Glomerular epithelialmesenchymal transdifferentiation in pauci-immune crescentic glomerulonephritis
Jean Bariety,
Gary S. Hill,
Chantal Mandet,
Theano Irinopoulou,
Christian Jacquot,
Alain Meyrier and
Patrick Bruneval
Université Paris VI, Faculté Broussais-Hotel Dieu, Hôpital Georges Pompidou and INSERM U430, Paris, France
Correspondence and offprint requests to: Patrick Bruneval, MD, Department of Pathology, Hôpital Georges Pompidou, 20 rue Leblanc, 75015 Paris, France. Email: patrick.bruneval{at}hop.egp.ap-hop-paris.fr
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Abstract
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Background. Among the cellular changes occurring in renal fibrosis, epithelialmesenchymal cell transdifferentiation or transition (EMT) is a phenomenon characterized in epithelial cells by loss of epithelial markers and acquisition of mesenchymal phenotype and of fibrosing properties.
Methods. To test the hypothesis that EMT is involved in human pauci-immune crescentic glomerulonephritis (PICGN), we studied 17 renal biopsies from 11 PICGN patients for: (i) proliferating cell nuclear antigen (PCNA) and cell cycle inhibitors (cyclin-dependent kinase inhibitors) p27 and p57; (ii) cell lineage phenotype markers: podocalyxin, synaptopodin and GLEPP-1 for podocytes; CD68 for macrophagic epitope; CD3 for T lymphocytes;
-smooth muscle actin (
-SMA) for myofibroblasts; vimentin for mesenchymal cells; and cytokeratins (CKs) for parietal epithelial cells (PECs); (iii) glomerular fibrosis by labelling collagens I, III and IV, and heat-shock protein 47 (HSP47), a marker of collagen-synthesizing cells; and (iv) co-localization of
-SMA, CK and HSP47 using confocal laser microscopy.
Results. The crescent cells proliferated greatly. They did not express p27 and p57. Different cell lineage markers could be identified in crescents: the major component was made of dysregulated PECs negative for CK, followed by PECs positive for CK, macrophagic cells and myofibroblasts. Furthermore, some cells co-expressed CK and
-SMA. This latter co-expression suggests a transitional phase in the dynamic phenomenon of EMT. Therefore, proliferative and dysregulated glomerular epithelial cells could be a possible cellular source of myofibroblasts via EMT. In addition, HSP47 labelled many crescent cells and frequently co-localized in CK-positive epithelial cells and in
-SMA-positive myofibroblasts, indicating that these cells were involved in glomerular accumulation of collagens.
Conclusion. EMT is a transient cellular phenomenon present in glomeruli in human PICGN contributing to the formation of myofibroblasts from epithelial cells and to glomerular fibrosis.
Keywords: cell transdifferentiation; crescentic glomerulonephritis; myofibroblasts; renal fibrosis
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Introduction
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In pauci-immune crescentic glomerulonephritis (PICGN), several cell types contribute to crescent formation and progression (from cellular to fibrous stages): epithelial cells, monocytemacrophages and myofibroblasts. Myofibroblasts are activated fibroblasts identified in humans and most animal species by the expression of
-smooth muscle actin (
-SMA) [1]. The identification of resting and activated fibroblasts by the specific marker fibroblast-specific protein 1 (FSP1) [2] is restricted to mice. Such a marker is missing in human tissues where the acquisition of mesenchymal phenotype in epithelial cells is characterized by positivity of vimentin or
-SMA [3]. Myofibroblasts have the ability to proliferate and to synthesize collagens. Myofibroblasts have been identified in experimental models [4] and human nephropathies [5,6] that evolve towards fibrosis. The myofibroblasts present in the crescents might have several origins: mesangial cells, periglomerular interstitial fibroblasts migrating into Bowmans space through a break in Bowmans capsule [7] or transdifferentiated glomerular epithelial cells. In effect, the capacity of epithelial cells to transdifferentiate into mesenchymal cells, particularly into
-SMA-positive myofibroblasts, has been documented both in vitro and in vivo in numerous organs, including the kidney [2,810]. Transdifferentiation is defined in differentiated cells by loss of the normal phenotype with acquisition of a new phenotype [8,11,12].
The purpose of this study was to test in 17 renal biopsies from 11 PICGN patients the hypothesis that epithelialmesenchymal cell transdifferentiation or transition (EMT) is involved in crescent formation and evolution.
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Materials and methods
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Patients (Table 1) and renal biopsies
The main clinical and biological data from the 11 patients are depicted in Table 1. At presentation, the 11 patients had a renal biopsy for a clinical picture of rapidly progressive glomerulonephritis. In six patients, a second biopsy was performed 412 months after the end of the induction treatment to assess an increase in serum creatinine or proteinuria in order to rule out a relapse of PICGN. Informed consent was obtained before renal biopsy.
The 17 renal biopsies were fixed in alcoholic Bouins solution and embedded in paraffin for histology and immunohistochemistry, and frozen for immunofluorescence. The number of normal or obsolescent glomeruli and of glomeruli exhibiting cellular, fibrocellular or fibrous crescents was counted among all glomeruli in all the biopsies. Cellular crescents contained three or more layers of cells without interposition of extracellular matrix identified on silver staining. Fibrocellular crescents contained a variable amount of extracellular matrix surrounding crescent cells. Fibrous crescents had a fibrous appearance with few or no admixed cells. Thus, in a given biopsy, an overlapping spectrum of crescentic lesions could be observed.
Controls
Controls consisted of normal portions of three kidneys removed for renal carcinoma in patients with no proteinuria, of renal biopsies from three nephrotic patients with minimal change disease, and from one donor kidney, minutes after unclamping the renal artery.
Immunohistochemistry
Markers.Cytokeratin (CK) polypeptides were labelled by C2562 monoclonal antibody (mAb) cocktail (Sigma Aldrich Chimie, St Quentin Fallavier, France), directed against nine CK types and used as a parietal epithelial cell (PEC) marker [13]. Normal podocytes are not labelled by this mAb.
-SMA was labelled by mAb 1A4 (Neomarkers, Union City, CA), a phenotypic marker of myofibroblasts [1,4]. Vimentin labelled by mAb V9 (Dako, Trappes, France) was used as a mesenchymal marker which is also characteristically positive on normal podocytes and not on normal PECs. Macrophage-associated epitope was detected using an anti-CD68 mAb, clone PGM1 (Dako) [14,15], and T lymphocytes by an anti-CD3 polyclonal antibody (pAb) (Dako). Podocytes were characterized using an anti-human podocalyxin mAb (MLC48A8, a gift of Pierre Ronco, MD, INSERM U 489, Hôpital Tenon, Paris, France), an anti-synaptopodin mAb, clone G1D4 (Progen and Biotechnik, Heidelberg, Germany), and an anti-glomerular epithelial protein-1 (anti-GLEPP-1) mAb (Biogenex, San Ramon, CA). Collagens were labelled by anti-collagen I and III pAb (Biogenesis, Poole, UK) for interstitial type collagens and by anti-collagen IV mAb (Biogenesis) for mesangial-basement membrane-type collagen. These antibodies were used as markers of glomerular fibrosis. Heat-shock protein 47 (HSP47), a collagen chaperone molecule, was detected by M16.10A1 mAb (Stressgen, Victoria, BC, Canada) and was used as an indicator of collagen synthesis by cells [2,3,6]. The cyclin-dependent kinase inhibitors (CKIs) p27 and p57 were detected using rabbit pAb (Santa Cruz Biotechnology, Santa Cruz, CA) [16,17]. For proliferating cell nuclear antigen (PCNA) immunohistochemistry, PC10 mAb (Dako) was used [15]. Antigen retrieval, visualization systems and negative procedures were performed as previously described [14,15].
Cell counts. For each renal biopsy, two observers assessed all the glomerular sections for CK,
-SMA, CD68, CD3 and PCNA labelling, and for polymorphonuclear cells. In the crescents, the percentage of the positive cells of each marker was counted in each glomerulus according to the type of lesion, i.e. cellular, fibrocellular or fibrous. The percentage of the unlabelled crescent cells was calculated as follows: 100% minus the percentage of the CK-, CD68-, CD3- and
-SMA-labelled cells.
Immunofluorescence for confocal laser microscopy. To assess co-localization of epitopes in the 17 renal biopsies, combined immunofluorescence for
-SMA and CK, CD68 and synaptopodin, CD68 and CK, HSP47 and CK, or HSP47 and
-SMA was performed as previously described [15]: briefly, the first antibody was revealed by using biotinylated anti-mouse IgG and streptavidincyanin-2 (Cy2; Amersham, Les Ulis, France), and the second antibody by Cy3-labelled anti-mouse IgG (Amersham). Between the two primary antibodies, after visualization of the first primary antibody, the sections were treated by heating in a microwave oven for 5 min at 750 W in citrate buffer (2 x SSC, pH 6.2) to avoid cross-reactivity with the second visualization system. For negative control procedures, the primary antibodies were replaced by normal mouse IgG (Dako) at concentrations similar to that of the primary antibodies. The sections were observed using a confocal microscope Leica TCS SP (Leica Microsystems, Heidelberg, Germany) powered with an argonkrypton laser beam, with excitation at 488 nm for Cy2 (detection: 500550 nm) and at 568 nm for Cy3 (detection: 580680 nm).
Statistics
On the one hand, the immunolabelled cell counts and on the other hand the glomerular lesion-type counts were compared between pre- and post-treatment biopsies in the six patients with sequential biopsies. The distribution of the immunolabelled cell counts was compared according to the glomerular lesion type.
2 tests and t-tests were used as appropriate, with P-values <0.05 considered significant.
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Results
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Standard pathology (Table 2)
The distribution of the glomerular lesions varied significantly over the course of successive biopsies. On pre-treatment biopsies, cellular and fibrocellular crescents predominated. On post-treatment biopsies, no relapse of PICGN was found and the cellular crescents had disappeared, whereas the fibrous crescents and the normal glomeruli had increased. The difference in distribution of lesions between the pre- and post-treatment biopsies was significant (for the group as a whole, P < 0.00001).
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Table 2. Distribution of types of glomerular lesions between biopsy 1 and biopsy 2: overall results in the six patients with sequential biopsies
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Polymorphonuclear cells were observed mainly on pre-treatment biopsies in crescents associated with necrosis. No glomerular immune deposits were observed by immunofluorescence. Large fibrin deposits in the glomerular urinary spaces and in the crescents were observed in all the crescentic glomeruli in the pre-treatment biopsies, contrasting with the absence or paucity of fibrin in post-treatment biopsies.
Immunohistochemistry characterizes crescent cell phenotypes (Table 3)
CK labelling. In cellular or fibrocellular crescents, cuboidal cells of epithelial appearance organized in a sheet-like fashion or in a pseudotubular pattern were either CK-positive or CK-negative (Figure 1AC). CK-negative, presumably epithelial cells, were the prominent crescent cell phenotype. This did not change from pre-treatment to post-treatment biopsies. CK-positive epithelial cells decreased on post-treatment biopsies and in fibrous crescents. In serial sections, these unlabelled epithelial-type cells did not appear to express any marker used except vimentin. In crescents, some spindle-shaped cells strongly expressed CK (Figure 1A). Besides the crescents, in the glomeruli, most of the PECs and some podocytes were positive. In the interstitium, isolated cells around atrophic tubules expressed CK.

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Fig. 1. (A) In a cellular crescent of a pre-treatment biopsy, C2562 cytokeratin (CK) labels layers of epithelial crescent cells some of which have a spindle shape (arrows). Magnification x150. (B) On a pre-treatment biopsy, crescent cells organize in pseudotubules (*) and are labelled with C2562 CK. Magnification x50. (C) On a pre-treatment biopsy, all the crescent cells (arrow) are CK negative, contrasting with the strongly positive tubular epithelial cells. Magnification x150. (D) On a pre-treatment biopsy, no crescent cell expresses -SMA (arrow). Note that in this cellular crescent without any -SMA expression, the crescent cells organize in a pseudotubular pattern (*). In the remaining glomerular tuft, activated mesangial cells are -SMA positive (arrowhead). Serial section with (G) and (H). Magnification x250. (E) On a pre-treatment biopsy, some spindle-shaped crescent cells express -SMA (arrow). Note that they are not in contact with Bowmans capsule and, furthermore, they are separated from it by several layers of epithelial cells. Mesangial cells (arrowhead) are positive in the remaining tuft. Magnification x200. (F) On a post-treatment biopsy, a fibrocellular crescent is organized in ectatic pseudotubules (*): -SMA-positive myofibroblasts surround the pseudotubules. Magnification x200. (G) On a pre-treatment biopsy, vimentin is strongly expressed in all the cells of this cellular type of crescent (arrow). Pseudotubule (*). In the remaining glomerular tuft, podocytes are labelled with vimentin, as well as glomerular free-floating cells (arrowhead). Serial section with (D) and (H). Magnification x250. (H) On a pre-treatment biopsy, in this cellular crescent (arrow), no cell expresses synaptopodin, a podocyte marker. Pseudotubule (*). In the remaining glomerular tuft, podocytes are labelled with synaptopodin. Serial section with (D) and (G). Magnification x250. (I) On a post-treatment biopsy, collagen I has accumulated in this fibrocellular cresent. Magnification x200. (J) On a pre-treatment biopsy, in this fibrocellular crescent, collagen IV labelling shows accumulation (arrows) around epithelial cells forming pseudotubules. The fibrous part of the crescent is only weakly labelled (*). Note the normal positivity of tubular and glomerular basement membranes. Magnification x150. (K) On a pre-treatment biopsy, in this cellular crescent, all the crescent cells are positive for HSP47 (arrow), as well as interstitial and pericapsular cells (arrowheads) and some tubular epithelial cells (*). Magnification x200. (L) On a post-treatment biopsy, HSP47 expression persists in some cells of this fibrous crescent (arrows). Magnification x250.
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-SMA labelling. In both crescentic and non-crescentic glomeruli, all the mesangial cells were strongly marked. In cellular crescents, no or only very rare cells were labelled (Figure 1D). In fibrocellular crescents, spindle cells situated between the layers of epithelial cells or around the pseudotubules were strongly labelled (Figure 1E and F). Abundant interstitial cells, often organized around the glomeruli, were positive. On serial sections, continuity of
-SMA-positive cells through breaks in Bowmans capsule from the interstitium into the crescents was rarely observed. The percentage of
-SMA-positive cells increased with evolution of crescents to fibrous-type lesions on pre-treatment biopsies and overall on post-treatment vs pre-treatment biopsies. Many interstitial cells expressed
-SMA.
Vimentin labelling. In the glomeruli, most and often all of the crescent cells were labelled (Figure 1G). Outside of the crescents, PECs along the Bowmans capsule, endocapillary cell, and podocytes that were still attached to the tuft expressed vimentin. Many interstitial cells were positive with vimentin.
Macrophagic epitope labelling. CD68-positive cells were distinctly fewer than CK-positive and CK-negative epithelial cells in cellular and fibrocellular crescents. Large round CD68-positive cells were observed in many tubular lumens and some glomerular urinary spaces. Numerous interstitial cells were labelled. The crescent type did not influence their percentage. However, on post-treatment vs pre-treatment biopsies, their number markedly diminished (P < 0.01).
Podocyte labelling. The three markers used (podocalyxin, synaptopodin and GLEPP-1) labelled no crescent cells. (Figure 1H).
CD3 labelling. Very few cells in crescents expressed CD3.
Collagen labelling. Interstitial-type collagens, prominently type I, appeared in glomeruli from the fibrocellular stage of crescents and were strongly positive in fibrous crescents and in obsolescent glomeruli (Figure 1). Collagen IV labelling was observed mainly around epithelial crescent cells and was weak in the fibrous part of crescents made of spindle-shaped cells (Figure 1J). All collagen types were detected constantly in extracellular location without any cellular labelling.
HSP47 labelling. Many crescent cells strongly expressed HSP47 in all types of crescents on pre-treatment biopsies (Figure 1K). Although HSP47 labelling decreased on post- vs pre-treatment biopsies (P < 0.01 for overall comparison), it persisted (Figure 1L). Finally, it did not disappear and was still present in obsolescent glomeruli. Some tubular epithelial cells were labelled.
CKI p27 and p57. No cells in the crescents expressed p27 or p57, although most of the nuclei of the identifiable normal podocytes were marked (Figure 2A and B).

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Fig. 2. (A) On a pre-treatment biopsy, in this cellular crescent (arrow), the nuclei of the crescent cells are negative for p57 CKI, contrasting with the positivity of podocyte nuclei in the preserved part of the glomerular tuft. Pseudotubule (*). Magnification x200. (B) On a post-treatment biopsy, in this fibrous crescent, the cobblestone cells are negative for p57 CKI (arrow). Note that in the preserved portion of the glomerular tuft, the podocytes are positive (arrowhead) as well as floating cells (double arrows). Magnification x150. (C) On a pre-treatment biopsy, PCNA labelling in a glomerulus without crescent shows that some PECs and a podocyte (arrow) are proliferating. Note that numerous tubular cells display positive nuclei. Magnification x130 (insert: x250). (D) On a pre-treatment biopsy, a high level of PCNA labelling is seen in this fibrocellular crescent (arrow) and in numerous PECs. Magnification x260.
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PCNA. In cellular and fibrocellular crescents, a very high percentage of cells showed PCNA positivity (Table 3). Outside of crescents, PCNA was expressed only in many PECs along the Bowmans capsule and in rare identifiable podocytes (Figure 2C and D). PCNA was also expressed in numerous tubular and interstitial cells. PCNA labelling was almost completely abolished on post-treatment biopsies (P < 0.00001).
Controls. The immunohistochemical markers labelled their expected cellular targets. In particular, CKI p27 and p57 labelled all the podocyte nuclei and very rarely PECs and tubular cell nuclei. PCNA was detected on some tubular epithelial cells, very rarely on PECs and not on podocytes.
Co-localization of epitopes by confocal laser microscopy
Detection of EMT. In all the pre-treatment biopsies, combined labelling with antibodies to
-SMA and CK showed a few cells expressing both myofibroblastic and epithelial epitopes (Figure 3A and B). This co-expression was found in some crescent cells in proximity to myofibroblasts expressing
-SMA and to epithelial cells expressing or not expressing CK (Figure 3C). On post-treatment biopsies, no co-expression was detected in crescents. Co-localization of
-SMA and CK was also observed in the tubular epithelium just inside the tubular basement membrane or in the peritubular interstitium (Figure 3D and E). No cell co-expressed CD68 and synaptopodin, or CD68 and CK. The controls were negative.
Detection of fibrosis. Many crescent cells co-expressed HSP47 and CK (Figure 4A and B), and HSP47 and
-SMA (Figure 4D). Many interstitial cells co-expressed HSP47 and CK (Figure 4C), and HSP47 and
-SMA (Figure 4E).
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Discussion
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Cellular and fibrocellular crescents are essentially formed of proliferating epithelial cells as previously reported [18]. These epithelial cells are derived from proliferated PECs, as suggested by: (i) strong PCNA positivity of the PECs all along the Bowmans capsule; (ii) presence of layers of epithelial cells, few in number, clearly joined to Bowmans capsule at some distance from the tuft; and (iii) labelling by C2562, a customary marker for PECs [13]. Crescents do not contain normal podocytes. However, it cannot be ruled out that modified podocytes could contribute to crescent formation. Indeed, modified human podocytes can proliferate [15] and lose p27 and p57 CKI. Conversely, normal podocytes from controls do not. Furthermore, proliferated and dysregulated human podocytes can change their phenotype. Thus, podocytes can undergo dysregulation or transdifferentiation, losing podocyte markers or expressing CK absent from normal podocytes. In some circumstances, podocytes can transdifferentiate into CD68-positive cells [14], but in our cases of PICGN no cells co-expressed synaptopodin and CD68 on confocal microscopy. Myofibroblasts are an important cell component of crescents, increasing with fibrosis development. Among crescent cells, many myofibroblasts and epithelial cells were positive for HSP47. This is in agreement with the involvement of these crescent cells in an active phenomenon of collagen synthesis [2,3,6]. Therefore, both epithelial cells and myofibroblasts contribute to collagen accumulation in PICGN. It should be noted that although the treatment induces a dramatic decrease in inflammation, necrosis and crescent cell proliferation, it does not completely stop fibrosis, which is an ongoing phenomenon in injured glomeruli.
The main issue in this study is that EMT contributes to the formation of myofibroblasts present in crescents in PICGN. The EMT phenomenon is accompanied by proliferation of epithelial cells, namely PECs, and by dysregulation of PECs as shown by acquisition of vimentin and loss of CK expression. Besides the glomerular EMT phenomenon, EMT was observed in certain tubular epithelial cells and in cells situated in the peritubular interstitium. CK and
-SMA co-expression suggests a transitional phase of the dynamic phenomenon in which transdifferentiating PECs, still expressing epithelial CK markers, acquire a myofibroblastic phenotype in the crescent. The low number of cells co-expressing both epitopes does not obviate the importance of the EMT phenomenon in generating myofibroblasts. This low number of co-expressing cells underestimates EMT, given first the time limitation of this transitional phase and, secondly, the loss of CK expression in many epithelial cells. That EMT is quite an early event in the glomerular fibrosis process is evident from the fact that it is only observed in cellular and fibrocellular proliferative crescents and only on pre-treatment biopsies.
The nature and the origin of myofibroblasts in crescents have been debated. On the one hand, they might be interstitial myofibroblasts invading the glomeruli through breaks in Bowmans capsule. Although this process has been widely recognized and documented [5,7], it seems unlikely to offer the entire explanation for the myofibroblasts found in the crescents. We found only a few crescentic glomeruli in which rupture could be recognized, but numerous glomeruli in which myofibroblasts were found in the glomerular crescents without rupture of Bowmans capsule (too numerous in fact for possible breaks in the capsule outside the plane of section to offer a tenable explanation for the absence of evident rupture). A second possibility is that some myofibroblasts of the crescents might be derived from activated mesangial cells which overexpress
-SMA, but migration of mesangial cells into the crescents has not been demonstrated, either in this study or, to the best of our knowledge, by others. Thus, in the rat, the mAb OX-7, a marker for mesangial cells, does not mark any cells in the crescents [4]. Finally, some myofibroblasts co-expressed
-SMA and CK, suggesting transdifferentiation from epithelial cells.
Thus, another cellular source for myofibroblasts in crescents could be the transdifferentiation of epithelial cells into myofibroblasts. This latter phenomenon is supported by our study and, furthermore, correlates with experimental data. Thus, in two types of crescentic glomerulonephritis, nephron reduction and anti-glomerular basement membrane antibody models [4], immunohistochemistry and in situ hybridization labellings demonstrated de novo expression of
-SMA by PECs. Some PECs no longer expressed E-cadherin, an epithelial cell marker, and others co-expressed both E-cadherin and
-SMA. Cellular crescents showed either no disruption or only local areas of disruption in the basal lamina of Bowmans capsule, suggesting that the myofibroblasts in the crescents were derived from transdifferentiation of proliferating glomerular epithelial cells. Moreover, the capacity of tubular epithelial cells to transdifferentiate into myofibroblasts is abundantly documented both in vitro and in vivo [710].
The mechanisms which lead to EMT in the kidney have been studied experimentally mainly on tubular epithelial cells. EMT can be achieved by changes in the extracellular matrix composition [8,9,19]. Cytokines and growth factors are also involved in EMT. In tubular epithelial cells, fibroblast growth factor (FGF) [20], transforming growth factor-ß (TGF-ß) [10] and FGF associated with TGF-ß [20] induced EMT. Similar results were obtained using interleukin-1 (IL-1) in a TGF-ß-dependent manner [21]. Hepatocyte growth factor (HGF) abrogated EMT-induced TGF-ß1 expression in tubular epithelial cells [22]. Loss of epithelial cell adhesion may also promote the EMT process. TGF-ß1 rapidly suppressed E-cadherin expression in cultured tubular epithelial cells before all the major events that characterize EMT [10]. It seems likely that similar mechanisms will be found to play a role in glomerular EMT.
In conclusion, this study provides, for the first time in man, evidence that EMT participates in the development and progression of glomerular and tubulointerstitial lesions in human PICGN.
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Acknowledgments
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This work was supported by grant CRC 97050 (Délégation de la Recherche Clinique, Assistance Publique, Paris, France), by the Association Claude Bernard and with the participation of the Association de Recherches Néphrologiques.
Conflict of interest statement. None declared.
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Received for publication: 18. 8.02
Accepted in revised form: 12. 3.03