The crucial study supporting EMT in kidney fibrosis is a cell fate tracing study published by Iwano and coworkers in 2002 (10
). The authors used the unilateral ureteral obstruction (UUO) model to induce kidney fibrosis in mice. They permanently labeled tubular cells with LacZ using the γGT promoter and showed that within a degenerating tubule, epithelial cells may acquire FSP1 positivity and that cells positive for both LacZ and FSP1 appeared to have settled in the fibrotic tissue surrounding the tubules. Moreover, colocalization of HSP47 in LacZ-positive cells located in the peritubular interstitium was taken as evidence that these cells were derived from epithelial cells and that they participated in the production of fibrous collagens.
It is our opinion that this study contains serious flaws. Later studies (33
) 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 . First, γGT is a brush border protein, expressed specifically in the proximal tubules. However, all tubules in the control (Figure A) 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+
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.
Figure 2 Reproduction of Figure 2 from the article by Iwano et al. (10).
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
) 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
). 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
); 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
). 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 ). 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.
Empty profile of a degenerating tubule.
In summary, none of these studies contain robust evidence for a contribution of EMT to kidney fibrosis. None of them met the crucial requirement of showing that in vivo, tubular cells/tubule-derived cells were capable of producing type I collagenous matrix, and none of them presented evidence for a migration of cells across the TBM. The cell culture studies did uncover a certain pattern of de-differentiation of tubular epithelial cells, including the upregulation of mesenchymal proteins that would be relevant to the EMT hypothesis. However, this pattern does not include evidence for the decisive final steps to a complete EMT, i.e., phenotype switch and ability to produce fibrous matrix; so we are left with only a “partial EMT” (3
) whose pathological relevance remains unclear.