Fate Tracing Reveals the Pericyte and Not Epithelial Origin of Myofibroblasts in Kidney Fibrosis

Understanding the origin of myofibroblasts in kidney is of great interest because these cells are responsible for scar formation in fibrotic kidney disease. Recent studies suggest epithelial cells are an important source of myofibroblasts through a process described as the epithelial-to-mesenchymal transition; however, confirmatory studies in vivo are lacking. To quantitatively assess the contribution of renal epithelial cells to myofibroblasts, we used Cre/Lox techniques to genetically label and fate map renal epithelia in models of kidney fibrosis. Genetically labeled primary proximal epithelial cells cultured in vitro from these mice readily induce markers of myofibroblasts after transforming growth factor β₁ treatment. However, using either red fluorescent protein or β-galactosidase as fate markers, we found no evidence that epithelial cells migrate outside of the tubular basement membrane and differentiate into interstitial myofibroblasts in vivo. Thus, although renal epithelial cells can acquire mesenchymal markers in vitro, they do not directly contribute to interstitial myofibroblast cells in vivo. Lineage analysis shows that during nephrogenesis, FoxD1-positive(⁺) mesenchymal cells give rise to adult CD73⁺, platelet derived growth factor receptor β⁺, smooth muscle actin-negative interstitial pericytes, and these FoxD1-derivative interstitial cells expand and differentiate into smooth muscle actin⁺ myofibroblasts during fibrosis, accounting for a large majority of myofibroblasts. These data indicate that therapeutic strategies directly targeting pericyte differentiation in vivo may productively impact fibrotic kidney disease.Understanding the origin and differentiation pathways of myofibroblasts in vivo is critical for identifying new therapeutic strategies for fibrosing disease. Myofibroblasts, contractile cells that deposit pathological extracellular matrix, were first believed to derive from a specialized perivascular cell known as the hepatic stellate cell when studied in the liver. In health these cells store retinoic acid in intracellular vesicles and cultured stellate cells possess all of the hallmarks of myofibroblasts in vitro.¹ In other organ systems, similar perivascular cells have been postulated to be the source of myofibroblasts, but have been hard to define.^2,3 Mesoderm-derived cells, when cultured in vitro, differentiate into cells with hallmarks of myofibroblasts, including most notably mesenchymal stem cells from bone marrow, as well as mesangial cells of the kidney, and cultured monocyte-derived macrophages.^4–6 Whether mesoderm-derived pericytes (also called perivascular fibroblasts) give rise to kidney myofibroblasts remains controversial, partly because primary epithelial cells when cultured in vitro can be induced to express some genes that are also expressed in myofibroblasts.^7–9 During carcinogenesis phenotypic alterations termed epithelial-to-mesenchymal transition (EMT) have been well characterized and promote cell migration, invasion, and metastasis.¹⁰ Further, a recent report suggests that other terminally differentiated cells such as endothelial cells can develop a myofibroblast phenotype in vitro and in vivo.^11,12It has been postulated that during kidney injury in vivo, epithelial cells undergo a phenotypic transition or can transdifferentiate into interstitial myofibroblasts by this same process of EMT.^13,14 Subsequent studies both in vivo and in vitro support this hypothesis.^15,16 The implication from these observations is that if the molecular mechanisms by which epithelial cells traverse the basement membrane and differentiate into myofibroblasts can be understood, novel antifibrotic strategies will be identified.Epithelial cells are known to respond to injury in several ways. They undergo morphological changes, lose polarity, acquire stress fibers, and migrate along the basement membrane.¹⁷ They up-regulate inflammatory genes and genes that enhance their ability to survive in a hostile environment.^18,19 In addition, they express some genes shared by embryonic mesenchymal cells transitioning to epithelium during nephrogenesis.^20–22 Thus it has been suggested that in response to injury epithelial cells undergo EMT, recapitulating primitive mesenchymal cells of the intermediate mesoderm.⁹ This, however, is misleading since intermediate mesoderm cells do not express inflammatory and cell-survival genes that injured adult epithelial cells up-regulate, and expression of a limited number of genes shared by embryonic mesenchyme such as α smooth muscle actin (SMA), by itself, does not define injured epithelial cells as mesenchymal.^23–25 Neoplastic epithelial cells have the capacity to metastasize, share some characteristics with myofibroblasts, and express or down-regulate key regulators of metastasis such as mts1 (S100A4 or FSP-1), Twist, Snail, and β-catenin, genes whose expression can also be activated in cultured epithelial cells.^26–28 Proponents of the hypothesis that myofibroblasts in inflammation and scarring derive from epithelial cells have drawn on these observations to extend the term EMT to mean epithelial-to-myofibroblast transition.Interstitial myofibroblasts are the principle source of interstitial collagens, including fibrillar collagens I and III. They are widely held to be the primary cell in the injured kidney that lays down the interstitial matrix that becomes fibrotic (For review see²⁹). Many myofibroblasts express the actin fiber, αSMA that correlates with contractile and activated morphology, and recent studies confirmed that in the fibrotic kidney more than 80% of these produce fibrillary collagen.³⁰ Although this is not specific to interstitial myofibroblasts (αSMA is also expressed by vascular smooth muscle cells), αSMA has long been used as a marker of myofibroblasts.Although it is widely accepted that primary epithelial cells cultured in vitro up-regulate genes that result in a myofibroblast phenotype,^9,25 and generate fibrillar collagens, the evidence that this occurs in vivo is less well-established. There are some published examples of epithelial cells transgressing intact or disrupted basement membrane or cells co-expressing established epithelial and fibroblast markers in vivo,^31–33 but histological snapshots do not prove a lineage relationship, and cells may express a variety of antigens during injury. In our own extensive studies of injured epithelial cells in kidney repair, we concluded that non-epithelial cells do not migrate from interstitium into the tubule.³⁴ Similarly, we have never observed a cell outside of the confines of the epithelial basement membrane that was positive for markers of epithelial injury. Explanations for a failure to make these observations in fixed tissues include the hypothesis that a cell exiting the confines of the basement membrane rapidly loses epithelial markers and only subsequently gains myofibroblast markers.³⁵ However, in vitro, epithelial cells can express both fibroblast markers and epithelial markers simultaneously.³⁶Because efforts to design new antifibrotic therapies require a rigorous understanding of the cellular origin of myofibroblasts in vivo, we have performed lineage analysis of both renal epithelial cells and interstitial stromal cells during fibrosis in vivo. Transgenic or knock-in mice with lineage-restricted expression of bacterial Cre recombinase were used for genetic tracking of three cell populations. The HoxB7-Cre driver is expressed exclusively in the mesonephric duct and its derivatives, resulting in labeling of collecting duct epithelium and ureteral epithelium of adult kidney.³⁷ In the Six2-Cre transgenic mouse, expression of Cre occurs in cap-mesenchyme and labels all non-ureteric, bud-derived, nephron epithelia, including podocytes, proximal tubule, loop of Henle, and connecting segment, but it does not label any interstitial cell population.^34,38 FoxD1 is a well characterized marker of renal stromal cells, but not epithelia, during development, and we used FoxD1-Cre knock-in mice to genetically label renal stroma.³⁹ We crossed these three Cre drivers against two different reporter lines to permanently and heritably label all epithelial cells of the entire nephron in adult mouse kidney or all stromal cells.^34,38 We demonstrate that, contrary to the prevailing model, kidney epithelial cells do not become myofibroblasts in vivo during fibrotic disease. Rather, we show by genetic tracing that myofibroblasts derive from interstitial pericytes/perivascular fibroblasts.