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Origin of myofibroblasts and cellular events triggering fibrosis

      Renal fibrosis is a major hallmark of chronic kidney disease that is considered to be a common end point of various types of renal disease. To date, the biological meaning of fibrosis during the progression of chronic kidney diseases is unknown and possibly depends on the cell type contributing to extracellular matrix production. During the past decade, the origin of myofibroblasts in the kidney has been intensively investigated. Determining the origins of renal myofibroblasts is important because these might account for the heterogeneous characteristics and behaviors of myofibroblasts. Current data strongly suggest that collagen-producing myofibroblasts in the kidney can be derived from various cellular sources. Resident renal fibroblasts and cells of hematopoietic origin migrating into the kidney seem to be the most important ancestors of myofibroblasts. It is likely that both cell types communicate with each other and also with other cell types in the kidney. In this review, we will discuss the current knowledge on the origin of scar-producing myofibroblasts and cellular events triggering fibrosis.

      Keywords

      Renal fibrosis consisting of interstitial fibrosis and/or glomerulosclerosis is frequently seen in end-stage renal disease and is considered to be a common end point of various types of renal disease. To date, it is unclear whether there is a causal relationship between fibrosis and chronic renal failure and whether fibrosis contributes to the development of chronic renal failure.
      The biological meaning of fibrosis is diverse, and it could be classified into at least four categories. First, after acute injury, recovery of renal structures might be incomplete and they thus may be replaced by fibrotic tissue (‘fibrosis as innocent fill-in’). In this case, prevention of fibrosis would not improve the course of the underlying disease. Second, an early appearance of fibrosis after acute or chronic injury could interfere with full regeneration of renal structures or even further damage renal structures (‘fibrosis as inhibitor of regeneration’). In this case, prevention of fibrosis would be an important part of the treatment. Third, fibrosis could be an intermediate state in the process of tissue regeneration, and could even be required for rebuilding or maintaining renal structures (‘fibrosis as mediator of reconstruction’). Although fibrosis is widely thought to compromise kidney structure and function,
      • Liu Y.
      Cellular and molecular mechanisms of renal fibrosis.
      ,
      • Bechtel W.
      • McGoohan S.
      • Zeisberg E.M.
      • et al.
      Methylation determines fibroblast activation and fibrogenesis in the kidney.
      some groups suggested that fibrosis may also support the healing process.
      • Kaissling B.
      • Lehir M.
      • Kriz W.
      Renal epithelial injury and fibrosis.
      • Sun D.F.
      • Fujigaki Y.
      • Fujimoto T.
      • et al.
      Possible involvement of myofibroblasts in cellular recovery of uranyl acetate-induced acute renal failure in rats.
      • Fujigaki Y.
      • Muranaka Y.
      • Sun D.
      • et al.
      Transient myofibroblast differentiation of interstitial fibroblastic cells relevant to tubular dilatation in uranyl acetate-induced acute renal failure in rats.
      Hishida et al. demonstrated that myofibroblasts emerge around injured tubules and disappear when tubular regeneration is completed, suggesting that myofibroblasts surrounding damaged tubules may support the structural integrity and regeneration of injured tubules.
      • Sun D.F.
      • Fujigaki Y.
      • Fujimoto T.
      • et al.
      Possible involvement of myofibroblasts in cellular recovery of uranyl acetate-induced acute renal failure in rats.
      ,
      • Fujigaki Y.
      • Muranaka Y.
      • Sun D.
      • et al.
      Transient myofibroblast differentiation of interstitial fibroblastic cells relevant to tubular dilatation in uranyl acetate-induced acute renal failure in rats.
      Fourth, if fibrosis is not removed after healing, connective tissue may accumulate over time and damage the kidney. In this case, treatment should focus on increasing fibrous tissue degradation after completion of healing (‘fibrosis as leftover after reconstruction’).
      Most likely, the role of fibrosis will differ between various types of renal disease (e.g., loss of nephrons following glomerular damage and tubulointerstitial disease following proteinuria, interstitial nephritis, tubular necrosis, or allograft rejection), and even within one disease fibrosis might fulfill more than one purpose.
      • Kaissling B.
      • Lehir M.
      • Kriz W.
      Renal epithelial injury and fibrosis.
      Merely identifying the presence of fibrosis during or after a certain renal disease will not reveal whether fibrosis is beneficial or detrimental for the recovery of renal function. If we have the tools to prevent matrix production or to increase extracellular matrix (ECM) removal, we can start to understand the role of fibrosis during various disease stages.
      We also need to know which cells contribute to ECM production, because fibrous tissue produced by one cell type might be beneficial, whereas matrix produced by another cell type might be harmful. Since the 1970s,
      • Nagle R.B.
      • Bulger R.E.
      Unilateral obstructive nephropathy in the rabbit. II. Late morphologic changes.
      the origin and the roles of scar-producing cells in renal fibrosis have been intensively investigated. These cells are called myofibroblasts, because of alpha smooth muscle actin (αSMA) expression and the similarities of their characteristics to those of fibroblasts.
      • Eyden B.
      The myofibroblast: an assessment of controversial issues and a definition useful in diagnosis and research.
      ,
      • Eyden B.
      • Banerjee S.S.
      • Shenjere P.
      • et al.
      The myofibroblast and its tumours.
      Although αSMA expression is confined to vascular smooth muscle cells in healthy kidneys, many αSMA-positive myofibroblasts emerge de novo in the interstitium of diseased kidneys and produce various types of ECM such as collagens, fibronectins, elastins, fibrillins, latent transforming growth factor β (TGF-β)-binding proteins, tenascins, and proteoglycans, which contribute to fibrosis.
      • Klingberg F.
      • Hinz B.
      • White E.S.
      The myofibroblast matrix: implications for tissue repair and fibrosis.
      The main features of myofibroblasts are an abundant rough endoplasmic reticulum, modestly developed peripheral myofilaments with focal densities, fibronexus junctions, and αSMA immunostaining.
      • Eyden B.
      The myofibroblast: an assessment of controversial issues and a definition useful in diagnosis and research.
      However, some reports indicate that certain αSMA-negative cells can also be considered myofibroblasts.
      • Hinz B.
      • Gabbiani G.
      Fibrosis: recent advances in myofibroblast biology and new therapeutic perspectives.
      A recent study using transgenic reporter mice expressing green fluorescent protein (GFP) under the control of collagen type I, α1 (col1α1) promoter, and enhancer (col1a1-GFP mouse) visualized the collagen-producing cells with GFP and demonstrated the close overlap between αSMA+ cells and GFP+ cells,
      • Lin S.L.
      • Kisseleva T.
      • Brenner D.A.
      • et al.
      Pericytes and perivascular fibroblasts are the primary source of collagen-producing cells in obstructive fibrosis of the kidney.
      supporting the importance of myofibroblasts as the target for antifibrotic therapeutic trials. Notably, 1% of col1α1-producing cells are αSMA negative, and 25% of αSMA+ cells are GFP negative,
      • Lin S.L.
      • Kisseleva T.
      • Brenner D.A.
      • et al.
      Pericytes and perivascular fibroblasts are the primary source of collagen-producing cells in obstructive fibrosis of the kidney.
      demonstrating the heterogeneity of ‘αSMA-positive myofibroblasts’.
      • Boor P.
      • Floege J.
      The renal (myo-)fibroblast: a heterogeneous group of cells.
      During the past decade, there has been considerable research on the origin of myofibroblasts in the kidney. Determining the precursor cells of renal myofibroblasts is important because these might account for the heterogeneous characteristics and behaviors of myofibroblasts. In this review, we will discuss the current understanding about the origin of scar-producing myofibroblasts and cellular events triggering fibrosis.

      CELLS RESPONSIBLE FOR FIBROSIS

      Until the mid-1990s, it was generally assumed that myofibroblasts were derived from resident fibroblasts.
      • Kriz W.
      • Kaissling B.
      • Le Hir M.
      Epithelial-mesenchymal transition (EMT) in kidney fibrosis: fact or fantasy?.
      Since that time, however, additional precursor cells of myofibroblasts in fibrotic kidneys have been reported, including circulating bone marrow–derived cells, or transition from either epithelial or endothelial cells (EMT or EndoMT; Figure 1). There is still considerable controversy regarding the contribution of various precursor cells to the development of myofibroblasts and renal fibrosis,
      • Kriz W.
      • Kaissling B.
      • Le Hir M.
      Epithelial-mesenchymal transition (EMT) in kidney fibrosis: fact or fantasy?.
      • Le Douarin N.M.
      • Teillet M.A.
      Experimental analysis of the migration and differentiation of neuroblasts of the autonomic nervous system and of neurectodermal mesenchymal derivatives, using a biological cell marking technique.
      • LeBleu V.S.
      • Taduri G.
      • O'Connell J.
      • et al.
      Origin and function of myofibroblasts in kidney fibrosis.
      • Campanholle G.
      • Ligresti G.
      • Gharib S.A.
      • et al.
      Cellular mechanisms of tissue fibrosis. 3. Novel mechanisms of kidney fibrosis.
      • Kramann R.
      • Dirocco D.P.
      • Humphreys B.D.
      Understanding the origin, activation and regulation of matrix-producing myofibroblasts for treatment of fibrotic disease.
      as summarized in Table 1. We will discuss in detail the role of resident fibroblasts, pericytes, bone marrow–derived fibrocytes, and EMT/EndoMT.
      Figure thumbnail gr1
      Figure 1Origin of scar-producing myofibroblasts. It has been controversial whether alpha smooth muscle actin (αSMA)-positive matrix-producing myofibroblasts are derived from resident fibroblasts, circulating bone marrow–derived fibrocytes, or transition from either epithelial or endothelial cells. Recent studies suggested that resident fibroblasts and bone marrow–derived fibrocytes are the two major sources of myofibroblasts in kidney fibrosis. Although pericytes are also reported to contribute to renal fibrosis, the difference and overlap with resident fibroblasts should be elucidated. In addition, the relationship between fibrocytes and monocytes needs to be clarified. EPO, erythropoietin.
      Table 1Controversy about the sources of myofibroblasts and their contribution to fibrosis
      Cell typeContribution to fibrosis (%) and modelsMethodsYearReferences
      Resident fibroblasts>90% Of myofibroblasts in UUO/othersLineage tracing with P0-Cre2011Asada et al.
      • Asada N.
      • Takase M.
      • Nakamura J.
      • et al.
      Dysfunction of fibroblasts of extrarenal origin underlies renal fibrosis and renal anemia in mice.
      50% Of myofibroblasts in UUOBMT, αSMA-RFP Tg mice2013LeBleu et al.
      • LeBleu V.S.
      • Taduri G.
      • O'Connell J.
      • et al.
      Origin and function of myofibroblasts in kidney fibrosis.
      Pericytes>90% Of myofibroblasts in UUOLineage tracing with FoxD1-Cre, FoxD-CreERT22010Humphreys et al.
      • Humphreys B.D.
      • Lin S.L.
      • Kobayashi A.
      • et al.
      Fate tracing reveals the pericyte and not epithelial origin of myofibroblasts in kidney fibrosis.
      BM-derived fibrocytes8.6% Of myofibroblasts in UUOBMT, pro-col1a2-luciferase/beta-Gal Tg mice2006Roufosse et al.
      • Roufosse C.
      • Bou-Gharios G.
      • Prodromidi E.
      • et al.
      Bone marrow-derived cells do not contribute significantly to collagen I synthesis in a murine model of renal fibrosis.
      27% Of myofibroblasts in ADRBMT, GFP Tg mice2007Li et al.
      • Li J.
      • Deane J.A.
      • Campanale N.V.
      • et al.
      The contribution of bone marrow-derived cells to the development of renal interstitial fibrosis.
      32% Of myofibroblasts in rat IRIBMT, R26-hPAP Tg mice2007Broekema et al.
      • Broekema M.
      • Harmsen M.C.
      • van Luyn M.J.
      • et al.
      Bone marrow-derived myofibroblasts contribute to the renal interstitial myofibroblast population and produce procollagen I after ischemia/reperfusion in rats.
      <0.1% Of myofibroblasts in UUOBMT, col1a1-GFP Tg mice2008Lin et al.
      • Lin S.L.
      • Kisseleva T.
      • Brenner D.A.
      • et al.
      Pericytes and perivascular fibroblasts are the primary source of collagen-producing cells in obstructive fibrosis of the kidney.
      35% Of myofibroblasts in UUOBMT, αSMA-RFP Tg mice2013LeBleu et al.
      • LeBleu V.S.
      • Taduri G.
      • O'Connell J.
      • et al.
      Origin and function of myofibroblasts in kidney fibrosis.
      40–50% Reduction in fibrosis in UUOAnti-CCL21 Ab, CCR7 null mice to reduce fibrocytes2006Sakai et al.
      • Sakai N.
      • Furuichi K.
      • Shinozaki Y.
      • et al.
      Fibrocytes are involved in the pathogenesis of human chronic kidney disease.
      20–30% Reduction in fibrosis in UUOIL-2 and TNF-α to reduce fibrocytes2009Niedermeier et al.
      • Niedermeier M.
      • Reich B.
      • Rodriguez Gomez M.
      • et al.
      CD4+ T cells control the differentiation of Gr1+ monocytes into fibrocytes.
      40–50% Reduction in fibrosis in UUOCXCL16-null mice to reduce fibrocytes2011Chen et al.
      • Chen G.
      • Lin S.C.
      • Chen J.
      • et al.
      CXCL16 recruits bone marrow-derived fibroblast precursors in renal fibrosis.
      20–30% Reduction in fibrosis in UUOCCR2-null mice, depletion of fibrocytes, BMT2013Reich et al.
      • Reich B.
      • Schmidbauer K.
      • Rodriguez Gomez M.
      • et al.
      Fibrocytes develop outside the kidney but contribute to renal fibrosis in a mouse model.
      30–50% Reduction in fibrosis in UUO/IRIAdiponectin-null mice to reduce fibrocytes2013Yang et al.
      • Yang J.
      • Lin S.C.
      • Chen G.
      • et al.
      Adiponectin promotes monocyte-to-fibroblast transition in renal fibrosis.
      EMT36% Of myofibroblasts in UUOLineage tracing with γ-GT-Cre2002Iwano et al.
      • Iwano M.
      • Plieth D.
      • Danoff T.M.
      • et al.
      Evidence that fibroblasts derive from epithelium during tissue fibrosis.
      No signs of EMT in UUOLineage tracing with Six2-Cre, HoxB7-Cre2010Humphreys et al.
      • Humphreys B.D.
      • Lin S.L.
      • Kobayashi A.
      • et al.
      Fate tracing reveals the pericyte and not epithelial origin of myofibroblasts in kidney fibrosis.
      No signs of EMT in UUOLineage tracing with Pax8-rtTA, LC12010Koesters et al.
      • Koesters R.
      • Kaissling B.
      • Lehir M.
      • et al.
      Tubular overexpression of transforming growth factor-β1 induces autophagy and fibrosis but not mesenchymal transition of renal epithelial cells.
      No signs of EMT in UUOLineage tracing with ksp-cadherin-Cre2010Li et al.
      • Li L.
      • Zepeda-Orozco D.
      • Black R.
      • et al.
      Autophagy is a component of epithelial cell fate in obstructive uropathy.
      <5% Of myofibroblasts in UUOLineage tracing with γ-GT-Cre2013LeBleu et al.
      • LeBleu V.S.
      • Taduri G.
      • O'Connell J.
      • et al.
      Origin and function of myofibroblasts in kidney fibrosis.
      EndoMT25% Of myofibroblasts in UUOExpression of CD31/αSMA, lineage tracing with tie2-Cre2008Zeisberg et al.
      • Zeisberg E.M.
      • Potenta S.E.
      • Sugimoto H.
      • et al.
      Fibroblasts in kidney fibrosis emerge via endothelial-to-mesenchymal transition.
      <10% Of myofibroblasts in UUOLineage tracing with cadherin 5-Cre2013LeBleu et al.
      • LeBleu V.S.
      • Taduri G.
      • O'Connell J.
      • et al.
      Origin and function of myofibroblasts in kidney fibrosis.
      Abbreviations: ADR, adriamycin-induced fibrosis; beta-Gal, beta-Galactosidase; BMT, bone marrow transplantation; EMT, epithelial–mesenchymal transition; EndoMT, endothelial–mesenchymal transition; GFP, green fluorescent protein; hPAP, human placental alkaline phosphatase; IRI, ischemia–reperfusion injury; RFP, red fluorescent protein; Tg, transgenic; UUO, unilateral ureteral obstruction.

      RESIDENT FIBROBLASTS

       Detection of resident fibroblasts

      Fibroblasts are versatile cells of mesenchymal origin that are embedded in the ECM and stroma of connective tissues and organs.
      • Boor P.
      • Floege J.
      The renal (myo-)fibroblast: a heterogeneous group of cells.
      ,
      • Chang H.Y.
      • Chi J.T.
      • Dudoit S.
      • et al.
      Diversity, topographic differentiation, and positional memory in human fibroblasts.
      Although fibroblasts are the most accessible normal mammalian cell type and can easily be isolated, cultured, and expanded in vitro, they remain poorly characterized at a molecular level. In practice, fibroblasts are usually identified by their elongated or spindle-shaped morphology, their anatomical localization in the interstitium, the expression of several markers such as PDGFR-β and CD73 (Figure 2), and the absence of markers for other cell lineages.
      • Boor P.
      • Floege J.
      The renal (myo-)fibroblast: a heterogeneous group of cells.
      Figure thumbnail gr2
      Figure 2Identification of resident fibroblasts. Resident fibroblasts are identified as the cells positive for PDGFR-β (a) in the interstitium of the kidney. Resident fibroblasts in the cortex are also positive for CD73 (b). Bars=10μm.

       Function of resident fibroblasts

      In the kidney, fibroblasts reside ubiquitously in the interstitium, which is the space between epithelial cell basement membranes and the nutritive capillaries.
      • Kaissling B.
      • Le Hir M.
      The renal cortical interstitium: morphological and functional aspects.
      They produce and organize the interstitial ECM and also communicate with other cell types including epithelial cells, endothelial cells, and circulating cells, having a crucial role in maintaining homeostasis. In addition, certain subpopulations of renal fibroblasts are specialized endocrine cells that produce erythropoietin (EPO) in response to hypoxia.
      • Sato Y.
      • Yanagita M.
      Renal anemia: from incurable to curable.
      Under physiological normoxic conditions, few EPO-producing fibroblasts are observed in the deep cortex and outer medulla of the kidney. Under hypoxic conditions, EPO-producing fibroblasts increase markedly and spread outward from the deep cortex toward the capsule.
      • Obara N.
      • Suzuki N.
      • Kim K.
      • et al.
      Repression via the GATA box is essential for tissue-specific erythropoietin gene expression.
      ,
      • Koury S.T.
      • Koury M.J.
      • Bondurant M.C.
      • et al.
      Quantitation of erythropoietin-producing cells in kidneys of mice by in situ hybridization: correlation with hematocrit, renal erythropoietin mRNA, and serum erythropoietin concentration.

       Origin of resident fibroblasts

      Although the interest in resident fibroblasts has increased over the years, the developmental origin of these cells has been poorly understood. In addition to the possible sources such as metanephric mesenchyme and uninduced intermediate mesenchyme,
      • Cullen-McEwen L.A.
      • Caruana G.
      • Bertram J.F.
      The where, what and why of the developing renal stroma.
      ,
      • Dressler G.R.
      The cellular basis of kidney development.
      a different source has been proposed. In 1974, xenotransplantation of a quail neural tube into a chick embryo revealed the contribution of neural crest–derived cells to the developing kidneys.
      • Le Douarin N.M.
      • Teillet M.A.
      Experimental analysis of the migration and differentiation of neuroblasts of the autonomic nervous system and of neurectodermal mesenchymal derivatives, using a biological cell marking technique.
      The neural crest is a unique vertebrate embryo structure, which migrates to many regions of the embryo and differentiates into a variety of local cells
      • Le Douarin N.M.
      The avian embryo as a model to study the development of the neural crest: a long and still ongoing story.
      (e.g., melanocytes, peripheral glial cells, and neurons).
      On the basis of these observations and the fact that EPO-producing cells and embryonic stroma express neuronal markers,
      • Obara N.
      • Suzuki N.
      • Kim K.
      • et al.
      Repression via the GATA box is essential for tissue-specific erythropoietin gene expression.
      ,
      • Sainio K.
      • Nonclercq D.
      • Saarma M.
      • et al.
      Neuronal characteristics in embryonic renal stroma.
      ,
      • Sariola H.
      • Holm K.
      • Henke-Fahle S.
      Early innervation of the metanephric kidney.
      Asada et al. hypothesized that neural crest–derived cells migrate into the embryonic kidney and differentiate into resident fibroblasts including EPO-producing cells. They conducted lineage-tracing studies with myelin protein zero (P0)-Cre transgenic mice (P0-Cre mice), which express Cre-recombinase in the migrating neural crest cells and neural crest–derived Schwann cells. P0-Cre lineage-labeled cells were observed in the kidney interstitium and expressed markers of resident fibroblasts, PDGFR-β, and CD73.
      • Asada N.
      • Takase M.
      • Nakamura J.
      • et al.
      Dysfunction of fibroblasts of extrarenal origin underlies renal fibrosis and renal anemia in mice.
      Most resident fibroblasts in the cortex and outer medulla, including EPO-producing cells, were lineage-labeled with P0-Cre. Recently, EPO production by certain subpopulations of neural crest cells has been reported.
      • Suzuki N.
      • Hirano I.
      • Pan X.
      • et al.
      Erythropoietin production in neuroepithelial and neural crest cells during primitive erythropoiesis.
      This observation led to the idea that this subpopulation might be the origin of P0-Cre-lineage-labeled resident fibroblasts in the kidney. Further study is required to show the relevance of these two populations.

       Dysfunction of resident fibroblasts causes renal fibrosis and renal anemia

      In three fibrosis models, unilateral ureteral obstruction (UUO), folic acid nephropathy, and severe ischemic–reperfusion (IR) injury, it is demonstrated that EPO-producing P0-Cre lineage–labeled resident fibroblasts are the main source of scar-producing myofibroblasts. In addition, EPO-producing fibroblasts also transform into myofibroblasts at the cost of EPO production and trans-differentiated myofibroblasts still possess EPO-producing capacity after the induction of severe anemia. Taken together, resident fibroblasts including EPO-producing cells in the healthy kidney and scar-producing myofibroblasts in renal fibrosis have the same origin and transit between one another depending on their microenvironment.
      Targeting P0-Cre lineage–labeled cells might represent a new therapeutic approach, which suppresses the generation of scar-producing myofibroblasts and restores EPO production.
      • Sato Y.
      • Yanagita M.
      Renal anemia: from incurable to curable.
      In the reversible short-term UUO model, Souma et al. demonstrated that activated myofibroblasts, which are derived from renal EPO-producing cells, can revert to their normal phenotype with EPO production,
      • Souma T.
      • Yamazaki S.
      • Moriguchi T.
      • et al.
      Plasticity of renal erythropoietin-producing cells governs fibrosis.
      indicating the functional plasticity of myofibroblasts.

       Regional heterogeneity of fibroblasts and myofibroblasts

      Recently, it was demonstrated that Wnt4 is expressed only in medullary myofibroblasts and not in cortical myofibroblasts.
      • Dirocco D.P.
      • Kobayashi A.
      • Taketo M.M.
      • et al.
      Wnt4/β-catenin signaling in medullary kidney myofibroblasts.
      Interestingly, the distribution of Wnt4-expressing myofibroblasts is different from that of P0-Cre lineage–labeled myofibroblasts in the cortex and outer medulla, suggesting the regional heterogeneity of myofibroblasts. A subset of medullary interstitial fibroblasts is also known to possess some unique characteristics that are similar to those of hepatic stellate cells. Hepatic stellate cells are liver-specific mesenchymal cells that store 50–80% of the body’s vitamin A as retinyl palmitate in cytoplasmic lipid droplets.
      • Senoo H.
      • Yoshikawa K.
      • Morii M.
      • et al.
      Hepatic stellate cell (vitamin A-storing cell) and its relative—past, present and future.
      Hepatic stellate cells have long been thought to be the primary source of myofibroblasts after liver injury,
      • Iwaisako K.
      • Brenner D.A.
      • Kisseleva T.
      What's new in liver fibrosis? The origin of myofibroblasts in liver fibrosis.
      which has been confirmed using genetic fate-mapping studies in mouse models of alcoholic and toxic liver fibrosis.
      • Kisseleva T.
      • Cong M.
      • Paik Y.
      • et al.
      Myofibroblasts revert to an inactive phenotype during regression of liver fibrosis.
      Vitamin A–storing cells also seem to contribute to renal fibrosis as cytoglobin/stellate cell activation–associated protein, a unique marker for the vitamin A–storing cell lineage, is expressed on cells that accumulate in UUO kidneys with incomplete colocalization with αSMA.
      • Kida Y.
      • Asahina K.
      • Inoue K.
      • et al.
      Characterization of vitamin A-storing cells in mouse fibrous kidneys using Cygb/STAP as a marker of activated stellate cells.

      PERICYTES

       Detection of pericytes

      Pericytes are contractile cells of mesenchymal origin that wrap around the microvessels.
      • Campanholle G.
      • Ligresti G.
      • Gharib S.A.
      • et al.
      Cellular mechanisms of tissue fibrosis. 3. Novel mechanisms of kidney fibrosis.
      Pericytes have a critical role in the stability and integrity of the microvessels, and they regulate vascular tone and capillary diameter to control microcirculation.
      • Herman I.M.
      • D'Amore P.A.
      Microvascular pericytes contain muscle and nonmuscle actins.
      ,
      • Pallone T.L.
      • Silldorff E.P.
      Pericyte regulation of renal medullary blood flow.
      Pericytes are defined as the cells embedded within the vascular basement membrane; however, this detection requires ultrastructure analysis, which is not practical.
      • Armulik A.
      • Genove G.
      • Betsholtz C.
      Pericytes: developmental, physiological, and pathological perspectives, problems, and promises.
      Instead, markers such as PDGFRβ and NG2 are used for pericyte detection, which are not specific for pericytes.
      • Armulik A.
      • Genove G.
      • Betsholtz C.
      Pericytes: developmental, physiological, and pathological perspectives, problems, and promises.

       Pericytes in fibrosis

      Humphreys et al. used lineage-tracing methods using FoxD1-Cre mice and indicator mice, in which the activation of the FoxD1 promoter induces the Cre-mediated recombination of reporter alleles and marks the cells arising from FoxD1-Cre–expressing cells with the expression of LacZ. The authors demonstrated that LacZ was expressed specifically in pericytes and that αSMA-positive cells in the interstitium originated from pericytes in both UUO and IR injury.
      • Humphreys B.D.
      • Lin S.L.
      • Kobayashi A.
      • et al.
      Fate tracing reveals the pericyte and not epithelial origin of myofibroblasts in kidney fibrosis.
      Recently, several studies have suggested a critical role for pericytes in the pathogenesis of tissue fibrosis in other organs. Hepatic stellate cells, as discussed in the previous section, are also known as the resident liver pericytes and the primary source of myofibroblasts in liver fibrosis.
      • Iwaisako K.
      • Brenner D.A.
      • Kisseleva T.
      What's new in liver fibrosis? The origin of myofibroblasts in liver fibrosis.
      In spinal cord scarring, a specific pericyte subtype gave rise to scar-forming stromal cells and contributed to tissue fibrosis.
      • Goritz C.
      • Dias D.O.
      • Tomilin N.
      • et al.
      A pericyte origin of spinal cord scar tissue.
      Moreover, perivascular mesenchymal cells of ADAM12-positive lineage were shown to transdifferentiate into myofibroblasts after acute muscle and dermis injury.
      • Dulauroy S.
      • Di Carlo S.E.
      • Langa F.
      • et al.
      Lineage tracing and genetic ablation of ADAM12(+) perivascular cells identify a major source of profibrotic cells during acute tissue injury.
      Research has focused on determining the developmental origin of pericytes. Recently, numerous lineage-tracing studies have revealed that pericytes have several different developmental origins.
      • Armulik A.
      • Genove G.
      • Betsholtz C.
      Pericytes: developmental, physiological, and pathological perspectives, problems, and promises.
      For example, vascular smooth muscle cells of the aorta and many of its branches originated from the secondary heart field, neural crest, somites, and splanchnic mesoderm,
      • Armulik A.
      • Genove G.
      • Betsholtz C.
      Pericytes: developmental, physiological, and pathological perspectives, problems, and promises.
      whereas pericytes in the thymus and the CNS are derived from the neural crest.
      • Etchevers H.C.
      • Vincent C.
      • Le Douarin N.M.
      • et al.
      The cephalic neural crest provides pericytes and smooth muscle cells to all blood vessels of the face and forebrain.
      • Korn J.
      • Christ B.
      • Kurz H.
      Neuroectodermal origin of brain pericytes and vascular smooth muscle cells.
      • Heglind M.
      • Cederberg A.
      • Aquino J.
      • et al.
      Lack of the central nervous system- and neural crest-expressed forkhead gene Foxs1 affects motor function and body weight.
      • Foster K.
      • Sheridan J.
      • Veiga-Fernandes H.
      • et al.
      Contribution of neural crest-derived cells in the embryonic and adult thymus.
      • Muller S.M.
      • Stolt C.C.
      • Terszowski G.
      • et al.
      Neural crest origin of perivascular mesenchyme in the adult thymus.
      Interestingly, pericytes in the CNS and renal pericytes share several characteristics under pathologic conditions. Schrimpf et al. explored the transcriptional changes between naïve pericytes and activated pericytes after UUO by microarray analysis. In the process of transdifferentiation into myofibroblasts, pericytes become not only matrix-secreting cells but also innate inflammatory effector cells in the kidney.
      • Schrimpf C.
      • Xin C.
      • Campanholle G.
      • et al.
      Pericyte TIMP3 and ADAMTS1 modulate vascular stability after kidney injury.
      This is in line with a recent report that pericytes in the brain transdifferentiate into inflammatory effector cells in response to the inflammatory microenvironment.
      • Stark K.
      • Eckart A.
      • Haidari S.
      • et al.
      Capillary and arteriolar pericytes attract innate leukocytes exiting through venules and 'instruct' them with pattern-recognition and motility programs.

       Pericytes and resident fibroblasts, distinct or overlapping populations?

      An important question that remains to be clarified is how resident fibroblasts labeled with P0-Cre differ from pericytes labeled with FoxD1-Cre, and to what degree these two populations overlap. Resident fibroblasts and pericytes are frequently confused because they share distinctive cellular markers, such as CD73 and PDGFRβ, and they both are found in the tubulointerstitial space.
      When P0-Cre lineage–labeled cells enter embryonic kidney during development, they transiently express FoxD1.
      • Asada N.
      • Takase M.
      • Nakamura J.
      • et al.
      Dysfunction of fibroblasts of extrarenal origin underlies renal fibrosis and renal anemia in mice.
      On the other hand, FoxD1 is expressed in the migrating neural crest.
      • Gomez-Skarmeta J.L.
      • de la Calle-Mustienes E.
      • Modolell J.
      • et al.
      Xenopus brain factor-2 controls mesoderm, forebrain and neural crest development.
      These data strongly suggest the possibility that these two cell populations are overlapping. The fact that pericytes in the brain, thymus, and the part of aorta are derived from neural crest also supports this idea.
      • Armulik A.
      • Genove G.
      • Betsholtz C.
      Pericytes: developmental, physiological, and pathological perspectives, problems, and promises.
      Elucidation of this point is crucial in terms of developing therapies to specifically target kidney myofibroblasts.

       Pericyte detachment: dual impact for CKD progression

      Histological studies using animal models have demonstrated that renal fibrosis and reduced peritubular capillary density occur under the same conditions and are associated with each other,
      • Choi Y.J.
      • Chakraborty S.
      • Nguyen V.
      • et al.
      Peritubular capillary loss is associated with chronic tubulointerstitial injury in human kidney: altered expression of vascular endothelial growth factor.
      • Seron D.
      • Alexopoulos E.
      • Raftery M.J.
      • et al.
      Number of interstitial capillary cross-sections assessed by monoclonal antibodies: relation to interstitial damage.
      • Bohle A.
      • Mackensen-Haen S.
      • Wehrmann M.
      Significance of postglomerular capillaries in the pathogenesis of chronic renal failure.
      suggesting that these two processes are mechanistically linked. Recently, it has been proposed that pericyte detachment underlies these two pathological conditions.
      • Campanholle G.
      • Ligresti G.
      • Gharib S.A.
      • et al.
      Cellular mechanisms of tissue fibrosis. 3. Novel mechanisms of kidney fibrosis.
      In response to kidney injury, pericytes promptly detach from peritubular capillaries, migrate into the interstitial space, and transdifferentiate into scar-forming myofibroblasts. In the absence of pericytes, peritubular capillaries are destabilized, which leads to capillary regression, rarefaction, and subsequent hypoxia in the interstitium.
      • Kida Y.
      • Duffield J.S.
      Pivotal role of pericytes in kidney fibrosis.
      Several studies have demonstrated that chronic hypoxia of the interstitium is the final common pathway by which CKD progresses to end-stage renal disease.
      • Mimura I.
      • Nangaku M.
      The suffocating kidney: tubulointerstitial hypoxia in end-stage renal disease.
      Hypoxia can activate fibroblasts and induce changes in ECM metabolism of the resident kidney cells, thereby leading to fibrogenesis.
      • Norman J.T.
      • Clark I.M.
      • Garcia P.L.
      Hypoxia promotes fibrogenesis in human renal fibroblasts.
      In addition, fibrosis itself hinders the efficient oxygen diffusion into the interstitium and promotes hypoxia.
      • Norman J.T.
      • Fine L.G.
      Intrarenal oxygenation in chronic renal failure.
      Thus, strategies to prevent pericyte detachment are potential novel therapeutic approaches for CKD, and further research is required.

       Pericyte activation: the role of injured tubules

      Although tubular epithelial cells may rarely transdifferentiate into myofibroblasts (this issue will be discussed in the EMT section), injured epithelial cells have important roles in the progression of renal fibrosis.
      • Kaissling B.
      • Lehir M.
      • Kriz W.
      Renal epithelial injury and fibrosis.
      ,
      • Prunotto M.
      • Budd D.C.
      • Gabbiani G.
      • et al.
      Epithelial-mesenchymal crosstalk alteration in kidney fibrosis.
      Grgic et al. have established a new mouse model of selective renal epithelial injury by using Cre-LoxP technology in combination with toxin receptor–mediated cell deletion
      • Grgic I.
      • Campanholle G.
      • Bijol V.
      • et al.
      Targeted proximal tubule injury triggers interstitial fibrosis and glomerulosclerosis.
      and have demonstrated that injured tubular epithelial cells can trigger renal fibrosis and capillary rarefaction.
      Recently, several mechanisms by which injured epithelial cells cause interstitial fibrosis have been proposed. First, injured epithelial cells produce a variety of proinflammatory chemokines and cytokines, such as interleukin (IL)-1, IL-6, monocyte chemotactic protein-1, or tumor necrosis factor (TNF)-α, which recruit and activate inflammatory cells and promote fibroblast differentiation into myofibroblasts.
      • Zeisberg M.
      • Neilson E.G.
      Mechanisms of tubulointerstitial fibrosis.
      Using various kidney injury models (moderate ischemic, severe ischemic, toxic, and obstructive), Yang et al. demonstrated that arresting the cell cycle at the G2/M checkpoint in kidney proximal tubular cells is sufficient to drive fibrosis.
      • Yang L.
      • Besschetnova T.Y.
      • Brooks C.R.
      • et al.
      Epithelial cell cycle arrest in G2/M mediates kidney fibrosis after injury.
      They showed that the expression of profibrotic cytokines such as TGF-β and connective tissue growth factor are upregulated in injured epithelial cells. They further demonstrated that prolonged G2/M arrest causes a profibrotic phenotype, whereas reversal of G2/M arrest rescues fibrosis in the unilateral ischemic injured kidney. In addition, they showed that G2/M-arrested proximal tubular epithelial cells activate the c-jun NH2-terminal kinase signaling cascade, which stimulates the production of profibrotic cytokine. Injured tubular epithelial cells also produce exosomes containing TGF-β1 mRNA and release them to promote proliferation, α-SMA expression, F-actin expression, and type I collagen production in neighboring fibroblasts.
      • Borges F.T.
      • Melo S.A.
      • Ozdemir B.C.
      • et al.
      TGF-β1-containing exosomes from injured epithelial cells activate fibroblasts to initiate tissue regenerative responses and fibrosis.
      Ding et al. demonstrated that Sonic Hedgehog is released from injured tubules, and it promotes myofibroblast activation and interstitial fibrosis through Sonic Hedgehog/Gli signaling.
      • Ding H.
      • Zhou D.
      • Hao S.
      • et al.
      Sonic Hedgehog signaling mediates epithelial-mesenchymal communication and promotes renal fibrosis.
      Another mechanism by which injured epithelial cells drive fibrosis is epigenetic modifications of fibroblasts, caused by molecules released by the injured tubule. Hypermethylation of RASAL1, an inhibitor of the Ras oncoprotein, in fibroblasts is identified as the cause of sustained fibrogenesis.
      • Bechtel W.
      • McGoohan S.
      • Zeisberg E.M.
      • et al.
      Methylation determines fibroblast activation and fibrogenesis in the kidney.
      Epigenetic RASAL1 silencing of fibroblasts lead to Ras hyperactivity, which resulted in sustained fibroblast activation. TGF-β1 silences RASAL1 expression by two mechanisms, direct transcriptional repression and epigenetic modification. In this manner, TGF-β1 produced by injured tubules contributes to sustained renal fibrosis.

      BONE MARROW–DERIVED CELLS (FIBROCYTES)

      Fibrocytes are defined as collagen-producing cells of hematopoietic origin. They were first described by Bucala et al. in the inflammatory exudate of subcutaneously implanted wound chambers
      • Bucala R.
      • Spiegel L.A.
      • Chesney J.
      • et al.
      Circulating fibrocytes define a new leukocyte subpopulation that mediates tissue repair.
      and were later found in various animal models of fibrosis (e.g., affecting lung, liver, heart, skin, and kidney),
      • Hashimoto N.
      • Jin H.
      • Liu T.
      • et al.
      Bone marrow-derived progenitor cells in pulmonary fibrosis.
      • Phillips R.J.
      • Burdick M.D.
      • Hong K.
      • et al.
      Circulating fibrocytes traffic to the lungs in response to CXCL12 and mediate fibrosis.
      • Kisseleva T.
      • Uchinami H.
      • Feirt N.
      • et al.
      Bone marrow-derived fibrocytes participate in pathogenesis of liver fibrosis.
      • Sakai N.
      • Wada T.
      • Yokoyama H.
      • et al.
      Secondary lymphoid tissue chemokine (SLC/CCL21)/CCR7 signaling regulates fibrocytes in renal fibrosis.
      • Niedermeier M.
      • Reich B.
      • Rodriguez Gomez M.
      • et al.
      CD4+ T cells control the differentiation of Gr1+ monocytes into fibrocytes.
      • Haudek S.B.
      • Xia Y.
      • Huebener P.
      • et al.
      Bone marrow-derived fibroblast precursors mediate ischemic cardiomyopathy in mice.
      • Mori L.
      • Bellini A.
      • Stacey M.A.
      • et al.
      Fibrocytes contribute to the myofibroblast population in wounded skin and originate from the bone marrow.
      as well as in human diseases.

       Detection of fibrocytes

      Several methods were used to show the existence of hematopoietic or bone marrow–derived fibrocytes in various organs including the kidney. The bone marrow origin of fibrocytes is commonly demonstrated by bone marrow transplantation using bone marrow from various types of reporter mice (e.g., ubiquitous expression of GFP or alkaline phosphatase) and subsequent detection of αSMA or collagen on reporter-positive cells.
      • Broekema M.
      • Harmsen M.C.
      • van Luyn M.J.
      • et al.
      Bone marrow-derived myofibroblasts contribute to the renal interstitial myofibroblast population and produce procollagen I after ischemia/reperfusion in rats.
      • Li J.
      • Deane J.A.
      • Campanale N.V.
      • et al.
      The contribution of bone marrow-derived cells to the development of renal interstitial fibrosis.
      • Jang H.S.
      • Kim J.I.
      • Jung K.J.
      • et al.
      Bone marrow-derived cells play a major role in kidney fibrosis via proliferation and differentiation in the infiltrated site.
      Alternatively, mice expressing GFP or other markers under the control of a specific promoter (e.g., collagen I-promoter or αSMA promoter) were used.
      • LeBleu V.S.
      • Taduri G.
      • O'Connell J.
      • et al.
      Origin and function of myofibroblasts in kidney fibrosis.
      ,
      • Roufosse C.
      • Bou-Gharios G.
      • Prodromidi E.
      • et al.
      Bone marrow-derived cells do not contribute significantly to collagen I synthesis in a murine model of renal fibrosis.
      Bone marrow transplantation does not fully ensure that only hematopoietic cells express the reporter, because other stem cells (e.g., mesenchymal stem cells) could also be transplanted and become reporter-positive. Ideally, bone marrow transplantation is combined with the detection of hematopoietic markers such as CD45 or CD11b on reporter-positive cells. An alternative approach for the detection of fibrocytes without bone marrow transplantation is double staining of cells using specific hematopoietic markers such as CD45, CD11b, and CD34 together with intracellular detection of collagen or αSMA.
      • Sakai N.
      • Wada T.
      • Yokoyama H.
      • et al.
      Secondary lymphoid tissue chemokine (SLC/CCL21)/CCR7 signaling regulates fibrocytes in renal fibrosis.
      ,
      • Niedermeier M.
      • Reich B.
      • Rodriguez Gomez M.
      • et al.
      CD4+ T cells control the differentiation of Gr1+ monocytes into fibrocytes.
      However, fibrocytes may lose some of these hematopoietic markers over time, such as during prolonged cell culture.
      • Phillips R.J.
      • Burdick M.D.
      • Hong K.
      • et al.
      Circulating fibrocytes traffic to the lungs in response to CXCL12 and mediate fibrosis.
      ,
      • Abe R.
      • Donnelly S.C.
      • Peng T.
      • et al.
      Peripheral blood fibrocytes: differentiation pathway and migration to wound sites.
      ,
      • Pilling D.
      • Fan T.
      • Huang D.
      • et al.
      Identification of markers that distinguish monocyte-derived fibrocytes from monocytes, macrophages, and fibroblasts.
      It is also important to include appropriate controls to ensure correct and specific detection of collagen or αSMA. Flow cytometric analysis of intracellular collagen type I in permeabilized cells is a reliable method, provided that appropriate controls with isotype control antibodies are included (Figure 3). Nonpermeabilized cells can be used to rule out binding of collagen on the cell surface, such as by integrins. In addition, one may use antibodies that are specific for procollagen I to rule out that intracellular collagen is derived from endocytosed or phagocytosed collagen. Procollagen I would only be detectable if a cell expresses collagen by itself. Procollagen-specific antibodies have been used in rats, mice, and humans.
      • Broekema M.
      • Harmsen M.C.
      • van Luyn M.J.
      • et al.
      Bone marrow-derived myofibroblasts contribute to the renal interstitial myofibroblast population and produce procollagen I after ischemia/reperfusion in rats.
      ,
      • Rydell-Tormanen K.
      • Andreasson K.
      • Hesselstrand R.
      • et al.
      Extracellular matrix alterations and acute inflammation; developing in parallel during early induction of pulmonary fibrosis.
      ,
      • Sakai N.
      • Furuichi K.
      • Shinozaki Y.
      • et al.
      Fibrocytes are involved in the pathogenesis of human chronic kidney disease.
      Flow cytometry also allows fibrocyte quantification within tissues, if single-cell suspensions are generated.
      Figure thumbnail gr3
      Figure 3Identification of fibrocytes. Splenocytes from mice with unilateral ureteral obstruction were stained with antibodies against CD45, CD11b, and collagen type I or isotype control. Only CD45+ cells are depicted. Intracellular staining of permeabilized cells reveals a distinct population of collagen-positive cells (a) in comparison with the isotype control (b). Extracellular staining of nonpermeabilized cells for collagen I (c) excludes the presence of collagen on the cell surface, as further control cells were digested before (d) or after permeabilization (d) with collagenase and then stained for intracellular collagen I.
      Detection of fibrocytes by two-color immunohistology or immunofluorescence (e.g., with antibodies against the hematopoietic marker CD45 and collagen) has the same limitations as described for flow cytometry above, and it also requires a good spatial resolution to distinguish between cells producing collagen and cells merely surrounded by fibrotic tissue. αSMA is a commonly used surrogate intracellular marker for cells that produce ECM (e.g., various types of collagen or fibronectin). However, as discussed in the Introduction, not all αSMA-positive cells produce ECM and not all matrix-producing cells also express αSMA,
      • Lin S.L.
      • Kisseleva T.
      • Brenner D.A.
      • et al.
      Pericytes and perivascular fibroblasts are the primary source of collagen-producing cells in obstructive fibrosis of the kidney.
      raising some concerns over whether αSMA is an ideal marker for matrix-producing cells.
      A further limitation of current studies comes from the uncertainty regarding how much matrix is produced or secreted by various collagen- or αSMA-positive cell types. Absolute or relative numbers of these cells do not necessarily correlate with the amount of matrix or collagen secreted by these cells. This limitation applies not only to fibrocytes but also to other cells such as myofibroblasts.

       Fibrocytes and monocytes: distinct or overlapping populations?

      A longstanding question in the field is the relationship between fibrocytes and monocytes, based on the observation that both cell types share a variety of surface markers, such as CD45, CD11b, CD16/32, CD68, major histocompatibility complex II, and fibroblast-specific protein 1.
      • Inoue T.
      • Plieth D.
      • Venkov C.D.
      • et al.
      Antibodies against macrophages that overlap in specificity with fibroblasts.
      • Osterreicher C.H.
      • Penz-Osterreicher M.
      • Grivennikov S.I.
      • et al.
      Fibroblast-specific protein 1 identifies an inflammatory subpopulation of macrophages in the liver.
      • Le Hir M.
      • Hegyi I.
      • Cueni-Loffing D.
      • et al.
      Characterization of renal interstitial fibroblast-specific protein 1/S100A4-positive cells in healthy and inflamed rodent kidneys.
      Fibrocytes could simply be a subpopulation of collagen-producing monocytes or macrophages, and indeed the production of various types of collagens has been found in human and murine macrophages.
      • Vaage J.
      • Harlos J.P.
      Collagen production by macrophages in tumour encapsulation and dormancy.
      • Vaage J.
      • Lindblad W.J.
      Production of collagen type I by mouse peritoneal macrophages.
      • Schnoor M.
      • Cullen P.
      • Lorkowski J.
      • et al.
      Production of type VI collagen by human macrophages: a new dimension in macrophage functional heterogeneity.
      Alternatively, under the influence of environmental and profibrotic stimuli, fibrocytes could develop from monocytes that have been attracted to sites of tissue damage. It is conceivable that surface markers would be downregulated or upregulated during this differentiation or transformation process, resulting in a cell population with a distinct set of surface markers.
      • Bertrand S.
      • Godoy M.
      • Semal P.
      • et al.
      Transdifferentiation of macrophages into fibroblasts as a result of Schistosoma mansoni infection.
      ,
      • Kuwana M.
      • Okazaki Y.
      • Kodama H.
      • et al.
      Human circulating CD14+ monocytes as a source of progenitors that exhibit mesenchymal cell differentiation.
      Pronounced changes in surface markers were observed when fibrocytes were generated in vitro from cultures of human PBMCs or murine leukocytes.
      • Phillips R.J.
      • Burdick M.D.
      • Hong K.
      • et al.
      Circulating fibrocytes traffic to the lungs in response to CXCL12 and mediate fibrosis.
      ,
      • Pilling D.
      • Fan T.
      • Huang D.
      • et al.
      Identification of markers that distinguish monocyte-derived fibrocytes from monocytes, macrophages, and fibroblasts.
      ,
      • Schmidt M.
      • Sun G.
      • Stacey M.A.
      • et al.
      Identification of circulating fibrocytes as precursors of bronchial myofibroblasts in asthma.
      After prolonged culture, even typical hematopoietic markers were gradually lost in a considerable fraction of fibrocytes. The concept that monocytes differentiate into fibrocytes is based on cell culture experiments with enriched or purified ‘monocytes’. A large number of groups have shown that cells expressing monocytic markers, such as CD14 or CD11b, develop into fibrocytes under appropriate culture conditions.
      • Niedermeier M.
      • Reich B.
      • Rodriguez Gomez M.
      • et al.
      CD4+ T cells control the differentiation of Gr1+ monocytes into fibrocytes.
      ,
      • Abe R.
      • Donnelly S.C.
      • Peng T.
      • et al.
      Peripheral blood fibrocytes: differentiation pathway and migration to wound sites.
      ,
      • Pilling D.
      • Buckley C.D.
      • Salmon M.
      • et al.
      Inhibition of fibrocyte differentiation by serum amyloid P.
      • Pilling D.
      • Gomer R.H.
      Differentiation of circulating monocytes into fibroblast-like cells.
      • Bellini A.
      • Mattoli S.
      The role of the fibrocyte, a bone marrow-derived mesenchymal progenitor, in reactive and reparative fibroses.
      • Chesney J.
      • Metz C.
      • Stavitsky A.B.
      • et al.
      Regulated production of type I collagen and inflammatory cytokines by peripheral blood fibrocytes.
      • Yang L.
      • Scott P.G.
      • Giuffre J.
      • et al.
      Peripheral blood fibrocytes from burn patients: identification and quantification of fibrocytes in adherent cells cultured from peripheral blood mononuclear cells.
      Factors and other cells (such as T cells) supporting this differentiation have been identified (see below), and several methods, including immunofluorescence, flow cytometry, reverse transcription PCR, and ELISA, have been used to demonstrate collagen production in the cultured cells. However, these experiments do not unequivocally demonstrate a differentiation of monocytes into collagen-producing fibrocytes, because fibrocytes are already detectable within human PBMCs or murine splenocytes, and the enrichment of monocytes with markers such as CD14 or CD11b could also result in the enrichment of fibrocytes. Monocytes, together with other cells (e.g., T cells) and soluble factors, can support the survival of fibrocytes in culture, their appearance as spindle-shaped cells, and their continued production of collagen. Recently, the relationship between monocytes and fibrocytes was analyzed in vivo using mice with renal fibrosis after UUO. As early as 3 days after ureteral obstruction, the number of monocytes and fibrocytes markedly increased in the kidney.
      • Reich B.
      • Schmidbauer K.
      • Rodriguez Gomez M.
      • et al.
      Fibrocytes develop outside the kidney but contribute to renal fibrosis in a mouse model.
      When monocytes were depleted using an antibody against CCR2 before ureteral ligation, resulting in the almost complete absence of Gr1+ and Gr1 monocytes in the kidney and the periphery, the increase in renal fibrocytes was the same. In addition, no improvement in fibrosis was observed. These data suggest that mature monocytes are not required for the development of fibrocytes in vivo or for the generation of renal fibrosis. The rapid appearance of fibrocytes in the UUO kidney after only 3 days suggests that fibrocytes migrate into the kidney rather than differentiate from infiltrating monocytes. Local proliferation is not involved in the expansion of bone marrow–derived fibrocytes in the kidney.
      • LeBleu V.S.
      • Taduri G.
      • O'Connell J.
      • et al.
      Origin and function of myofibroblasts in kidney fibrosis.

       Fibrocytes in fibrosis

      Considering the limitations in detecting fibrocytes described above, several groups have concluded that bone marrow–derived, most likely hematopoietic, cells produce collagen and are present in various types of renal disease in mice and man. Recently, bone marrow chimeric αSMA reporter mice were used to demonstrate that about 35% of the αSMA-positive cells are derived from the bone marrow, whereas 50% seem to be derived from resident renal fibroblasts in the model of UUO.
      • LeBleu V.S.
      • Taduri G.
      • O'Connell J.
      • et al.
      Origin and function of myofibroblasts in kidney fibrosis.
      Epithelial and endothelial cells made only a minor contribution. No data were presented to distinguish between hematopoietic cells and mesenchymal stem cells in bone marrow–derived cells. Other studies used co-staining of hematopoietic markers, mainly CD45, CD11b, and CD34 together with intracellular collagen I, for the detection of fibrocytes in UUO kidneys.
      • Sakai N.
      • Wada T.
      • Yokoyama H.
      • et al.
      Secondary lymphoid tissue chemokine (SLC/CCL21)/CCR7 signaling regulates fibrocytes in renal fibrosis.
      ,
      • Niedermeier M.
      • Reich B.
      • Rodriguez Gomez M.
      • et al.
      CD4+ T cells control the differentiation of Gr1+ monocytes into fibrocytes.
      About 20% of the collagen-positive cells expressed the hematopoietic marker CD45, indicating that these cells are of hematopoietic origin.
      • Niedermeier M.
      • Reich B.
      • Rodriguez Gomez M.
      • et al.
      CD4+ T cells control the differentiation of Gr1+ monocytes into fibrocytes.
      Using similar approaches, bone marrow–derived collagen-producing cells or fibrocytes were also detected in renal fibrosis models, including Alport syndrome,
      • LeBleu V.S.
      • Taduri G.
      • O'Connell J.
      • et al.
      Origin and function of myofibroblasts in kidney fibrosis.
      adriamycin-induced fibrosis,
      • Li J.
      • Deane J.A.
      • Campanale N.V.
      • et al.
      The contribution of bone marrow-derived cells to the development of renal interstitial fibrosis.
      IR,
      • Broekema M.
      • Harmsen M.C.
      • van Luyn M.J.
      • et al.
      Bone marrow-derived myofibroblasts contribute to the renal interstitial myofibroblast population and produce procollagen I after ischemia/reperfusion in rats.
      and chronic angiotensin infusion model.
      • Sakai N.
      • Wada T.
      • Matsushima K.
      • et al.
      The renin-angiotensin system contributes to renal fibrosis through regulation of fibrocytes.
      In some reporter mice, luciferase/galactosidase or GFP was driven by a fragment of the col1a2 or the col1a1 promoter to detect collagen I–expressing cells.
      • Lin S.L.
      • Kisseleva T.
      • Brenner D.A.
      • et al.
      Pericytes and perivascular fibroblasts are the primary source of collagen-producing cells in obstructive fibrosis of the kidney.
      ,
      • Roufosse C.
      • Bou-Gharios G.
      • Prodromidi E.
      • et al.
      Bone marrow-derived cells do not contribute significantly to collagen I synthesis in a murine model of renal fibrosis.
      In both cases, only a minor fraction of bone marrow–derived cells in the kidney were positive for the reporter. The genetic approach is limited in that the activity of the promoter fragment may be restricted to some cell types. In addition, the reporter expression could be downregulated by certain cell types or interfere with cellular function, migration, and survival, making it difficult to interpret negative data obtained with reporter mice.
      In human renal disease, fibrocytes were identified using pro-collagen I and CD45 costaining, and they were found mainly in the interstitium in various types of glomerulonephritis and chronic kidney disease.
      • Sakai N.
      • Furuichi K.
      • Shinozaki Y.
      • et al.
      Fibrocytes are involved in the pathogenesis of human chronic kidney disease.
      The number of fibrocytes correlated with the severity of tubulointerstitial damage and fibrosis, as well as with the number of infiltrating CD68+ macrophages. In another study involving biopsies from patients with glomerulonephritis, CD34+, αSMA+ spindle-shaped cells were detected in the renal interstitium.
      • Okon K.
      • Szumera A.
      • Kuzniewski M.
      Are CD34+ cells found in renal interstitial fibrosis?.
      In patients with chronic allograft nephrophathy and a pronounced interstitial fibrosis, a significant proportion of myofibroblasts were derived from the recipient.
      • Grimm P.C.
      • Nickerson P.
      • Jeffery J.
      • et al.
      Neointimal and tubulointerstitial infiltration by recipient mesenchymal cells in chronic renal-allograft rejection.
      Identifying interference with fibrocytes or fibrocyte function is important in understanding their role in renal disease. Depletion of these cells could be one possible approach. The high Gr1-expression level allowed depletion of fibrocytes with an antibody against Gr-1, and bone marrow chimeric transgenic mice expressing the diphtheria toxin receptor under the control of the CD11b promoter are also suitable.
      • Reich B.
      • Schmidbauer K.
      • Rodriguez Gomez M.
      • et al.
      Fibrocytes develop outside the kidney but contribute to renal fibrosis in a mouse model.
      Both approaches are effective, but neither is specific for fibrocytes. Nevertheless, a clear reduction of renal fibrosis was observed, suggesting that fibrocytes contribute to the production of collagen I in the kidney. An alternative approach is the specific deletion of αSMA-positive cells in combination with bone marrow transplantation or inhibition of TGF-β signaling in αSMA-positive cells.
      • LeBleu V.S.
      • Taduri G.
      • O'Connell J.
      • et al.
      Origin and function of myofibroblasts in kidney fibrosis.
      In summary, a large number of data suggest that fibrocytes contribute to renal fibrosis, either directly by production of ECM or indirectly by supporting the expansion and matrix production of resident cells in the kidney.

       Fibrocyte migration

      Currently, fibrocytes are thought to develop in the bone marrow from myeloid precursor cells, but not from mature monocytes.
      • Reich B.
      • Schmidbauer K.
      • Rodriguez Gomez M.
      • et al.
      Fibrocytes develop outside the kidney but contribute to renal fibrosis in a mouse model.
      Therefore, fibrocyte and monocyte migration might not follow the same rules. It is generally accepted that monocytes require CCR2 to migrate out of the bone marrow, and consequently that CCR2−/- mice have fewer Gr-1+ and Gr-1 monocytes in the peripheral blood and lymphatic organs and larger numbers in the bone marrow.
      • Tsou C.L.
      • Peters W.
      • Si Y.
      • et al.
      Critical roles for CCR2 and MCP-3 in monocyte mobilization from bone marrow and recruitment to inflammatory sites.
      In contrast, fibrocyte migration out of the bone marrow is CCR2-independent, because CCR2−/- mice have the same numbers of fibrocytes in the spleen and peripheral blood as do wild-type mice.
      • Reich B.
      • Schmidbauer K.
      • Rodriguez Gomez M.
      • et al.
      Fibrocytes develop outside the kidney but contribute to renal fibrosis in a mouse model.
      It is currently unknown as to which factors regulate fibrocyte generation and release from the bone marrow.
      Fibrocyte migration from the peripheral blood into fibrotic organs was shown to depend on various chemokine receptors. CCR2, CCR7, and CXCR6 are involved in fibrocyte migration into the kidney, as shown by inhibition or genetic ablation of receptors and ligands.
      • Sakai N.
      • Wada T.
      • Yokoyama H.
      • et al.
      Secondary lymphoid tissue chemokine (SLC/CCL21)/CCR7 signaling regulates fibrocytes in renal fibrosis.
      ,
      • Reich B.
      • Schmidbauer K.
      • Rodriguez Gomez M.
      • et al.
      Fibrocytes develop outside the kidney but contribute to renal fibrosis in a mouse model.
      ,
      • Chen G.
      • Lin S.C.
      • Chen J.
      • et al.
      CXCL16 recruits bone marrow-derived fibroblast precursors in renal fibrosis.
      ,
      • Xia Y.
      • Entman M.L.
      • Wang Y.
      Critical role of CXCL16 in hypertensive kidney injury and fibrosis.
      Fibrocyte migration into other organs such as the lung, liver, heart, and skin is mediated by CCR2, CCR5, CCR7, and CXCR4.
      • Hashimoto N.
      • Jin H.
      • Liu T.
      • et al.
      Bone marrow-derived progenitor cells in pulmonary fibrosis.
      ,
      • Phillips R.J.
      • Burdick M.D.
      • Hong K.
      • et al.
      Circulating fibrocytes traffic to the lungs in response to CXCL12 and mediate fibrosis.
      ,
      • Abe R.
      • Donnelly S.C.
      • Peng T.
      • et al.
      Peripheral blood fibrocytes: differentiation pathway and migration to wound sites.
      ,
      • van Deventer H.W.
      • Wu Q.P.
      • Bergstralh D.T.
      • et al.
      C-C chemokine receptor 5 on pulmonary fibrocytes facilitates migration and promotes metastasis via matrix metalloproteinase 9.
      • Chu P.Y.
      • Mariani J.
      • Finch S.
      • et al.
      Bone marrow-derived cells contribute to fibrosis in the chronically failing heart.
      • Ishida Y.
      • Kimura A.
      • Kondo T.
      • et al.
      Essential roles of the CC chemokine ligand 3-CC chemokine receptor 5 axis in bleomycin-induced pulmonary fibrosis through regulation of macrophage and fibrocyte infiltration.
      • Moore B.B.
      • Kolodsick J.E.
      • Thannickal V.J.
      • et al.
      CCR2-mediated recruitment of fibrocytes to the alveolar space after fibrotic injury.
      • Moore B.B.
      • Murray L.
      • Das A.
      • et al.
      The role of CCL12 in the recruitment of fibrocytes and lung fibrosis.
      • Sun L.
      • Louie M.C.
      • Vannella K.M.
      • et al.
      New concepts of IL-10-induced lung fibrosis: fibrocyte recruitment and M2 activation in a CCL2/CCR2 axis.
      • Scholten D.
      • Reichart D.
      • Paik Y.H.
      • et al.
      Migration of fibrocytes in fibrogenic liver injury.
      • Xu J.
      • Lin S.C.
      • Chen J.
      • et al.
      CCR2 mediates the uptake of bone marrow-derived fibroblast precursors in angiotensin II-induced cardiac fibrosis.
      However, none of these chemokine receptors and ligands are fibrocyte-specific and exclusively interfere with fibrocyte migration. Reduced migration of other leukocyte populations may indirectly affect fibrocyte numbers or fibrosis in the kidney.

       Factors regulating fibrocyte development

      Several factors were shown to influence the appearance of fibrocytes, when human PBMCs or murine leukocytes were taken into culture. Some of these factors were also analyzed using models of renal or other organ fibrosis.
      Human fibrocyte development is facilitated by helper cells, especially T cells.
      • Abe R.
      • Donnelly S.C.
      • Peng T.
      • et al.
      Peripheral blood fibrocytes: differentiation pathway and migration to wound sites.
      ,
      • Yang L.
      • Scott P.G.
      • Giuffre J.
      • et al.
      Peripheral blood fibrocytes from burn patients: identification and quantification of fibrocytes in adherent cells cultured from peripheral blood mononuclear cells.
      Studies with murine splenocytes confirmed that nonactivated CD4+ T cells enhance the appearance of collagen-producing fibrocytes in culture.
      • Niedermeier M.
      • Reich B.
      • Rodriguez Gomez M.
      • et al.
      CD4+ T cells control the differentiation of Gr1+ monocytes into fibrocytes.
      The requirement for CD4+ T cells is also evident in vivo. In the absence of CD4+ T cells, reduced numbers of fibrocytes and less fibrosis were detectable in the UUO kidneys.
      • Niedermeier M.
      • Reich B.
      • Rodriguez Gomez M.
      • et al.
      CD4+ T cells control the differentiation of Gr1+ monocytes into fibrocytes.
      ,
      • Tapmeier T.T.
      • Fearn A.
      • Brown K.
      • et al.
      Pivotal role of CD4+ T cells in renal fibrosis following ureteric obstruction.
      Studies conducted with cytokine-deficient mice have demonstrated a positive correlation between fibrosis and Th-2 responses and a negative correlation between fibrosis and Th-1 responses.
      • Cheever A.W.
      • Williams M.E.
      • Wynn T.A.
      • et al.
      Anti-IL-4 treatment of Schistosoma mansoni-infected mice inhibits development of T cells and non-B, non-T cells expressing Th2 cytokines while decreasing egg-induced hepatic fibrosis.
      • Chiaramonte M.G.
      • Donaldson D.D.
      • Cheever A.W.
      • et al.
      An IL-13 inhibitor blocks the development of hepatic fibrosis during a T-helper type 2-dominated inflammatory response.
      • Pesce J.
      • Kaviratne M.
      • Ramalingam T.R.
      • et al.
      The IL-21 receptor augments Th2 effector function and alternative macrophage activation.
      • Reiman R.M.
      • Thompson R.W.
      • Feng C.G.
      • et al.
      Interleukin-5 (IL-5) augments the progression of liver fibrosis by regulating IL-13 activity.
      • Wynn T.A.
      Fibrotic disease and the T(H)1/T(H)2 paradigm.
      IL-2, TNF-α, IFN-γ, and IL-12 were found to inhibit the development of human and/or murine fibrocytes.
      • Niedermeier M.
      • Reich B.
      • Rodriguez Gomez M.
      • et al.
      CD4+ T cells control the differentiation of Gr1+ monocytes into fibrocytes.
      ,
      • Shao D.D.
      • Suresh R.
      • Vakil V.
      • et al.
      Pivotal Advance: Th-1 cytokines inhibit, and Th-2 cytokines promote fibrocyte differentiation.
      In vivo, the combination of IL-2 and TNF-α was able to reduce the number of fibrocytes and the severity of fibrosis in UUO kidneys. In addition, serum amyloid P and cross-linked IgG delay the differentiation of human and murine fibrocytes in culture
      • Pilling D.
      • Buckley C.D.
      • Salmon M.
      • et al.
      Inhibition of fibrocyte differentiation by serum amyloid P.
      ,
      • Crawford J.R.
      • Pilling D.
      • Gomer R.H.
      Improved serum-free culture conditions for spleen-derived murine fibrocytes.
      ,
      • Pilling D.
      • Tucker N.M.
      • Gomer R.H.
      Aggregated IgG inhibits the differentiation of human fibrocytes.
      by binding to Fc-γ receptors.
      • Crawford J.R.
      • Pilling D.
      • Gomer R.H.
      FcγRI mediates serum amyloid P inhibition of fibrocyte differentiation.
      Systemic administration of human serum amyloid P reduced fibrosis in models of lung and renal fibrosis (UUO and IR injury).
      • Castano A.P.
      • Lin S.L.
      • Surowy T.
      • et al.
      Serum amyloid P inhibits fibrosis through FcγR-dependent monocyte-macrophage regulation in vivo.
      ,
      • Pilling D.
      • Roife D.
      • Wang M.
      • et al.
      Reduction of bleomycin-induced pulmonary fibrosis by serum amyloid P.
      Cytokines supporting fibrocyte development are TGF-β, endothelin-1, and IL-13.
      • Phillips R.J.
      • Burdick M.D.
      • Hong K.
      • et al.
      Circulating fibrocytes traffic to the lungs in response to CXCL12 and mediate fibrosis.
      ,
      • Abe R.
      • Donnelly S.C.
      • Peng T.
      • et al.
      Peripheral blood fibrocytes: differentiation pathway and migration to wound sites.
      ,
      • Schmidt M.
      • Sun G.
      • Stacey M.A.
      • et al.
      Identification of circulating fibrocytes as precursors of bronchial myofibroblasts in asthma.
      ,
      • Hong K.M.
      • Belperio J.A.
      • Keane M.P.
      • et al.
      Differentiation of human circulating fibrocytes as mediated by transforming growth factor-β and peroxisome proliferator-activated receptor γ.
      TGF-β and endothelin-1 enhance the release of collagen and fibronectin by human fibrocytes. IL-13, a well-known profibrotic cytokine, also increases the number of fibrocytes in combination with macrophage colony-stimulating factor and the release of collagen by fibrocytes.
      • Niedermeier M.
      • Reich B.
      • Rodriguez Gomez M.
      • et al.
      CD4+ T cells control the differentiation of Gr1+ monocytes into fibrocytes.
      ,
      • Crawford J.R.
      • Pilling D.
      • Gomer R.H.
      Improved serum-free culture conditions for spleen-derived murine fibrocytes.
      The role of other cytokines (e.g., IL-10 and IL-4) is somewhat contradictory. IL-4 seems to stimulate fibrocyte development in humans but not in mice.
      • Niedermeier M.
      • Reich B.
      • Rodriguez Gomez M.
      • et al.
      CD4+ T cells control the differentiation of Gr1+ monocytes into fibrocytes.
      ,
      • Shao D.D.
      • Suresh R.
      • Vakil V.
      • et al.
      Pivotal Advance: Th-1 cytokines inhibit, and Th-2 cytokines promote fibrocyte differentiation.
      ,
      • Crawford J.R.
      • Pilling D.
      • Gomer R.H.
      Improved serum-free culture conditions for spleen-derived murine fibrocytes.
      A strong impact on fibrocyte development in vitro and in vivo was seen with the immunosuppressive drug cyclosporine A that is known to induce renal fibrosis.
      • Liptak P.
      • Ivanyi B.
      Primer: histopathology of calcineurin-inhibitor toxicity in renal allografts.
      Cyclosporine A downregulates the expression of cytokines such as TNF-α, IFN-γ, and IL-2 but upregulates the expression of the profibrotic TGF-β,
      • Li B.
      • Sehajpal P.K.
      • Khanna A.
      • et al.
      Differential regulation of transforming growth factor β and interleukin 2 genes in human T cells: demonstration by usage of novel competitor DNA constructs in the quantitative polymerase chain reaction.
      which may partially explain the profibrotic effects of cyclosporine A. In contrast, the mammalian target of rapamycin-inhibitor did not enhance fibrocyte development.
      • Niedermeier M.
      • Reich B.
      • Rodriguez Gomez M.
      • et al.
      CD4+ T cells control the differentiation of Gr1+ monocytes into fibrocytes.
      Additional factors that support fibrocyte development are semaphorin 7A, angiotensin II type 1 receptor, and adiponectin.
      • Sakai N.
      • Wada T.
      • Matsushima K.
      • et al.
      The renin-angiotensin system contributes to renal fibrosis through regulation of fibrocytes.
      ,
      • Gan Y.
      • Reilkoff R.
      • Peng X.
      • et al.
      Role of semaphorin 7a signaling in transforming growth factor β1-induced lung fibrosis and scleroderma-related interstitial lung disease.
      ,
      • Yang J.
      • Lin S.C.
      • Chen G.
      • et al.
      Adiponectin promotes monocyte-to-fibroblast transition in renal fibrosis.
      In a chronic angiotensin II infusion model, blockade of the angiotensin II type 1 receptor reduced renal fibrosis and fibrocyte numbers in the kidney. Adiponectin enhanced the monocyte-to-fibroblast transition in vitro and the number of renal fibrocytes in UUO and IR injury models.
      • Yang J.
      • Lin S.C.
      • Chen G.
      • et al.
      Adiponectin promotes monocyte-to-fibroblast transition in renal fibrosis.
      Overall, development, migration, survival, and collagen production of fibrocytes seem to be tightly regulated. The role of these factors in vivo needs to be better defined to understand whether they directly act on fibrocytes or on other cells that influence fibrocytes.

      EMT AND ENDO-MT

      EMT is a molecular mechanism, whereby terminally differentiated epithelial cells transform into mesenchymal cells with increased migratory potential and drastic changes in their gene-expression profiles.
      • Kalluri R.
      • Weinberg R.A.
      The basics of epithelial-mesenchymal transition.
      EMT is found in the context of embryonic development
      • Greenburg G.
      • Hay E.D.
      Epithelia suspended in collagen gels can lose polarity and express characteristics of migrating mesenchymal cells.
      and in adult cancer cells as a molecular program for tumor invasion and metastasis.
      • Thiery J.P.
      Epithelial-mesenchymal transitions in tumour progression.
      ,
      • Nakaya Y.
      • Sheng G.
      EMT in developmental morphogenesis.
      In 2002, EMT was analyzed in kidney fibrosis with cell-lineage tracing and staining of fibroblast-specific protein 1 as a marker for fibroblasts.
      • Strutz F.
      • Okada H.
      • Lo C.W.
      • et al.
      Identification and characterization of a fibroblast marker: FSP1.
      ,
      • Iwano M.
      • Plieth D.
      • Danoff T.M.
      • et al.
      Evidence that fibroblasts derive from epithelium during tissue fibrosis.
      On the basis of colocalization of epithelial and mesenchymal markers, many studies have reported that EMT is a source of myofibroblasts and contributes to organ fibrosis.
      • Kalluri R.
      • Weinberg R.A.
      The basics of epithelial-mesenchymal transition.
      In vitro studies also showed that cultured epithelial cells incubated with profibrotic factors such as TGF-β acquire mesenchymal features and lose epithelial features.
      • Zeisberg M.
      • Hanai J.
      • Sugimoto H.
      • et al.
      BMP-7 counteracts TGF-β1-induced epithelial-to-mesenchymal transition and reverses chronic renal injury.
      ,
      • Ivanova L.
      • Butt M.J.
      • Matsell D.G.
      Mesenchymal transition in kidney collecting duct epithelial cells.
      However, with the advent of cell fate tracing technologies, a number of conflicting data about the ‘in vivo’ existence of EMT have been published.
      • Humphreys B.D.
      • Lin S.L.
      • Kobayashi A.
      • et al.
      Fate tracing reveals the pericyte and not epithelial origin of myofibroblasts in kidney fibrosis.
      ,
      • Koesters R.
      • Kaissling B.
      • Lehir M.
      • et al.
      Tubular overexpression of transforming growth factor-β1 induces autophagy and fibrosis but not mesenchymal transition of renal epithelial cells.
      In addition, a variety of studies by renal pathologists using more traditional techniques such as electron microscopy have also failed to observe epithelial cells traversing the tubular basement membrane.
      • Picard N.
      • Baum O.
      • Vogetseder A.
      • et al.
      Origin of renal myofibroblasts in the model of unilateral ureter obstruction in the rat.
      Recently, LeBleu et al. reported that the contribution of EMT to myofibroblasts is less than 5%,
      • LeBleu V.S.
      • Taduri G.
      • O'Connell J.
      • et al.
      Origin and function of myofibroblasts in kidney fibrosis.
      suggesting that the contribution of EMT to fibrosis is less significant than previously thought.
      It has been suggested that endothelial cells can also undergo a phenotypic transition similar to EMT, referred to as endothelial–mesenchymal transition (EndoMT). Recent studies using cell-lineage analysis have demonstrated that EndoMT may be an important mechanism in the pathogenesis of cardiac, pulmonary, and kidney fibrosis.
      • Zeisberg E.M.
      • Tarnavski O.
      • Zeisberg M.
      • et al.
      Endothelial-to-mesenchymal transition contributes to cardiac fibrosis.
      • Zeisberg E.M.
      • Potenta S.E.
      • Sugimoto H.
      • et al.
      Fibroblasts in kidney fibrosis emerge via endothelial-to-mesenchymal transition.
      • Hashimoto N.
      • Phan S.H.
      • Imaizumi K.
      • et al.
      Endothelial-mesenchymal transition in bleomycin-induced pulmonary fibrosis.
      • Li J.
      • Qu X.
      • Bertram J.F.
      Endothelial-myofibroblast transition contributes to the early development of diabetic renal interstitial fibrosis in streptozotocin-induced diabetic mice.
      However, some limitations in the detection strategies are discussed,
      • Kramann R.
      • Dirocco D.P.
      • Humphreys B.D.
      Understanding the origin, activation and regulation of matrix-producing myofibroblasts for treatment of fibrotic disease.
      and more studies are needed to clarify the contribution to tissue fibrosis.

      CONCLUSION

      Currently available data strongly suggest that collagen-producing myofibroblasts in the kidney can be derived from various cellular sources. Resident renal fibroblasts and cells of hematopoietic origin migrating into the kidney seem to be the most important ancestors of myofibroblasts. It is likely that both cell types communicate with each other and with other cell types in the kidney. These could be tubular epithelial cells, resident dendritic cells/macrophages, or leukocytes infiltrating the kidney during injury. The interaction of all of these cell types will determine how much collagen and matrix is produced, whether production exceeds degradation, and whether the timing of both processes is adequate to restore the integrity of the kidney. Currently, our research heavily depends on rodent models of renal fibrosis. UUO, which recapitulates various key features of fibrotic responses together with tubular damage, is the most intensively used model.
      • Chevalier R.L.
      • Forbes M.S.
      • Thornhill B.A.
      Ureteral obstruction as a model of renal interstitial fibrosis and obstructive nephropathy.
      ,
      • Eddy A.A.
      • Lopez-Guisa J.M.
      • Okamura D.M.
      • et al.
      Investigating mechanisms of chronic kidney disease in mouse models.
      UUO is advantageous because it causes significant fibrosis in a short period of time, it does not require administration of toxins, and it is highly reproducible. Although surgically challenging, fibrosis can also be reversed by release of the ureter clamp,
      • Cochrane A.L.
      • Kett M.M.
      • Samuel C.S.
      • et al.
      Renal structural and functional repair in a mouse model of reversal of ureteral obstruction.
      making this model even more attractive. The primary limitation of this model is the rare occurrence of ureteral obstruction as a trigger of renal fibrosis in humans. We therefore need to determine whether the sequence of events in the UUO model can be applied to other types of kidney fibrosis. In addition, a precise comparison of characteristics between animal fibrosis models and human kidney fibrosis is definitely essential.
      Once we have identified when and in which diseases the reduction of fibrosis will improve kidney regeneration and function, we can adequately design clinical trials (e.g., for chronic allograft nephrophathy or diabetic nephropathy). Promising data with pirfenidone in overt diabetic nephropathy
      • Sharma K.
      • Ix J.H.
      • Mathew A.V.
      • et al.
      Pirfenidone for diabetic nephropathy.
      suggest that antifibrotic treatment might be a reasonable approach to preserve renal function in selected kidney diseases.

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