁺ cells that coexpress Col1a1-GFP before (Con) and 7 days (Hypochlorite) after injury (n=6 per group). DAPI, 4′,6-diamidino-2-phenylindole. Bar, 20 μm. Original magnification, ×630.

Figure 6.

Col1a2-RFP⁺ fate-mapped SM fibroblasts are the major precursors of myofibroblasts in the peritoneum after hypochlorite injury. (A) Experimental schema for cohort labeling and hypochlorite injury in Col1a2-CreERT^Tg;ROSA26^fstdTomato mice. Analysis is performed on the peritoneal covering of liver 7 or 10 days after injury. (B) Active proliferation of Col1a2-RFP⁺ SM fibroblasts after injury is shown by Ki67 expression (arrows). (C) Images show numerous Col1a2-RFP⁺;αSMA⁺ myofibroblasts (arrowheads) after injury. Col1a2-RFP⁺;αSMA^– SM fibroblasts and DAPI⁺;Col1a2-RFP^–;αSMA^– MCs at the peritoneal surface are indicated by arrows and asterisks, respectively. The graph beneath shows the percentage of αSMA⁺ myofibroblasts coexpressing the fate marker Col1a2-RFP (n=6 per group). (D) Three-dimensional images with YZ and XZ stacks show numerous Col1a2-RFP⁺;αSMA⁺ myofibroblasts after injury. Cells are indicated as in C. (E) Images show that cytokeratin⁺ MCs (arrowheads) do not express the fate marker Col1a2-RFP. (F) Three-dimensional images with YZ and XZ stacks show numerous Col1a2-RFP⁺ cells beneath cytokeratin⁺ MCs after injury. Cytokeratin⁺ MCs (arrowheads) do not express the fate marker Col1a2-RFP. DAPI, 4′,6′-diamidino-2-phenylindole. Bar, 20 μm. Original magnification, ×630.

Figure 7.

TGF-β1 induces αSMA expression in cultured SM fibroblasts. (A) Peritoneal Col1a1-GFP⁺ SM fibroblasts are isolated and cultured from normal Col1a1-GFP^Tg mice after intraperitoneal retention of trypsin-EDTA. The bright-field image shows SM fibroblasts at passage two cultured in the treated cell culture dish. (B) Col1a1-GFP⁺ SM fibroblasts cultured in the chamber slide lack expression of αSMA in culture medium alone (CON), whereas they activate expression of αSMA in the presence of TGF-β1 for 2 days. (C) Col1a1-GFP⁺ SM fibroblasts are cultured in the presence or absence of TGF-β1 for 24 hours. Quantitative PCR shows that Acta2 and Col1a1 are upregulated by TGF-β1. The expression levels are normalized by Gapdh and expressed as the mean±SEM (n=3). ^†P<0.01. Bar, 25 μm in A; 20 μm in B. Original magnification, ×200 in A; ×630 in B.

Figure 8.

The illustration indicates the major fates of MCs and SM fibroblasts after peritoneal injury.