EMT is a process well known in ontogeny. In an early embryo, the epithelial layer (epiblast) gives rise to most, if not all, mesodermal structures of the body. Also, later in ontogeny, epithelial cells (e.g., cells of the somites) transform into mesenchymal cells. Thus, in ontogeny, mesenchymal cells emerge from epithelial cells. In recent reviews, this type of EMT has been classified as type 1 (3).

In pathological processes, EMT has been described to occur in carcinogenesis. In that realm, the dedifferentiation of cells with loss of epithelial and acquisition of mesenchymal features is also termed EMT (4) and has been recently classified as type 3 EMT (3).

Another type of pathological EMT (classified as type 2) has been proposed to occur in a parenchymal organ (liver, lung, or kidney) when specific cells are lost in the course of a disease and are replaced by fibrotic tissue. This hypothesis is highly controversial. It postulates that parenchymal cells (hepatocytes, alveolar cells, or renal tubular cells undergo transition into myofibroblasts, which then produce collagenous matrix contributing to organ fibrosis (3, 5–7).

We will discuss only type 2 EMT in the context of kidney fibrosis here, listing the pros and cons of the hypothesis that renal tubular cells, in the setting of parenchymal injury, undergo transition into mesenchymal cells, which participate in the development of fibrosis.

The transition of a tubular epithelial cell into a myofibroblast is thought to be initiated by cell injury. At an early stage of tubular degeneration, some cells will respond to stimuli of autocrine and/or paracrine origin by downregulating their epithelial and upregulating a mesenchymal genetic program. The new cells are thought to produce proteases that dissolve the TBM locally, allowing cells to move through the TBM and to settle in the peritubular interstitium. Here they promote fibrosis by producing type I collagenous matrix (3, 8).

In light of our current knowledge, the use of FSP1/S100A4 antibodies as a specific marker for fibroblasts is not tenable. The interstitium of the healthy kidney contains an extensive network of fibroblasts (Figure ​(Figure1),1), which do not express FSP1/S100A4 (15, 16); also, myofibroblasts do not express FSP1/S100A4 (17). The FSP1/S100A4-positive cells in the renal interstitium belong rather to the immune system (14, 15, 18–20). In addition, the endothelial cells of some renal microvessels express FSP1/S100A4 (15, 19). These findings are in agreement with the detection of S100A4 in inflammatory cells and in some endothelia in humans (21).

Similarly, another marker used to indicate EMT, vimentin, cannot be taken as an unequivocal marker for mesenchymal cells. In rat and human material, it was shown (22) that vimentin is very commonly expressed in injured tubular cells not only during tubular degeneration in chronic diseases, but also during regeneration in acute tubular injury. Later studies (15, 23, 24) showed that the expression of vimentin in injured tubular cells displayed a clear basolateral, thus an epithelial, pattern. In a model of transiently injured tubular cells (23), epithelial repair took place by local proliferation of surviving cells. Vimentin expression was maintained at a high level until day 5 after ischemia; thereafter, it decreased concurrently with recovery. This observation supports the conclusion by Gröne and colleagues (22) that vimentin may be regarded as an indicator of tubular regenerating activity.

α-SMA is the characteristic actin isoform found in vascular smooth muscle cells, pericytes, and myofibroblasts (25, 26). Proximal tubular cells contain a cytoskeleton with prominent F-actin microfilaments that do not stain with antibodies against α-SMA. In some studies of renal disease models with fibrosis expression, α-SMA has been found in tubular cells, and that finding was considered as evidence for EMT (27–29). However, in most other studies α-SMA was exclusively found in interstitial cells around injured tubules, whereas the tubules themselves remained α-SMA–negative (15, 17, 19, 30–33). The same observation was made in biopsies of patients with nephritic syndrome (34).

Since direct detection of collagen synthesis by immunolabeling has not yet been achieved, proteins that participate in its synthesis, HSP 47 (a collagen-specific molecular chaperone) and prolyl-4-hydroxylase, have been used as surrogate markers. Expression of HSP47 in injured tubules has been taken as evidence for synthesis of collagen by tubular cells and thus for EMT (10, 11). However, HSP47 is not specific for collagen type I (35) but is equally involved in collagen type IV synthesis, and thus in the synthesis of TBM collagen.

The loss of proteins that are typically found only in epithelia has also been used to indicate EMT. Most frequently, the loss of keratin, β-catenin, E-cadherin, and the kidney-specific cadherin 16 (36) has been invoked in this respect (37). When tubules undergo decomposition, the cells progressively lose their intercellular junctional contacts, and also their contacts with the basement membrane. Thus, it is not surprising that they lose some of the junctional proteins. This, however, by no means constitutes evidence of a mesenchymal phenotype.

It is our opinion that this study contains serious flaws. Later studies (33, 38) showed that the immunofluorescent labeling of LacZ is prone to artefacts, which may account for some of the following inconsistencies in the crucial Figure 2 of the study by Iwano and coworkers (10), reproduced here as Figure ​Figure2.2. First, γGT is a brush border protein, expressed specifically in the proximal tubules. However, all tubules in the control (Figure ​(Figure2A)2A) appear equally labeled, although two profiles (both at 7:00 from the glomerulus, one of them partially covered by “fibroblasts”) are definitely not proximal. Second, the LacZ labeling is homogeneous in all tubular cells. However, insertion of a transgene using Cre recombinase usually yields variable levels of expression. Third, the merged pictures showing the colocalization of LacZ and FSP1 or HSP47 after UUO exhibit a complete overlap in location and strength, quite obvious in panels E, F, and G, but also in panels B and D. This is possible only under the very unlikely condition that the levels of expression of the two proteins are in a constant ratio at all sites where coexpression takes place. Fourth, the FSP1 antibody is not suitable to identify fibroblasts, as is illustrated in panel A, where there are three green-labeled (FSP1⁺) structures, two of them designated as fibroblasts. However, on the basis of structural studies (39), the area shown in panel A contains more fibroblasts. In addition, the labeled cell at 8:00 from the glomerulus is a round cell hardly compatible with a fibroblast. Last, in panel C, seven arrows point to LacZ⁺FSP1⁺ structures, i.e., cells that have undergone EMT, suggesting a high frequency of such an event. This is hardly compatible with the fact that cells in the process of EMT have so far not been found in structural studies.

The study by Iwano and coworkers (10) stimulated widespread activity in the field. Numerous studies were undertaken aiming at elucidating details of this new mechanism. Most of them were cell culture studies (12, 40–45) that clearly showed that renal tubular epithelial cells when exposed to profibrotic cytokines (especially TGF-β1) lose cell polarity, downregulate epithelial markers (E-cadherin, β-catenin, zonula occludens protein 1), acquire mesenchymal features (become spindle-shaped and mobile, produce MMP-2), and express mesenchymal markers (vimentin, α-SMA). This pattern of changes was taken to be a common signature for EMT and was proposed to be valid as indirect evidence of EMT in vivo (5, 46). In a recent review supporting the EMT hypothesis (3), it was conceded that the phenotypic alterations in vitro characterize a “partial EMT,” lacking the final transition to a “fully fibroblastic phenotype.” These studies do not contribute any relevant evidence for a role of EMT in renal fibrosis, which clearly requires a “fully fibroblastic phenotype.”

Among the experimental in vivo studies supporting EMT, there were a few that used routine techniques of light and electron microscopy and claimed to show morphological alterations in proof of EMT (11, 36, 40); however, the quality of the morphological data was not sufficient for that purpose. Neither a cell with characteristics of a fibroblast within the TBM, nor a cell crossing the TBM, nor gaps in the TBM were properly documented.

In a second category of pro-EMT articles, EMT was assumed as an established fact, and the study was designed to uncover details of this mechanism in the context of renal fibrosis. Data that were compatible with EMT were considered as evidence for EMT. This type of circular reasoning has been applied in several studies manipulating the activity of TGF-β in models of renal fibrosis (41, 47, 48). According to these studies, the reduction of fibrosis in response to suppression of the TGF-β pathway reflected a blocking effect on EMT. Other possible effects were not considered.

Similar reasoning was found in the study that used a tamoxifen-inducible Snail1 transgenic mouse model (36) that expressed significant levels of the protein in the kidney. The Snail family of transcription factors induces EMT in vitro, shown in a mouse mammary gland–derived epithelial cell line (NMuMG) (36). Induction of Snail1 in the above in vivo model resulted in an increase in interstitial fibrous collagen. This was interpreted only in the context of EMT, although the authors did not provide any specific evidence of EMT. In a recent study (49), activation of the Notch pathway (involving the Snail transcription factors) was shown to induce upregulation of mesenchymal markers in vitro but not in vivo.

Venkov and coworkers (12) showed in a proximal tubule epithelial cell line that the transcription factor CBF-A led to downregulation of epithelial-specific proteins and upregulation of mesenchymal-specific proteins including FSP1. In an attempt to investigate a possible role of CFB-A in vivo, the authors examined the localization of CFB-A and the expression of FSP1 in the UUO model in FSP1/GFP mice. Their data are confusing; we cannot retrace how the authors came to the conclusion that these pictures show FSP1-positive cells that were derived from CBF-A–stimulated tubular cells and settled in the interstitium (Figure ​(Figure3).3). In the text the authors simply state, “This series of images suggests that the nuclei of many tubular cells in the UUO kidney are enriched for CBF-A and potentially positioned for an EMT event.” Thus, the authors do not really claim to show EMT in the UUO model.

As has been shown in previous studies (1, 53–55), the process of tubular degeneration occurs in a certain sequence. First, cells simplify their structural elaborations, losing their surface differentiation, but maintaining cell polarity even after collapse of the lumen. The process of decomposition generally starts with the accumulation of lysosomal elements indicating autophagy (19, 50). In this stage, the degenerating cells generally lose their fixation to the TBM, frequently forming strands of dying cells. There is no noticeable invasion of the tubule by inflammatory cells; thus, autophagy and fratricide are the most likely mechanisms accounting for the disappearance of the tubular cells.

It is important to remember that during all these stages of degeneration, the integrity of the TBM — even if it becomes collapsed and highly folded — is preserved (Figure ​(Figure3).3). This survival of the TBM represents the crucial prerequisite for the high regeneration capacity of the tubular epithelium in acute renal failure.

Over the years, we have studied tubular degeneration associated with fibrosis in various rat and mouse models. Since at the time of most of these studies EMT was not yet in discussion, we did not look for it. However, we described the specific pattern of tubular degeneration eventually leading to almost cell-free remnants of intact, frequently thickened TBM (54, 56, 57).

In two recent studies (50, 58), we deliberately searched in a large number of electron micrographs for cells crossing the TBM from inside the tubular compartment to outside. We did not find such cells. Similar efforts were undertaken in an model of acute renal failure (59); also, in this study cells crossing the TBM were not encountered.

These data only show that any cells in transit were not found; they do not prove that such cells do not exist. Yet after 15 years of EMT research, cells in transit from inside an injured tubule through the TBM into the peritubular space have only been shown in schematics. Not a single picture of a cell in transit has ever been published, much less any regular structural evidence that might give significance to this mechanism.

In the model of TGF-β–induced renal fibrosis (50), we also addressed the question of whether tubular cells underwent EMT and produced collagen still within the tubular compartment. From this study, Figure ​Figure33 shows almost cell-free tubular remnants with massive deposition of fibrous collagen in their surroundings, but without a single fiber inside. Cells with a mesenchymal morphology were never encountered inside the TBM.

Nevertheless, there is a widespread resistance to the idea that local resident interstitial cells are the major, if not the only, source for the fibrosis-producing myofibroblasts. These interstitial cells are traditionally termed fibroblasts. They are the ubiquitous cells that perform a scaffolding function interconnecting the various organ-specific substructures — in the kidney, these are the nephrons and the supplying capillaries. They perform this function by focal contacts of their cytoplasmic processes with neighboring fibroblasts as well as with capillaries and tubules (refs. 39, 68, and Figure ​Figure4).4). In addition, they produce the fibrous matrix, which serves together with these cells as a common scaffold throughout the renal parenchyma.

Interstitial fibroblasts in the kidney cortex can be unequivocally detected by electron microscopy and fluorescence microscopy by their strong expression of ecto-5′-nucleotidase (CD73) (16, 33, 39, 68, 69). Some unique ultrastructural features, such as the abundance of rough ER (RER) and F-actin filaments and an association with collagen fibrils, make identification in the electron microscope easy.

In the UUO model, Picard and coworkers (17) investigated the alterations of fibroblasts upon conversion into myofibroblasts. Relevant changes were seen already on the first day following ligature consisting of the appearance of bundles of actin microfilaments and of abundant cisterns of RER. Double immunofluorescence revealed the gradual expression of α-SMA exclusively in ecto-5′-nucleotidase–positive cells. Thus, the conversion of a fibroblast into a myofibroblast was documented in a continuous sequence of steps (Figure ​(Figure5).5). A corresponding continuous sequence was also documented after uranyl acetate–induced acute renal failure by transmission and scanning electron microscopy (59).

There are several other recent in vivo studies showing that the myofibroblasts emerge from proliferation of local interstitial cells. Lin and coworkers (65) used a coll1a1-GFP transgenic mouse line to identify pericytes/fibroblasts as the major contributors to the myofibroblast population in UUO. In a cell fate study, using transgenic mice in which LacZ was expressed specifically in pericytes/fibroblasts, Humphreys and coworkers (33) found that the totality of α-SMA–expressing cells in the interstitium originated from pericytes/fibroblasts both in UUO and after ischemia-reperfusion injury. A further study (32) showed similar results in an accelerated model of angiotensin II–induced renal fibrosis.

These studies certainly do not rule out a source of myofibroblasts from other cell populations, but they show that the transition of fibroblasts into myofibroblasts occurs on a large scale and may occur very early after injury, long before tubular degeneration.

A final point remains to be discussed: the controversy over whether these resident interstitial cells should be termed fibroblasts or pericytes. The fact that renal interstitial fibroblasts have frequent contacts with capillaries (Figure ​(Figure4)4) led some authors to identify these cells as pericytes (33, 65). However, the same cells have frequent contacts with tubules and other interstitial cells. In total, they are identified in the renal cortex by the expression of ecto-5′-nucleotidase (CD73) (16, 33) as well as of PDGFRβ (65). If we term these interstitial cells pericytes, the peritubular interstitium of the kidney would be devoid of any fibroblasts.