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Lineage Tracing Reveals Distinctive Fates for Mesothelial Cells and Submesothelial Fibroblasts during Peritoneal Injury Yi-Ting Chen,*†‡ Yu-Ting Chang,* Szu-Yu Pan,† Yu-Hsiang Chou,*† Fan-Chi Chang,*† Pei-Ying Yeh,* Yuan-Hung Liu,§ Wen-Chih Chiang,† Yung-Ming Chen,† Kwan-Dun Wu,† Tun-Jun Tsai,† Jeremy S. Duffield,| and Shuei-Liong Lin*† *Graduate Institute of Physiology, College of Medicine, and †Renal Division, Department of Internal Medicine, National Taiwan University Hospital, National Taiwan University, Taipei, Taiwan; ‡Department of Internal Medicine, E-DA Hospital, I-Shou University, Kaohsiung, Taiwan; §Department of Cardiovascular Medicine, Far Eastern Memorial Hospital, New Taipei City, Taiwan; and |Institute for Stem Cell and Regenerative Medicine, and Kidney Research Institute, University of Washington, Seattle, Washington

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 a-smooth muscle actin–positive myofibroblasts that accumulate progressively in models of peritoneal fibrosis induced by sodium hypochlorite, hyperglycemic dialysis solutions, or TGF-b1. 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 a-smooth muscle actin under the influence of TGF-b1, only submesothelial fibroblasts expressed a-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. J Am Soc Nephrol 25: 2847–2858, 2014. doi: 10.1681/ASN.2013101079

Many patients with kidney failure rely on the peritoneal membrane to perform life-saving dialysis.1–3 In addition to changes in permeability of the peritoneal membrane, the dialysis process itself frequently triggers a fibrosing process that progressively reduces membrane function resulting in dialysis failure, sometimes with high patient mortality.4–7 In a small percentage of patients, severe fibrosis occurs primarily in the visceral peritoneum, resulting in encapsulating peritoneal sclerosis (EPS), a catastrophic complication with obscure pathogenesis and a high mortality rate.6,7 Such dialysis failure is characterized by progressive peritoneal fibrosis that can be seen as with J Am Soc Nephrol 25: 2847–2858, 2014

thickening of basal lamina and accumulation of a-smooth muscle actin (aSMA) + myofibroblasts.4,5,8–10

Received October 15, 2013. Accepted March 1, 2014. Published online ahead of print. Publication date available at www.jasn.org. Correspondence: Dr. Shuei-Liong Lin, Graduate Institute of Physiology, College of Medicine, National Taiwan University, No. 1, Jen-Ai Road Section 1, Taipei, Taiwan 100. Email: linsl@ntu. edu.tw Copyright © 2014 by the American Society of Nephrology

ISSN : 1046-6673/2512-2847

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The peritoneum is composed of mesothelium, basal lamina, and submesothelial (SM) connective tissue.11–13 The mesothelium consists of a single layer of flattened mesothelial cells (MCs) that lines the peritoneal cavity and internal organs.12,14–16 In many circumstances such as organ development or tissue injury repair, MCs are the cellular source of growth factors including TGF-b1, PDGF, and vascular endothelial growth factor, which support cell proliferation and differentiation of parenchymal and stromal cells as well as angiogenesis.8,17–22 By contrast, SM connective tissue containing plexuses of blood vessels, lymphatic channels, and scattered fibroblasts has drawn much less attention.22–24 A number of studies suggested MCs as the major source of myofibroblasts through the epithelial–mesenchymal transition (EMT) during peritoneal fibrosis.8,18,25–27 However, these studies relied predominantly on in vitro experiments to show that MCs can be stimulated to express aSMA and produce matrix proteins outside of the body under the influence of profibrotic agents such as TGFb1.8,25–28 Nevertheless, confirmatory studies of mesothelial EMT in vivo are lacking even though costaining of cytokeratin and aSMA was previously shown.8,18 The contribution of SM fibroblasts to myofibroblasts in vivo is not clear despite some studies in vitro have suggested.22–24 A conditional cell lineage analysis using WT1CreERT2/+ mice demonstrated that Wilms’ tumor-1 (WT1)+ septum transversum mesenchyme gives rise to MCs, SM fibroblasts covering the liver, and hepatic stellate cells within the liver during hepatic development.12,13 Using WT1CreERT2/+ mice, a recent study reported that WT1+ MCs may differentiate into myofibroblasts in liver injury.29 WT1 expression in MCs is observed in both embryonic development and adult peritoneum; however, the expression of WT1 by SM fibroblasts is not clearly defined in the adult peritoneum.12,13,21,30–32 Hence, the progenitors of myofibroblasts in the injured liver and the peritoneum remain controversial despite these studies. Because efforts to design new antifibrotic therapies require a rigorous understanding of the cellular origin of myofibroblasts in vivo, we performed lineage tracing of both MCs and SM fibroblasts in models of peritoneal fibrosis induced by sodium hypochlorite solution, hyperglycemic dialysis solution, or adenovirus-expressing TGF-b1 (AdTGF-b1). Although these models are more akin to EPS than the progressive thickening peritoneum seen in humans on peritoneal dialysis, they represent robust tools to study the pathogenesis of peritoneal fibrosis in the laboratory.33–36 Contrary to the prevailing model, our findings indicate that peritoneal myofibroblasts derive from SM fibroblasts and peritoneal membrane disruption is repaired by surviving MCs.

RESULTS Expanded Population of Myofibroblasts in Models of Peritoneal Fibrosis

Using Col1a1-GFP transgenic reporter mice (Col1a1-GFPTg) expressing enhanced green fluorescent protein (GFP) under 2848

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the regulation of the Col1a1 promoter and enhancers, we studied the collagen-producing cells in normal and injured peritoneum. Col1a1-GFP–positive cells were cytokeratin–, PDGF receptor-b (PDGFRb)+, and vimentin+, and lay beneath the nidogen+ basal lamina (Figure 1, A–C). We called these cells SM fibroblasts. Seven days after hypochlorite injury, the population of Col1a1-GFP+ cells expanded in the peritoneum of the liver, omentum, and abdominal wall (Supplemental Figure 1). In addition, we noted diffuse thickening of the basal lamina to become scar tissue (Figure 1, D and E). Because the preparation of peritoneum covering solid organs for immunofluorescence study was easier, we showed the findings of peritoneal covering of liver unless otherwise specified. After hypochlorite injury, cytokeratin+ staining was not detected at many surfaces of the peritoneum, suggesting the loss of MCs. At peritoneal surfaces in which MCs remained attached after injury, the cytokeratin+ MCs could now be seen to express Col1a1-GFP, in sharp contrast with the healthy state (Figure 1, A, D, and F). Cytokeratin+ MCs did not express detectable aSMA (Figure 1F). Myofibroblasts, defined by coexpression of aSMA and Col1a1-GFP, accumulated markedly within the thickened basal lamina between MCs and SM fibroblasts (Figure 1, E and F), and expressed both PDGFRb and vimentin (Figure 1, G and H). The observations that injured cytokeratin+ MCs generated Col1a1 transcripts and that aSMA+ myofibroblasts accumulated in the thickened laminin+ scar were reproduced in Col1a1-GFPTg mice 10 days after intraperitoneal injection of AdTGF-b1 (Supplemental Figure 2). The Fate Marker Activated in WT1CreERT2/+ Mice Identified MCs and a Small Population of SM Fibroblasts

Permanent expression red fluorescence protein (RFP) was activated in WT1-expressing cells by somatic DNA recombination in adult WT1CreERT2/+;ROSA26fstdTomato (WT1-RFP) mice. Activation was induced conditionally by oral tamoxifen administration so that a cohort of cells was permanently labeled only during tamoxifen exposure (Figure 2A). On the peritoneal surface, 83.3% of the cytokeratin+ MCs underwent somatic recombination and were referred to as WT1-RFP+ MCs (Figure 2, B and C, Supplemental Figure 3). In addition, WT1-RFP+;cytokeratin– cells, representing 23.6% of all WT1-RFP+ cells, were seen beneath the mesothelium in keeping with labeling of SM fibroblasts (Figure 2B). In addition to notable expression in SM fibroblasts, vimentin was detectable in normal WT1-RFP+ MCs (Figures 1C and 2D), suggesting that MCs normally expressed this mesenchymal protein. To confirm that WT1-RFP+ cells beneath the mesothelium were SM fibroblasts, we generated WT1CreERT2/+;ROSA26fstdTomato;Col1a1-GFPTg mice. Of the Col1a1-GFP+ SM fibroblasts, 17.6% expressed WT1-RFP, indicating that CreERT2 at the WT1 locus activated ROSA26fstdTomato in a small population of SM fibroblasts (Figure 2E). Furthermore, 74.8% of all WT1-RFP+ cells were Col1a1-GFP– and were on the peritoneal surface, in keeping with them being MCs (Figure 2E). FACS analysis showed similar results (Figure 2F). J Am Soc Nephrol 25: 2847–2858, 2014

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Injured Peritoneum Was Remesothelialized by WT1-RFP+ MCs

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 (a1) (Col1a1) promoter and enhancers are SM fibroblasts (arrows), not cytokeratin+ MCs (arrowheads), in normal peritoneum of Col1a1-GFPTg mice. (B and C) Col1a1-GFP+ SM fibroblasts (arrows) express PDGFRb and vimentin in normal peritoneum. Cells (arrowheads) above Col1a1-GFP+ SM fibroblasts, suggesting MCs are PDGFRb– and weak vimentin+. (D) Cell numbers of Col1a1-GFP+ collagen– producing cells and thickness of the nidogen+ scar in Col1a1-GFPTg mice increase within 4 days after intraperitoneal injection of hypochlorite. Cytokeratin+ MCs after hypochlorite injury (arrowheads) also express Col1a1-GFP. (E) Myofibroblasts, characterized by aSMA+ 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 aSMA. (F) Cytokeratin+ MCs (arrowheads) express Col1a1-GFP, not aSMA, 7 days after hypochlorite injury. (G) Col1a1-GFP+ cells on the peritoneal surface, suggesting injured MCs (arrowheads), do not express PDGFRb 7 days after hypochlorite injury. (H) Vimentin is expressed in Col1a1-GFP+ cells 7 days after hypochlorite injury. Arrowheads indicate Col1a1-GFP+

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Peritoneal fibrosis was initiated by hypochlorite in WT1CreERT2/+;ROSA26fstdTomato mice 2 weeks after the last dose of tamoxifen and the fate of the cohort of WT1-RFP+ cells was mapped (Figure 3A). Within 10 days after injury, peritoneal surfaces were partially covered by WT1-RFP+ or cytokeratin+ MCs (Figure 3, B and C), but many surfaces were devoid of MCs (Figure 3B). WT1RFP expression was noted in 85.6% of cytokeratin+ MCs (Figure 3B). There was no dilution of the proportion of cytokeratin+ MCs with the fate marker WT1-RFP before and after injury (Figures 2B and 3B). Active proliferation in WT1-RFP+ MCs was confirmed by positive Ki67 staining (Figure 3D). No recombination of somatic DNA in the absence of tamoxifen administration was detected before and after injury (Supplemental Figure 4). In contrast with the active involvement of surviving MCs in remesothelialization after hypochlorite injury (Figure 3, B–D), only a few WT1-RFP+ cells coexpressing aSMA were found within the thickened laminin+ scar (Figure 3, E and F, Supplemental Figure 5). Most of the WT1-RFP+;a SMA– cells were found on the peritoneal surfaces, indicating that they were MCs as those observed in Figures 2 and 3C, the others were beneath the thickened scar and therefore SM fibroblasts (Figure 3, E and F, Supplemental Figure 5). The percentage of aSMA+ cells expressing WT1RFP was 15.9%, representing a proportion similar to that of Col1a1-GFP+ SM fibroblasts expressing WT1-RFP (Figures 2, E and F, and 3, E and F). Furthermore, WT1-RFP+ cells within and beneath the thickened scar expressed PDGFRb, but WT1-RFP+ MCs did not (Supplemental Figure 6), collectively suggesting that the minor population of WT1-RFP+ SM fibroblasts is the source of the minor population of WT1-RFP+ myofibroblasts.

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 mm. Original magnification, 3630.

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To validate the fate of WT1 lineage of cells in peritoneal injury, we studied a second model of peritoneal fibrosis induced by daily intraperitoneal injection, for 2 weeks, of dialysis solution containing 4.25% glucose and 40 mM of the glucose degradation product methylglyoxal (Supplemental Figure 7A). Although the injury was much milder than that induced by hypochlorite, aSMA+ myofibroblasts was detectable (Supplemental Figure 7B). Similar to the observation in the hypochlorite-induced model, WT1-RFP+ cells were noted on the peritoneal surface or beneath the thickened scar (Supplemental Figure 7, B and C). A low percentage (16.5%) of aSMA+ myofibroblasts coexpressing WT1-RFP was detected within the laminin+ scar (Supplemental Figure 7B). Similar to the findings in the hypochlorite-induced model, .85% of the cytokeratin+ MCs at the peritoneal surface were WT1-RFP+ before and after injury (Supplemental Figure 7C). TGF-b1 Upregulated aSMA in WT1RFP+ MCs In Vitro but Not In Vivo

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 WT1CreERT2/+;ROSA26fstdTomato and WT1CreERT2/+;ROSA26fstdTomato;Col1a1-GFPTg 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 WT1CreERT2/+;ROSA26fstdTomato 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 (mean6SEM, n=6). (C) Three-dimensional images with XZ stacks show cytokeratin+;WT1-RFP+ MCs (arrowheads) on the surface of normal peritoneum of WT1CreERT2/+;ROSA26fstdTomato 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 WT1CreERT2/+;ROSA26fstdTomato 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 WT1CreERT2/+;ROSA26fstdTomato; Col1a1-GFPTg 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 WT1CreERT2/+;ROSA26fstdTomato;Col1a1-GFPTg mice after intraperitoneal retention of trypsin-EDTA. The

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We induced a third model of peritoneal fibrosis by intraperitoneal injection of AdTGF-b1 in WT1CreERT2/+;ROSA26fstdTomato mice (Figure 4A). Before AdTGF-b1 administration, 82.6% of cytokeratin+ MCs were labeled with the WT1-RFP fate marker. Ten days after AdTGF-b1 administration, marked accumulation of aSMA+ myofibroblasts was seen in the peritoneum (Figure 4, B and C). In addition to WT1RFP+;aSMA– SM fibroblasts beneath the thickened laminin+ scar, occasional scattered WT1-RFP+;aSMA+ myofibroblasts were noted within the thickened scar but these amounted to only 14.6% of aSMA+ myofibroblasts (Figure 4, B and C). aSMA was not detected in WT1-RFP+ MCs on the peritoneal surface (Figure 4C). In peritoneal areas in which coverage by MCs remained intact, .85% of cytokeratin+ MCs were WT1-RFP+ (Figure 4D).

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 mm. Original magnification, 3630.

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To determine whether primary MCs had the capacity to express aSMA in vitro, which some investigators have used to define the mesothelial EMT process, we isolated and cultured WT1-RFP+;PDGFRb-APC– MCs from WT1CreERT2/+;ROSA26fstdTomato mice after tamoxifen pretreatment (Supplemental Figure 8). MCs in culture medium alone lacked detectable aSMA (Figure 4E). In the presence of TGF-b1 for 2 days, cultured MCs activated expression of aSMA (Figure 4E), indicating that although MCs in vivo do not express aSMA, they are capable of activating this protein in vitro. In contrast with the upregulation of mesenchymal genes (Acta2 and Col1a1) by TGF-b1 in cultured MCs, TGF-b1 suppressed Gpm6a (glycoprotein m6a, a MC marker) (Supplemental Figure 9). However, Krt8 (cytokeratin8) was upregulated by TGF-b1 (Supplemental Figure 9). SM Fibroblasts Were the Major Precursors of Peritoneal Myofibroblasts

Figure 3. Injured peritoneum is remesothelialized by surviving MCs. (A) The experimental schema shows cohort labeling followed by hypochlorite injury in WT1CreERT2/+; ROSA26fstdTomato 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+;aSMA+ myofibroblasts (asterisks) are within the thickened laminin+ scar. The graph beneath shows the percentage of aSMA+ 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+;aSMA+ myofibroblasts (asterisks) are extremely rare. Bar, 20 mm. Original magnification, 3200 in B upper panel; 3630 in B lower panel and C–F.

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To determine whether SM fibroblasts were the primary source of aSMA+ myofibroblasts, we generated Col1a2-CreERT Tg ; ROSA26 fstdTomato ;Col1a1-GFP Tg mice in which cells expressing the Col1a2 chain of collagen I could undergo somatic recombination to express the tdTomato RFP permanently after tamoxifen administration (Col1a2-RFP+ cells). Dynamic expression of the Col1a1 chain reported by the Col1a1-GFP transgene was used to define SM fibroblasts. The cohort of Col1a1-GFP+ SM fibroblasts induced by tamoxifen to express Col1a2-RFP was 64.2% in Col1a2CreERTTg;ROSA26fstdTomato;Col1a1-GFPTg mice (the numbers of Col1a2-RFP+ cells and Col1a1-GFP+ cells were 2.8 and 4.4 cells per field at 3630 magnification, respectively) (Figure 5, A, B, and D). Two weeks after cohort labeling, hypochlorite injury was induced. After 1 week, the numbers of Col1a2-RFP+ cells and Col1a1-GFP+ cells increased (11.7 and 18.5 cells per field at 3630 magnification, respectively); the proportion of Col1a1-GFP+ cells that coexpressed Col1a2-RFP was 63.0% (Figure 5, A, C, and D). The absence of a significant decrease in the proportion of Col1a1-GFP+ cells coexpressing Col1a2-RFP indicated that SM fibroblasts, rather than other cell sources, were the major precursors of collagen-producing cells in the injured Lineage Tracing during Peritoneal Injury

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Figure 4. Overexpression of TGF-b1 in the peritoneum induces peritoneal fibrosis in WT1CreERT2/+;ROSA26fstdTomato mice. (A) Experimental schema for cohort labeling in WT1CreERT2/+;ROSA26fstdTomato mice followed by injection of AdTGF-b1 transgenic expression. Analysis is performed on the peritoneal covering of liver 10 days after AdTGF-b1. (B) Low-powered (upper panel) and high-powered images (lower panel) show large areas of the peritoneal surface devoid of WT1-RFP+ MCs. Occasional WT1RFP+ cells (SM fibroblasts, arrows) are found beneath the accumulated aSMA+ myofibroblasts. Rare WT1-RFP+;aSMA+ myofibroblasts (asterisks) are identified. (C) Surviving WT1-RFP+ MCs (arrowheads) on the surface of injured peritoneum do not express aSMA. Rare WT1-RFP+;aSMA+ myofibroblasts (asterisks), amounting to 14.6% of aSMA+ myofibroblasts, are seen exclusively within the thickened laminin+ scar. WT1-RFP+;aSMA– SM fibroblast (arrows) is located beneath the scar. (D) Images show WT1-RFP+;cytokeratin+ MCs on the peritoneal surface after AdTGF-b1 (arrowheads). A WT1-RFP+;cytokeratin– SM fibroblast (arrows) is seen. (E) MCs isolated and cultured from peritoneal WT1RFP+;PDGFRb-APC– cells of WT1CreERT2/+;ROSA26fstdTomato mice after cohort labeling lack expression of aSMA in culture medium alone (CON), whereas activate expression of aSMA in the presence of TGF-b1 for 2 days. Scale bar, 20 mm. Original magnification, 3200 in B upper panel; 3630 in B lower panel and C–E.

peritoneum. To be sure that these SM fibroblasts became aSMA+ myofibroblasts, we induced peritoneal fibrosis by hypochlorite injection in Col1a2-CreERTTg;ROSA26fstdTomato mice 2 weeks after tamoxifen treatment to label a 64.2% cohort of SM fibroblasts (Figure 6A). Before hypochlorite injury, Col1a2-RFP+ SM fibroblasts were beneath cytokeratin+ MCs (Supplemental Figure 10). Seven days after hypochlorite injection, many Col1a2RFP+ cells entered the cell cycle evidenced by expression of Ki67 (Figure 6B). Col1a2-RFP + cells accumulated within the 2852

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thickened peritoneum and accounted for .60% of aSMA+ myofibroblasts, confirming SM fibroblasts as the major precursors of peritoneal myofibroblasts (Figure 6, C and D, Supplemental Figure 11) and also supporting WT1-RFP+ SM fibroblasts as the progenitors of WT1-RFP+;aSMA+ myofibroblasts in WT1CreERT2/+;ROSA26fstdTomato mice after peritoneal injury (Figures 3, E and F, and 4, B and C, Supplemental Figures 5–7). Although remesothelialization occurred, no cytokeratin+ MCs coexpressed Col1a2-RFP, indicating that the injured peritoneum is not remesothelialized by SM fibroblasts (Figure 6, E and F, Supplemental Figure 12) and also excluding the possible contribution of WT1RFP+ SM fibroblasts to remesothelialization (Figure 3, B and C). Without cohort labeling by tamoxifen, ,0.01% of cells expressed RFP before or after hypochlorite injury, indicating that “leaky” somatic recombination could not be responsible for the appearance of Col1a2RFP+ cells after injury (Supplemental Figure 13). In separate cohorts of Col1a2-CreERTTg; ROSA26fstdTomato mice, we induced a second model of fibrosis by AdTGF-b1 injection (Supplemental Figure 14A). Ten days after viral administration, we observed marked expansion of the Col1a2-RFP+ SM fibroblasts, which again accounted for 62.5% of myofibroblasts, indicating in this second model that SM fibroblasts are the major source of myofibroblasts during peritoneal fibrosis (Supplemental Figure 14B). To determine whether primary SM fibroblasts had the capacity to express aSMA in vitro, we isolated and cultured Col1a1-GFP+ SM fibroblasts from normal Col1a1-GFPTg mice (Figure 7A). SM fibroblasts in culture medium alone lacked detectable aSMA (Figure 7B). In the presence of TGF-b1 for 2 days, SM fibroblasts activated expression of aSMA (Figure 7B). Quantitative PCR (QPCR) showed the increase of Acta2 and Col1a1 by TGF-b1 (Figure 7C).

Imatinib Reduced Peritoneal Myofibroblasts and Fibrosis

Because SM fibroblasts, not MCs, express PDGFRb and SM fibroblasts are the precursors of scar-forming myofibroblasts, we blocked PDGFR signaling during the hypochlorite model using the PDGFR tyrosine kinase inhibitor imatinib (Supplemental Figure 15A). Imatinib significantly attenuated peritoneal adhesion, thickening of fibrotic peritoneum, and J Am Soc Nephrol 25: 2847–2858, 2014

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these peritoneal fibrosis models derive from SM fibroblast precursors, rather than from an alternate cell precursor. Although conditional labeling of all SM fibroblasts would be desirable, the labeling of 65% of cells is the technical limit of labeling using these genetic tools in adult mice. We mapped the fate of MCs using a similar strategy to that for mapping the fate of SM fibroblasts. The transcriptional regulator WT1 is expressed by adult MCs and therefore the conditional Cre enzyme knocked into the WT1 locus was used to map MCs. Our conditional labeling strategy resulted in labeling a cohort of .80% of all MCs. It also labeled a minor population (approximately 15%) of SM fibroblasts. In response to peritoneal injury, the cohort of somatically + Figure 5. SM fibroblasts are the major precursors of collagen-producing cells during labeled WT1 cells did not expand. In fact, peritoneal fibrosis after hypochlorite injury. (A) Experimental schema for cohort labeling of there was a reduction in cell number. The Col1a2+ cells and hypochlorite peritoneal injury in Col1a2-CreERTTg;ROSA26fstdTomato; proportion of myofibroblasts that derived Col1a1-GFPTg mice. Analysis is performed on the peritoneal covering of liver before and from WT1-labeled cells was approximately 7 days after hypochlorite injury. (B and C) Images show tamoxifen-induced cohort la- 15%, a number consistent with these myofibeling of Col1a1-GFP+ SM fibroblasts with Col1a2-RFP before (B) and 7 days after hy- broblasts deriving from the WT1-labeled SM pochlorite injury (C). Arrowheads and arrows indicate Col1a1-GFP+ with and without fibroblasts in the normal adult, rather than coexpression of Col1a2-RFP, respectively. Numerous DAPI+ nuclei without labeling of from the cohort of WT1-labeled MCs. RFP or GFP noted at the surface of normal peritoneum suggest MCs (asterisks) (B). (D) Although our data did not support the The graph shows the percentages of Col1a1-GFP+ cells that coexpress the fate reporter transition of MCs to myofibroblasts in vivo, + Col1a2-RFP and the proportion of Col1a2-RFP cells that coexpress Col1a1-GFP before MCs were found to express Col1a1 after peri(Con) and 7 days (Hypochlorite) after injury (n=6 per group). DAPI, 49,6-diamidino-2toneal injury. Therefore, the role of MCs in phenylindole. Bar, 20 mm. Original magnification, 3630. peritoneal fibrosis cannot be excluded because they produce Col1a1 after injury. Although EMT results in differentiation of epithelial cells to miaccumulation of aSMA+ myofibroblasts despite no change in gratory mesenchymal cells in the setting of cancer metastasis and the remesothelialization induced by hypochlorite (Suppledevelopment, recent fate mapping experiments in mice using mental Figure 15, B–E). conditional somatic recombination techniques led to a reappraisal of the importance of EMT as an explanation for the appearance of myofibroblasts in multiple organs as well as to a new DISCUSSION appreciation for discrete mesenchymal cells known as resident fibroblasts or pericytes as the major origin of myofibroblasts in These studies report an extensive population of Col1a1+;Col1a2+; many tissues, including the liver, muscle, skin, intestine, lung, PDGFRb+ SM fibroblasts lying below the basal lamina of the spinal cord, and kidney.37–44 Our studies stand in contrast with normal peritoneal membrane, which produce collagen protein in healthy states. By mapping the fate of cohorts of conditional, previous studies that reported MCs as an important source of somatically labeled SM fibroblasts, our studies show that they peritoneal myofibroblasts8,25–27; rather, our studies support SM +; transdifferentiate into an expanding population of Col1a1 fibroblasts as the major source of myofibroblasts during peritoneal fibrosis. Our studies represent an advance of the previous Col1a2+;PDGFRb+;aSMA+ myofibroblasts in the region of pathstudies that analyzed MC responses to peritoneal injury for two ologic matrix production. These cells thus are a major source of myofibroblasts in three different models of peritoneal fibrosis and reasons. First, we used conditional labeling of cohorts of cells with robust Cre-expressing mouse lines, whereas previous studies used are an important new cellular target for fibrosing diseases of the less robust methods including immunohistochemistry and cell peritoneum. Our labeling strategies did not label all SM fibroculture in vitro. Second, we described a poorly appreciated popblasts, but we successfully labeled cohorts of approximately 65% of these discrete SM fibroblast cells and they expanded in disease ulation of SM fibroblasts that were not considered in those previous studies. Although variations in experimental conditions cannot settings to become approximately 65% of all myofibroblasts. The be excluded as a cause for differing results, our mapping strategy fact that the proportion of cells did not change from healthy to provides the most robust approach reported to date. disease states suggests that the vast majority of myofibroblasts in J Am Soc Nephrol 25: 2847–2858, 2014

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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-CreERTTg;ROSA26fstdTomato 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+;aSMA+ myofibroblasts (arrowheads) after injury. Col1a2-RFP+;aSMA– SM fibroblasts and DAPI+;Col1a2RFP–;aSMA– MCs at the peritoneal surface are indicated by arrows and asterisks, respectively. The graph beneath shows the percentage of aSMA+ myofibroblasts coexpressing the fate marker Col1a2-RFP (n=6 per group). (D) Three-dimensional images with YZ and XZ stacks show numerous Col1a2-RFP+;aSMA+ 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, 49,69diamidino-2-phenylindole. Bar, 20 mm. Original magnification, 3630.

PDGFRb signaling by myofibroblasts was shown to be pathologic in models of kidney, liver, and lung fibrosis. 43,45,46 The restricted expression of PDGFRb to SM 2854

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fibroblasts and myofibroblasts in peritoneal fibrosis suggested that PDGFRb blockade might be an attractive therapeutic strategy. Blockade of PDGFRb signaling by the tyrosine kinase–inhibitor imatinib provides supportive evidence that SM fibroblast–derived myofibroblasts are critical cells in fibrogenesis, and provides new avenues for therapeutic discovery. However, we emphasize that the result is far more applicable to EPS than to the vast majority of peritoneal dialysis patients in whom fibrosis is a late clinical event. Our data indicate that the injured peritoneum was remesothelialized by surviving MCs, not by SM fibroblasts. These studies did not support WT1-labeled SM fibroblasts as stem cells for mesothelial repair. This mechanism of mesothelial regeneration is similar to kidney epithelial regeneration, which was previously shown to be from intrinsic surviving tubular epithelial cells rather than epithelial progenitors.47 Because MCs express different growth factor receptors from SM fibroblasts and myofibroblasts (e.g., EGF receptor, as previously reported29), interventions that activate specific receptor signaling for mesothelial repair may promote more effective remesothelialization after injury. In conclusion, we used comprehensive genetic lineage analysis to clarify the role of MCs and SM fibroblasts in the repair of mesothelium and generation of myofibroblasts during peritoneal fibrosis. We provide lineage tracing evidence that SM fibroblasts are the major myofibroblast precursors in peritoneal fibrosis and surviving MCs are the principal cells for remesothelialization after injury (Figure 8). These findings need to be recapitulated in more clinically relevant models.

CONCISE METHODS Animals

Col1a1-GFP transgenic mice were generated and validated as previously described on the C57BL/6 background whose Col1a1-expressing cells expressed GFP.37 Col1a2-CreERT transgenic mice were generated using a 6-kb Col1a2 enhancer to drive the expression of a cDNA encoding CreERT.48 WT1CreERT2/+ mice obtained from The Jackson Laboratory (Bar Harbor, ME) were generated by knocking a cDNA encoding CreERT2 into

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Figure 7. TGF-b1 induces aSMA expression in cultured SM fibroblasts. (A) Peritoneal Col1a1-GFP+ SM fibroblasts are isolated and cultured from normal Col1a1-GFPTg 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 aSMA in culture medium alone (CON), whereas they activate expression of aSMA in the presence of TGF-b1 for 2 days. (C) Col1a1-GFP+ SM fibroblasts are cultured in the presence or absence of TGF-b1 for 24 hours. Quantitative PCR shows that Acta2 and Col1a1 are upregulated by TGF-b1. The expression levels are normalized by Gapdh and expressed as the mean6SEM (n=3). †P,0.01. Bar, 25 mm in A; 20 mm in B. Original magnification, 3200 in A; 3630 in B.

the WT1 locus.49 B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J reporter mice (referred as ROSA26fstdTomato) were obtained from The Jackson Laboratory. All studies were carried out under a protocol approved by the Institutional Animal Care and Use Committee of National Taiwan University College of Medicine.

Mouse Models of Peritoneal Fibrosis

Adultmice (aged 14 weeks) were used for induction ofperitoneal fibrosis. Hypochlorite-induced peritoneal fibrosis was induced by an intraperitoneal injection of 100 ml/kg body weight of normal saline with 0.05% sodium hypochlorite.33,34 Dialysis solution–induced peritoneal fibrosis was induced by a daily intraperitoneal injection of 50 ml/kg body weight of dialysis solution by mixing Dianeal containing 4.25% glucose (Baxter Healthcare, Singapore) with 40 mM methylglyoxal (Sigma-Aldrich, St. Louis, MO) to accelerate fibrosis within 2 weeks.35 On the basis of evidence that TGF-b1 was the major profibrotic cytokine in tissue fibrosis, TGF-b1–induced peritoneal fibrosis was induced by an intraperitoneal injection of cesium chloride gradient purified AdTGF-b1 at a dose of 1.53108 plaque forming units diluted in 100 ml PBS.17,36,39,50 Control virus at the same dose was used as the control.

Imatinib Administration in a Mouse Model of Peritoneal Fibrosis Wild-type C57BL/6 mice were administered PBS or imatinib mesylate (50 mg/kg; Novartis Pharmaceuticals Co., Basel, Switzerland) J Am Soc Nephrol 25: 2847–2858, 2014

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Figure 8. The illustration indicates the major fates of MCs and SM fibroblasts after peritoneal injury.

through oral gavage 2 hours before an intraperitoneal injection of 100 ml/kg body weight of normal saline with 0.05% sodium hypochlorite, and then once a day until analysis on day 10 (n=6 for each group). The peritoneal adhesion score was quantified by scoring 1 point for each presence of an adhesion between the abdominal wall and intestine, intestine and intestine, intestine and omentum, omentum and kidney, and kidney and liver according to a previously described method.34 The peritoneal adhesion score was expressed as the mean of total scores for each mouse. Peritoneal membrane thickness and cell numbers of aSMA+ myofibroblasts were quantified using Masson’s trichrome stain and immunofluorescence, respectively, in the peritoneal covering of liver using the method described below in the subsection on tissue preparation and histology.

Temporal Induction of Cre Activity by Tamoxifen Administration A tamoxifen base in olive oil (10 mg/ml) (Sigma-Aldrich) was prepared by sonication. Mice were administered 1 mg daily through oral gavage for 5 days every week at the age of 10 and 11 weeks.29,48 After washing period for 2 weeks, mice were subjected to peritoneal injury.

Tissue Preparation and Histology Mouse tissues includingthe liver, omentum, and anterior abdominal wall were prepared and stained as previously described.37,38 Primary antibodies against the following proteins were used for immunolabeling in 5 mm-thick cryosections: aSMA-Cy3, aSMA-FITC, laminin, and cytokeratin (Sigma-Aldrich), as well as Ki67 (Abcam, Inc., Cambridge, UK), vimentin and nidogen (Santa Cruz Biotechnology, Santa Cruz, CA), and PDGFRb (gift from Dr. Stallcup). Fluorescence conjugated secondary antibody labeling (Jackson Immunoresearch Laboratories, West Grove, PA), colabeled with 49,6-diamidino-2-phenylindole, and mounting with Vectashield were carried out as previously described.37,38 Conventional and confocal images were taken with an Zeiss Axio Imager A1 Microscope with AxioVision Software and a Zeiss Laser Scanning 780 Microscope with Zen 2011 Software, respectively (Carl Zeiss, Jena, Germany). Images were processed using Adobe Photoshop software. Lineage Tracing during Peritoneal Injury

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GFP+ and RFP+ cells were identified by positive nuclear and cytoplasmic fluorescence; Ki67+ cells was identified by positive nuclear fluorescence; and aSMA+, cytokeratin+, vimentin+, and PDGFRb+ cells were identified by .75% of the cell area immediately surrounding the nuclei (detected by 49,69-diamidino-2-phenylindole) staining positive with fluorescence. To avoid bias caused by focal alterations, the numbers of cells with specific staining in the peritoneal membrane of livers were quantified in 10 sections of 3630 magnification from every 10th section of each mouse (five randomly selected images per section). The peritoneal surface was divided into 25 equal parts and the percentage of the peritoneal surface with cytokeratin+ MCs was calculated in 10 sections from every 10th section for each mouse (five randomly selected images per section). Masson’s trichrome stain was performed in 4-mm–thick paraffin sections and the peritoneal membrane thickness on the liver surface was measured in 10 sections from every 10th section for each mouse (five randomly selected images per section). The cell numbers of aSMA+ myofibroblasts, percentage of peritoneal surface with cytokeratin+ MCs, and peritoneal membrane thickness of each group were averaged and expressed as the mean6SEM.

ACKNOWLEDGMENTS The authors acknowledge Dr. C.P. Denton (University College London, London, UK) and Dr. A. Leask (University of Western Ontario, ON, Canada) for Col1a2-CreERTTg mice, Dr. W. Stallcup (Burnham Institute, CA) for anti-PDGFRb antibody, Dr. P.J. Margetts (McMaster University, ON, Canada) for AdTGF-b1, the Department of Medical Research of National Taiwan University Hospital for equipment support, the Imaging Core Facility and Cell Sorting Core Facility in the First Core Laboratory, and the Transgenic Mouse Core Facility in the Center for Genomic Medicine of National Taiwan University College of Medicine. J.S.D. is supported by grants from the National Institutes of Health (DK94768, DK93493, DK84077, and TR000504). S.-L.L. is supported by grants from the National Science Council (101-2321-B-002-060, 101-2314-B-002-084, 102-2628-B-002-015, and 102-2321-B002045), the E-Da Hospital/National Taiwan University Hospital Joint Research Program (102-EDN07), and the Mrs. Hsiu-Chin Lee Kidney Research Foundation.

Purification and Culture of MCs and SM Fibroblasts

Two weeks after the last dose of tamoxifen, WT1CreERT2/+;ROSA26fstdTomato mice were injected intraperitoneally with 10 ml of 0.125% trypsin and 0.05% EDTA (Life Technologies, Carlsbad, CA). After digestion for 30 minutes, the intraperitoneal cell suspension was collected by syringe. After centrifugation, cells were resuspended in 5 ml of PBS/1% BSA, and filtered (40 mm). MCs were purified by isolating WT1-RFP+; PDGFRb-APC– cells (eBioscience, San Diego, CA) using a FACSAria cell sorter (BD Biosciences, San Jose, CA). Col1a1-GFP+ SM fibroblasts were purified from peritoneal cells of normal Col1a1-GFPTg mice after intraperitoneal injection with trypsin/EDTA as described above. Purified cells were then cultured in DMEM containing 10% FBS. Cells at passages two to four were used for experiments. Cultured cells were stimulated with TGF-b1 (5 ng/ml) or no additional treatment. Two days later, cells were fixed with 4% paraformaldehyde for 15 minutes, washed with PBS, and then labeled with antibodies for aSMA. Total RNA was extracted from cells in the presence or absence of TGF-b1 for 24 hours and QPCR was performed using previously described methods.50 The specific primer pairs used in QPCR are listed in Supplemental Table 1.

Detection of TGF-b1 in Peritoneal Effluent Ten days after intraperitoneal injection of adenovirus, 3 ml of PBS was instilled in the peritoneum, and samples were collected 15 minutes later. The fluid was centrifuged at 1500 rpm for 5 minutes and the supernatant was frozen at 220°C. This effluent was assayed for total TGF-b1 concentration using ELISA according to the manufacturer’s instructions (R&D Systems, Minneapolis, MN).

Statistical Analyses Data are expressed as the mean6SEM. Statistical analyses were carried out using GraphPad Prism software (GraphPad Software, La Jolla, CA). The statistical significance was evaluated by one-way ANOVA. P,0.05 was considered significant. 2856

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DISCLOSURES J.S.D. is on the Scientific Advisory Board for Regulus Therapeutics and Promedior Inc., a cofounder of Muregen LLC.

REFERENCES 1. Grassmann A, Gioberge S, Moeller S, Brown G: ESRD patients in 2004: Global overview of patient numbers, treatment modalities and associated trends. Nephrol Dial Transplant 20: 2587–2593, 2005 2. Wu MS, Wu IW, Shih CP, Hsu KH: Establishing a platform for battling end-stage renal disease and continuing quality improvement in dialysis therapy in Taiwan - Taiwan Renal Registry Data System (TWRDS). Acta Nephrol 25: 148–153, 2011 3. Kramer A, Stel VS, Abad Diez JM, Alonso de la Torre R, Bouzas Caamaño E, Čala S, Cao Baduell H, Castro de la Nuez P, Cernevskis H, Collart F, Couchoud C, de Meester J, Djukanovic L, Ferrer-Alamar M, Finne P, Fogarty D, de los Ángeles García Bazaga M, Garneata L, Golan E, Gonzalez Fernández R, Heaf JG, Hoitsma A, Ioannidis GA, Kolesnyk M, Kramar R, Leivestad T, Limido A, Lopot F, Macario F, Magaz Å, Martín-Escobar E, Metcalfe W, Noordzij M, Ots-Rosenberg M, Palsson R, Piñera C, Postorino M, Prutz KG, Ratkovic M, Resic H, Rodríguez Hernández A, Rutkowski B, Serdengeçti K, Yebenes TS, Spustová V, Stojceva-Taneva O, Tomilina NA, van de Luijtgaarden MWM, van Stralen KJ, Wanner C, Jager KJ: Renal replacement therapy in Europe– a summary of the 2010 ERA–EDTA Registry Annual Report. Clin Kidney J 6: 105–115, 2013 4. Mateijsen MA, van der Wal AC, Hendriks PM, Zweers MM, Mulder J, Struijk DG, Krediet RT: Vascular and interstitial changes in the peritoneum of CAPD patients with peritoneal sclerosis. Perit Dial Int 19: 517– 525, 1999 5. Williams JD, Craig KJ, Topley N, Von Ruhland C, Fallon M, Newman GR, Mackenzie RK, Williams GT; Peritoneal Biopsy Study Group: Morphologic changes in the peritoneal membrane of patients with renal disease. J Am Soc Nephrol 13: 470–479, 2002 6. Korte MR, Sampimon DE, Betjes MGH, Krediet RT: Encapsulating peritoneal sclerosis: The state of affairs. Nat Rev Nephrol 7: 528–538, 2011 7. Chang FC, Huang TM, Li WY, Lin SL: Café-au-lait ascites in encapsulating peritoneal sclerosis. Kidney Int 79: 1261, 2011

J Am Soc Nephrol 25: 2847–2858, 2014

www.jasn.org

8. Margetts PJ, Bonniaud P, Liu L, Hoff CM, Holmes CJ, West-Mays JA, Kelly MM: Transient overexpression of TGF-b1 induces epithelial mesenchymal transition in the rodent peritoneum. J Am Soc Nephrol 16: 425–436, 2005 9. Devuyst O, Margetts PJ, Topley N: The pathophysiology of the peritoneal membrane. J Am Soc Nephrol 21: 1077–1085, 2010 10. Aroeira LS, Aguilera A, Selgas R, Ramírez-Huesca M, Pérez-Lozano ML, Cirugeda A, Bajo MA, del Peso G, Sánchez-Tomero JA, JiménezHeffernan JA, López-Cabrera M: Mesenchymal conversion of mesothelial cells as a mechanism responsible for high solute transport rate in peritoneal dialysis: Role of vascular endothelial growth factor. Am J Kidney Dis 46: 938–948, 2005 11. Michailova KN: A combined electron microscopic investigation of the peritoneal mesothelium in the rat. Eur J Morphol 33: 265–277, 1995 12. Asahina K, Tsai SY, Li P, Ishii M, Maxson RE Jr, Sucov HM, Tsukamoto H: Mesenchymal origin of hepatic stellate cells, submesothelial cells, and perivascular mesenchymal cells during mouse liver development. Hepatology 49: 998–1011, 2009 13. Asahina K, Zhou B, Pu WT, Tsukamoto H: Septum transversum-derived mesothelium gives rise to hepatic stellate cells and perivascular mesenchymal cells in developing mouse liver. Hepatology 53: 983–995, 2011 14. Colmont CS, Raby A-C, Dioszeghy V, Lebouder E, Foster TL, Jones SA, Labéta MO, Fielding CA, Topley N: Human peritoneal mesothelial cells respond to bacterial ligands through a specific subset of Toll-like receptors. Nephrol Dial Transplant 26: 4079–4090, 2011 15. Amenta PS, Harrison D: Expression and potential role of the extracellular matrix in hepatic ontogenesis: A review. Microsc Res Tech 39: 372–386, 1997 16. Topley N, Jörres A, Luttmann W, Petersen MM, Lang MJ, Thierauch KH, Müller C, Coles GA, Davies M, Williams JD: Human peritoneal mesothelial cells synthesize interleukin-6: Induction by IL-1 beta and TNF alpha. Kidney Int 43: 226–233, 1993 17. Offner FA, Feichtinger H, Stadlmann S, Obrist P, Marth C, Klingler P, Grage B, Schmahl M, Knabbe C: Transforming growth factor-beta synthesis by human peritoneal mesothelial cells. Induction by interleukin-1. Am J Pathol 148: 1679–1688, 1996 18. Patel P, West-Mays J, Kolb M, Rodrigues JC, Hoff CM, Margetts PJ: Platelet derived growth factor B and epithelial mesenchymal transition of peritoneal mesothelial cells. Matrix Biol 29: 97–106, 2010 19. Liu L, Shi CX, Ghayur A, Zhang C, Su JY, Hoff CM, Margetts PJ: Prolonged peritoneal gene expression using a helper-dependent adenovirus. Perit Dial Int 29: 508–516, 2009 20. Gerwin BI, Lechner JF, Reddel RR, Roberts AB, Robbins KC, Gabrielson EW, Harris CC: Comparison of production of transforming growth factor-b and platelet-derived growth factor by normal human mesothelial cells and mesothelioma cell lines. Cancer Res 47: 6180–6184, 1987 21. Onitsuka I, Tanaka M, Miyajima A: Characterization and functional analyses of hepatic mesothelial cells in mouse liver development. Gastroenterology 138: 1525–1535, e1–e6, 2010 22. Sakai N, Chun J, Duffield JS, Wada T, Luster AD, Tager AM: LPA1induced cytoskeleton reorganization drives fibrosis through CTGFdependent fibroblast proliferation. FASEB J 27: 1830–1846, 2013 23. Beavis MJ, Williams JD, Hoppe J, Topley N: Human peritoneal fibroblast proliferation in 3-dimensional culture: Modulation by cytokines, growth factors and peritoneal dialysis effluent. Kidney Int 51: 205–215, 1997 24. Fang CC, Lai MN, Chien CT, Hung KY, Tsai CC, Tsai TJ, Hsieh BS: Effects of pentoxifylline on peritoneal fibroblasts and silica-induced peritoneal fibrosis. Perit Dial Int 23: 228–236, 2003 25. Yang AH, Chen JY, Lin JK: Myofibroblastic conversion of mesothelial cells. Kidney Int 63: 1530–1539, 2003 26. Yáñez-Mó M, Lara-Pezzi E, Selgas R, Ramírez-Huesca M, DomínguezJiménez C, Jiménez-Heffernan JA, Aguilera A, Sánchez-Tomero JA,

J Am Soc Nephrol 25: 2847–2858, 2014

27.

28.

29.

30.

31.

32.

33. 34.

35.

36.

37.

38.

39.

40.

41. 42.

43.

BASIC RESEARCH

Bajo MA, Alvarez V, Castro MA, del Peso G, Cirujeda A, Gamallo C, Sánchez-Madrid F, López-Cabrera M: Peritoneal dialysis and epithelialto-mesenchymal transition of mesothelial cells. N Engl J Med 348: 403– 413, 2003 Bajo MA, Pérez-Lozano ML, Albar-Vizcaino P, del Peso G, Castro M-J, Gonzalez-Mateo G, Fernández-Perpén A, Aguilera A, SánchezVillanueva R, Sánchez-Tomero JA, López-Cabrera M, Peter ME, Passlick-Deetjen J, Selgas R: Low-GDP peritoneal dialysis fluid (‘balance’) has less impact in vitro and ex vivo on epithelial-to-mesenchymal transition (EMT) of mesothelial cells than a standard fluid. Nephrol Dial Transplant 26: 282–291, 2011 Wong TYH, Phillips AO, Witowski J, Topley N: Glucose-mediated induction of TGF-b 1 and MCP-1 in mesothelial cells in vitro is osmolality and polyol pathway dependent. Kidney Int 63: 1404–1416, 2003 Li Y, Wang J, Asahina K: Mesothelial cells give rise to hepatic stellate cells and myofibroblasts via mesothelial-mesenchymal transition in liver injury. Proc Natl Acad Sci U S A 110: 2324–2329, 2013 Walker C, Rutten F, Yuan X, Pass H, Mew DM, Everitt J: Wilms’ tumor suppressor gene expression in rat and human mesothelioma. Cancer Res 54: 3101–3106, 1994 Chau Y-Y, Brownstein D, Mjoseng H, Lee W-C, Buza-Vidas N, Nerlov C, Jacobsen SE, Perry P, Berry R, Thornburn A, Sexton D, Morton N, Hohenstein P, Freyer E, Samuel K, van’t Hof R, Hastie N: Acute multiple organ failure in adult mice deleted for the developmental regulator Wt1. PLoS Genet 7: e1002404, 2011 Parenti R, Perris R, Vecchio GM, Salvatorelli L, Torrisi A, Gravina L, Magro G: Immunohistochemical expression of Wilms’ tumor protein (WT1) in developing human epithelial and mesenchymal tissues. Acta Histochem 115: 70–75, 2013 Levine S, Saltzman A: Abdominal cocoon: An animal model for a complication of peritoneal dialysis. Perit Dial Int 16: 613–616, 1996 Huang JW, Yen CJ, Wu HY, Chiang CK, Cheng HT, Lien YC, Hung KY, Tsai TJ: Tamoxifen downregulates connective tissue growth factor to ameliorate peritoneal fibrosis. Blood Purif 31: 252–258, 2011 Kitamura M, Nishino T, Obata Y, Furusu A, Hishikawa Y, Koji T, Kohno S: Epigallocatechin gallate suppresses peritoneal fibrosis in mice. Chem Biol Interact 195: 95–104, 2012 Margetts PJ, Hoff C, Liu L, Korstanje R, Walkin L, Summers A, Herrick S, Brenchley P: Transforming growth factor b-induced peritoneal fibrosis is mouse strain dependent. Nephrol Dial Transplant 28: 2015–2027, 2013 Lin SL, Kisseleva T, Brenner DA, Duffield JS: Pericytes and perivascular fibroblasts are the primary source of collagen-producing cells in obstructive fibrosis of the kidney. Am J Pathol 173: 1617–1627, 2008 Humphreys BD, Lin SL, Kobayashi A, Hudson TE, Nowlin BT, Bonventre JV, Valerius MT, McMahon AP, Duffield JS: Fate tracing reveals the pericyte and not epithelial origin of myofibroblasts in kidney fibrosis. Am J Pathol 176: 85–97, 2010 Koesters R, Kaissling B, Lehir M, Picard N, Theilig F, Gebhardt R, Glick AB, Hähnel B, Hosser H, Gröne HJ, Kriz W: Tubular overexpression of transforming growth factor-beta1 induces autophagy and fibrosis but not mesenchymal transition of renal epithelial cells. Am J Pathol 177: 632–643, 2010 Asada N, Takase M, Nakamura J, Oguchi A, Asada M, Suzuki N, Yamamura K, Nagoshi N, Shibata S, Rao TN, Fehling HJ, Fukatsu A, Minegishi N, Kita T, Kimura T, Okano H, Yamamoto M, Yanagita M: Dysfunction of fibroblasts of extrarenal origin underlies renal fibrosis and renal anemia in mice. J Clin Invest 121: 3981–3990, 2011 Göritz C, Dias DO, Tomilin N, Barbacid M, Shupliakov O, Frisén J: A pericyte origin of spinal cord scar tissue. Science 333: 238–242, 2011 Rock JR, Barkauskas CE, Cronce MJ, Xue Y, Harris JR, Liang J, Noble PW, Hogan BL: Multiple stromal populations contribute to pulmonary fibrosis without evidence for epithelial to mesenchymal transition. Proc Natl Acad Sci U S A 108: E1475–E1483, 2011 Kisseleva T, Cong M, Paik Y, Scholten D, Jiang C, Benner C, Iwaisako K, Moore-Morris T, Scott B, Tsukamoto H, Evans SM, Dillmann W, Glass CK,

Lineage Tracing during Peritoneal Injury

2857

BASIC RESEARCH

44.

45.

46.

47.

www.jasn.org

Brenner DA: Myofibroblasts revert to an inactive phenotype during regression of liver fibrosis. Proc Natl Acad Sci U S A 109: 9448–9453, 2012 Dulauroy S, Di Carlo SE, Langa F, Eberl G, Peduto L: Lineage tracing and genetic ablation of ADAM12(+) perivascular cells identify a major source of profibrotic cells during acute tissue injury. Nat Med 18: 1262– 1270, 2012 Chen YT, Chang FC, Wu CF, Chou YH, Hsu HL, Chiang WC, Shen J, Chen YM, Wu KD, Tsai TJ, Duffield JS, Lin SL: Platelet-derived growth factor receptor signaling activates pericyte-myofibroblast transition in obstructive and post-ischemic kidney fibrosis. Kidney Int 80: 1170– 1181, 2011 Hung C, Linn G, Chow Y-H, Kobayashi A, Mittelsteadt K, Altemeier WA, Gharib SA, Schnapp LM, Duffield JS: Role of lung pericytes and resident fibroblasts in the pathogenesis of pulmonary fibrosis. Am J Respir Crit Care Med 188: 820–830, 2013 Humphreys BD, Valerius MT, Kobayashi A, Mugford JW, Soeung S, Duffield JS, McMahon AP, Bonventre JV: Intrinsic epithelial cells repair the kidney after injury. Cell Stem Cell 2: 284–291, 2008

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48. Zheng B, Zhang Z, Black CM, de Crombrugghe B, Denton CP: Liganddependent genetic recombination in fibroblasts: A potentially powerful technique for investigating gene function in fibrosis. Am J Pathol 160: 1609–1617, 2002 49. Zhou B, Ma Q, Rajagopal S, Wu SM, Domian I, Rivera-Feliciano J, Jiang D, von Gise A, Ikeda S, Chien KR, Pu WT: Epicardial progenitors contribute to the cardiomyocyte lineage in the developing heart. Nature 454: 109–113, 2008 50. Wu CF, Chiang WC, Lai CF, Chang FC, Chen YT, Chou YH, Wu TH, Linn GR, Ling H, Wu KD, Tsai TJ, Chen YM, Duffield JS, Lin SL: Transforming growth factor b-1 stimulates profibrotic epithelial signaling to activate pericyte-myofibroblast transition in obstructive kidney fibrosis. Am J Pathol 182: 118–131, 2013

This article contains supplemental material online at http://jasn.asnjournals. org/lookup/suppl/doi:10.1681/ASN.2013101079/-/DCSupplemental.

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