Transdifferentiation of Epithelial Glomerular Cells
Authors: Jean Barie[Combining Acute Accent]ty, Patrick Bruneval, Gary S. Hill, Chantal Mandet, Christian Jacquot, Alain Meyrier
Journal: Journal of the American Society of Nephrology (2003)

Cell transdifferentiation is characterized by loss of some phenotypes along with acquisition of new phenotypes in differentiated cells. Differentiated cells are endowed with the capacity of transforming into cells of a different type having other functions (1,2). Gene expression in differentiated cells has long been considered an irreversible phenomenon that is established at the time of replication. Given that, although repressed, the same genetic framework is present in all cell types, a change in gene expression among differentiated cells was predictable in particular conditions. In fact, the differentiated state of a given cell is not irreversible. It depends on the up- and downregulation exerted by specific molecules (3,4). Apart from organogenesis, malignant cell development, and tumor progression, the best documented examples of cell transdifferentiation concern transdifferentiation of epithelial cells into mesenchymatous cells (EMT). Such a transdifferentiation of epithelial cells into myofibroblasts has been identified as a factor fostering fibrosis in various organs such as the liver (5,6), the lung (7), and the kidney (8,9). Myofibroblasts exhibit features common to both fibroblasts and myocytes and may be considered as activated fibroblasts that express α-smooth muscle actin (α-SMA) (10). Their ability to proliferate and to synthesize extracellular matrix (interstitial type collagens) is remarkable (7,11,12). In the human kidney, we have been the first to describe transdifferentiation of glomerular epithelial cells into myofibroblastic or into macrophagic cells (13–15). EMT-Like Changes of Podocytes during Nephrogenesis Apart from the collecting duct cells, all renal cells are derived from the metanephrogenic mesenchyme, a stem cell population that has the capacity to differentiate in either epithelial or interstitial cells (2). Immature glomerular epithelial cells originate from the metanephric mesenchyme after induction by the ureteric bud. Glomerular development is divided into four stages: renal vesicle, S-shaped stage, capillary loop stage, and maturing glomeruli. Glomerular parietal and visceral epithelium (podocytes) can be distinguished after the S-shaped stage. During nephrogenesis and in mature glomeruli, parietal epithelial cells (PEC) express desmosomal proteins and cytokeratin (CK), which are markers of an epithelial phenotype. Conversely, the podocyte differentiates from an epithelial to a mesenchymal phenotype (16,17). Immature podocyte precursor cells at the S-shaped body stage are simple cuboidal cells expressing epithelial markers (desmosomal proteins and CK) with apical tight junctions. These precursors cells, actively dividing, express Ki67 and proliferating cell nuclear antigen (PCNA) proliferation markers. As the developing glomerulus evolves from the S-shaped body stage to the capillary loop stage and finally to the mature glomerulus, podocytes acquire their characteristic architecture. Their features include foot process formation and replacement of apical tight junctions by basal slit diaphragms. Podocytes reorganize their actin cytoskeleton and extend the ordered array of actin-based foot processes. They lose their mitotic activity and no longer express proliferation markers. De novo expression of cyclin-dependent kinase inhibitors (CDKI) p21, p27, and p57, which prevent cell proliferation by inhibiting the activation of CDK, coincides with the cessation of podocyte proliferation (18,19). Transition from the S-shaped body stage to the capillary loop stage represents the transdifferentiation of an epithelial to a mesenchymal phenotype that is characterized by the disappearance of epithelial markers (desmosomal proteins, CK) and the reappearance of vimentin, a characteristic intermediate filament protein of mesenchymal cells. This maturation is associated with expression of mature podocyte markers (14,16,20) including the Wilms tumor protein (WT-1), common acute lymphoblastic leukemia antigen, C3b receptor (CR1), glomerular epithelial protein-1 (GLEPP-1), podocalyxin, and synaptopodin. WT-1, which was weakly expressed in all cells at early stages, becomes restricted to podocyte nuclei. Synaptopodin associated with actin filaments, linked to the formation of foot processes, is restricted to the sole of the podocytes. GLEPP-1 and podocalyxin, the latter being the major sialomucin of the glycocalyx, are located at the apical part of the podocytes and of the foot processes. Podocalyxin, complexed with ezrin, which mediates its link to the actin cytoskeleton, plays an important role in maintaining the foot process architecture by virtue of its highly negatively charged ectodomain (21). From the capillary loop stage, podocyte proteins are detected at the slit diaphragm level or in foot processes close to the slit diaphragm (22). Podocyte proteins consist of nephrin; podocin; CD2AP; P cadherin; Z0-1; α-, β-, and γ-catenin; and α-actinin-4. They play a crucial role in the functions of the glomerular filtration barrier. The fibroblast growth factor (FGF) (16,17) and Pod-1 (23) signalings seem to control EMT-like changes of podocytes during nephrogenesis. Glomerular EMT in Crescentic Glomerulonephritis Several cell types, including monocyte-macrophages, epithelial cells, and myofibroblasts, have been implicated in crescent formation and progression toward fibrosis. Crescents seem to evolve through three stages—cellular, fibrocellular, and fibrous—with much heterogeneity even in the same patient (24). The nature and origin of myofibroblastic cells responsible for eliciting crescents have been debated. They might originate from mesangial cells (25), from periglomerular fibroblasts migrating into Bowman's space through a gap in Bowman's capsule (26,27), or from transdifferentiated epithelial cells. Glomerular EMT in Human Pauci-Immune Crescentic Glomerulonephritis We tested the hypothesis that EMT is involved in human pauci-immune crescentic glomerulonephritis (CGN) (15). We studied 18 pretreatment and posttreatment renal biopsies from 11 patients with pauci-immune CGN. In seven of them, a renal biopsy was carried out before and after treatment. All of these biopsies were studied for (1) the proliferation marker PCNA and CDKI p27 and p57 and (2) the cellular markers podocalyxin, synaptopodin, and GLEPP-1 for podocytes; α-SMA for myofibroblasts; and CK (C2562) for PEC (28). Vimentin, a mesenchymal marker, was considered as an additional marker for podocytes because vimentin is normally expressed on podocytes but not on PEC. Confocal laser microscopy was used to assess colocalization of α-SMA and CK. On pretreatment biopsies, crescent cells expressed the proliferation marker PCNA in 33 ± 10% of the cells, along with lack of CDKI expression in crescents, whereas p27 and p57 CDKI expression persisted in the sound areas of the glomerular tuft. Most—sometimes all—of the crescent cells were labeled with vimentin, as were endocapillary cells. Different cell phenotypes could be identified in the crescents (Figure 1): PEC positive for C2562 CK, their customer marker (26 ± 17%), dedifferentiated epithelial cells that were not labeled by C2562 CK antibodies or by any of the markers used except vimentin (an acquired phenotype usually not observed in normal PEC), macrophagic cells (16 ± 6%), myofibroblasts (9 ± 4%,) and fewer than 1% of cells coexpressing CK and α-SMA (Figure 2). That last coexpression suggests a transitional phase of the dynamic phenomenon in which transdifferentiating PEC still expressing epithelial CK markers acquire a myofibroblastic epitope in the crescent. Figure 1. : Immunohistochemistry can identify different phenotypes among crescent cells, although they exhibit the same pattern of spindle cells. These spindle-shaped cells are either cytokeratin (CK)-positive (a; arrows) or α-smooth muscle actin (α-SMA)-positive (b; arrows). Human pauci-immune crescentic glomerulonephritis in a Wegener patient. Initial pretreatment biopsy labeled by C2562 anti-CK antibody (a) or 1A4 anti–α-SMA antibody. Magnification, ×300.Figure 2. Confocal laser microscopy analysis of immunofluorescent labeling on crescent cells. Rare crescent cells (arrow) show coexpression of α-SMA and CK, suggesting a transitional phase of the dynamic phenomenon in which transdifferentiating parietal epithelial cells (PEC) still expressing epithelial CK markers acquire a myofibroblastic epitope (α-SMA) in the crescent. Human pauci-immune crescentic glomerulonephritis in a Wegener patient. Initial pretreatment biopsy showing pseudotubule organization (*) in a fibrocellular crescent. Labeling by 1A4 anti–α-SMA antibody (a; green cyanin-2 fluorescence) and C2562 anti-CK antibody (b; red cyanin-3 fluorescence). (c) Merged images. Magnification, ×800.Figure 3. Confocal laser microscopy analysis of immunofluorescent labeling on glomerular epithelial cells. Coexpression of the macrophagic epitope CD68 and of the epithelial maker AE1/AE3 CK on still-attached podocytes (arrows) and PEC (arrowhead) suggesting a transdifferentiation process from epithelial type into macrophagic type. Posttransplantation relapse of FSGS. Labeling by anti-CD68 antibody (a; green cyanin-2 fluorescence) and AE1/AE3 anti-CK antibody (b; red cyanin-3 fluorescence). (c) Merged images. Magnification, ×250. (From Bariety et al. J Am Soc Nephrol 12: 261–274, 2001).Figure 4. Confocal laser microscopy analysis of immunofluorescent labeling on glomerular epithelial cells. Coexpression of the macrophagic epitope CD68 and of the podocyte maker podocalyxin on glomerular cells (arrows) suggesting transdifferentiation of podocytes into macrophagic cells. Posttransplantation relapse of FSGS. Merged image of the labeling by anti-CD68 antibody (green cyanin-2 fluorescence) and by anti-podocalyxin antibody (red cyanin-3 fluorescence). Magnification, ×800. (From Bariety et al. J Am Soc Nephrol 12: 261–274, 2001).Figure 5. Immunohistochemical labeling of dysregulated podocytes expressing CD68 macrophagic epitope (arrow). Note that a few parietal epithelial cells express this marker, indicating that they are also involved in a process of epithelial to macrophagic transdifferentiation. The renal biopsy with collapsing glomerulopathy in an HIV-negative patient is labeled by the PGM1 anti-CD68 antibody. Magnification, ×500Crescents do not contain normal podocytes. However, the possibility that modified and/or hyperplastic podocytes might participate in crescent development cannot be ruled out. Thus, the podocytes that proliferate are dysregulated (14,20,29) and no longer express their normal phenotypes. Furthermore, they acquire new phenotypes (14), particularly CK expression. The possibility remains that some CD68-positive cells might be transdifferentiated podocytes, as has recently been described (14). However, in our cases of pauci-immune CGN, no cells coexpressed synaptopodin and C68 on confocal microscopy. In crescents, PEC were intermingled with a major component of epithelial cells that had lost their CK customary epitopes, with cells coexpressing CK and α-SMA and with mature myofibroblasts. Therefore, PEC could be a possible cellular source for myofibroblasts originating from EMT. In addition, EMT was observed in rare tubular epithelial cells that coexpressed C2562 CK and α-SMA. Posttreatment lesions were qualitatively similar to the pretreatment lesions, but there was a marked shift toward fibrotic lesions with increased α-SMA and decreased CK labeled cells. No cell coexpressed CK and α-SMA in the crescents. Glomerular EMT in Rat Crescentic Glomerulonephritis The correlate of these human phenomena was described in two experimental types of CGN, one related to nephron reduction and the other to anti–glomerular basement membrane (GBM) antibody glomerulonephritis (30). In these experiments, immunohistochemistry and in situ hybridization labelings demonstrated de novo expression of α-SMA by PEC. Some PEC no longer expressed E-cadherin, a rat epithelial cell marker, and others coexpressed both E-cadherin and α-SMA. There was a marked increase in both TGF-β1 and FGF-2 expression by PEC in association with glomerular crescent formation. Cellular crescents showed either no disruption or only local areas of disruption in the basal lamina of Bowman's capsule, suggesting that the myofibroblasts in the crescents are derived from transdifferentiation of proliferating glomerular epithelial cells rather than by migration of interstitial myofibroblasts into Bowman's space. Mechanisms of EMT The mechanisms that lead to EMT are still poorly understood and are probably diverse. In the kidney, EMT was essentially studied experimentally on tubular epithelial cells. EMT can be achieved by changes in the extracellular matrix composition: tubular epithelial cells grown in three-dimensional collagen type I promotes EMT (2,3,31–33) with loss of CK expression and acquisition of mesenchymal morphology and phenotype, including fibroblast-specific protein-1 (FSP-1) and vimentin. This suggests that direct interaction between epithelial cells and the interstitial extracellular matrix could induce EMT. Type IV collagen contributes to the maintenance of the epithelial phenotype of cultured proximal tubular cells, whereas type I collagen promotes EMT. Inhibition of type IV collagen assembly by the α1-NC1 domain upregulated the production of TGF-β in proximal tubular cells and induced EMT (34,35). The transdifferentiated epithelial cells exhibited fibroblast-like morphology, increased expression of FSP-1 and vimentin, decreased CK expression, and increased synthesis of collagen I, which stabilizes the mesenchyme phenotype of the transdifferentiated cells. EMT could be blocked by anti–TGF-β1 antibodies. These data suggest that changes in the basement membrane architecture can lead to upregulation of TGF-β1, which contributes to EMT that accompanies renal fibrosis. Cytokines, growth factors, and adhesion molecules are involved in EMT. In tubular epithelial cells, FGF (36), TGF-β (37–39), and FGF associated with TGF-β (33) induced de novo expression of α-SMA, loss of the epithelial marker E-cadherin, change from an epithelial (cuboidal) appearance to a myofibroblastic (spindle shaped) morphology, along with build-up of interstitial matrix. The typical cobblestone pattern of cultured epithelial cells was replaced by a spindle-shaped fibroblast-like appearance with cytoplasmic projections at the front end, large bundles of actin microfilaments, and dense bodies. All of these effects were blocked in NRK-52 E cells, a normal rat kidney epithelial cell line, by a neutralizing antibody to TGF-β1 (38). Similar results were obtained using IL-1. Addition of a neutralizing antibody to TGF-β blocked the effects of IL-1 on EMT, suggesting a TGF-β dependence (40). EMT in tubular epithelial cells has been demonstrated in vivo. In DBA/2-pcy mice, a model of polycystic kidney disease (41), epithelial cells in remnant tubules lost expression of CK but expressed FSP-1 and HSP47, a marker for collagen synthesis. In unilateral renal obstruction, epithelial cells exhibited features of EMT (39,42). In the same experimental model, TGF-β1 receptor expression was increased in renal tubules. Hepatocyte growth factor blocked EMT and prevented interstitial fibrosis in the obstructed kidney. In vitro hepatocyte growth factor abrogated EMT-induced TGF-β1 expression in tubular epithelial cells (43). Loss of epithelial cell adhesion may also promote the EMT process. E-cadherin, an adhesion receptor found within adherens-type junctions, plays a role in maintaining the polarity and the structural integrity of renal epithelial cells. The addition of antibodies to E-cadherin induced disaggregation of the MDCK kidney epithelial cell line and their reversion to fibroblast-like cells (44,45). TGF-β1 rapidly suppressed E-cadherin expression in cultured tubular epithelial cells before all of the major events that characterize EMT (39). All of these human and experimental studies on EMT suggest that EMT may participate in the development and the progression of glomerular and tubulointerstitial fibrosis. Transdifferentiation of Glomerular Epithelial Cells into Macrophagic Cells Transdifferentiation of Glomerular Epithelial Cells in Posttransplantation Relapse of Primary FSGS Relapse of primary FSGS on transplanted kidneys offers a privileged model for studying the early and later lesions that characterize this glomerulopathy. We studied 18 renal biopsies from 6 cases of primary nephrotic FSGS that had relapsed after transplantation (14). The glomerular lesions comprised the cellular, the collapsing, and the scar variants of FSGS and showed shedding of large round cells into Bowman's space and within the tubular lumens. With the use of immunohistochemical identification of glomerular cells and of free migrating cells, some phenotypic changes suggesting transdifferentiation were found. Some podocytes identified by podocyte markers (podocalyxin, synaptopodin, GLEPP-1) were detached from the tuft and were free in the urinary spaces. Loss of normal podocyte epitopes (podocalyxin, synaptopodin, GLEPP-1, WT1, CR1) was observed on the podocytes in the cellular variant and on the cobblestone-like epithelial cells that covered the scar lesions outside the synechiae. Podocytes acquired expression of various CK (identified by the AE1/AE3, C2562, CK22, and AEL-KS2 monoclonal antibodies) that were not found in the podocytes of normal glomeruli. However, PEC expressed AE1/AE3 CK that were rarely found on the PEC of normal glomeruli. Expression of macrophagic epitopes, identified by the PGM1 (CD68) and the HAM56 monoclonal antibodies, was observed on numerous cells located at the periphery of the tuft or free in the urinary space. That these cells that express epitopes specific for the monocyte-macrophage lineage were endowed with macrophagic attributes was shown by expression of 25F9, which characterizes macrophage maturation, and even more convincingly by expression of HLA-DR and CD16, which characterize macrophagic cell activation. The best argument for a process of cell transdifferentiation occurring in the glomerular epithelial cells stems from confocal laser microscopic examination. Using this technique, we observed coexpression of (1) CD68 + AE1/AE3 CK (Figure 3) (2), podocalyxin + CD68 (Figure 4), and (3) podocalyxin + AE1/AE3 CK on cells that were still attached to the glomerular tuft or that drifted in Bowman's space and within the tubular lumens. These findings strongly suggest that some "dysregulated" podocytes, occasionally some PEC, and possibly some tubular epithelial cells undergo a process of transdifferentiation. This process of transdifferentiation was especially striking in podocytes that acquired macrophagic and CK epitopes, which are not expressed in normal adult and fetal podocytes. Transdifferentiation of Podocytes in Idiopathic Collapsing Glomerulopathy Collapsing glomerulopathy, a severe variant of FSGS, is characterized by collapse of most, if not all, of the glomerular tufts with proliferation of modified podocytes. In the most damaged glomeruli, the hyperplastic podocytes show swelling, vacuolization, multinucleation, cobblestone alignment around the glomerular tuft, and pseudocrescent formation in Bowman's space. Some of these podocytes are separated from the GBM and seem to be drifting in Bowman's space. We studied eight cases of idiopathic collapsing glomerulopathy. There were five black and three white patients. All were HIV negative (13). Podocytes were identified by the presence of podocalyxin, of vimentin, a protein of the podocyte cytoskeleton, and of CR1. To characterize further the phenotypic expression of swollen, vacuolated, cobblestone-like, or detached podocytes, we used three anti-human monoclonal antibodies with different specificities for macrophage-associated epitopes: an anti-CD68 monoclonal antibody (clone PGM1,) an anti-CD68 monoclonal antibody (clone KP1), and an anti-human macrophage antibody (clone M18). Along with dislodging, podocytes exhibited phenotypic transformation. Most of those still attached to the GBM still expressed podocalyxin, CR1, and vimentin, whereas some expressed CD68 (Figure 5). Once assuming cobblestone-like alignment or detachment from the tuft and moving free in the urinary space, they lost normal podocyte epitopes and acquired a macrophage-associated phenotype. Our findings are substantiated by experiments based on normal rat glomerular cell cultures. Glomerular epithelial cells are able to acquire in vitro the ability to process and present antigens (46). Whole glomeruli in culture were isolated by a graded sieving technique. Cells identified as being derived from podocytes changed into macrophages that migrated from the glomeruli. Once cultured, podocytes lost both the ultrastructural appearance and some immunohistochemical podocyte markers, and they acquired morphologic and functional characteristics of macrophages (47). The significance and the consequences of such intriguing phenomena regarding transdifferentiation of glomerular epithelial cells into macrophagic cells are a matter of obvious interest. There is no doubt that these observations should—and will—foster further studies that hopefully will unravel their implications in the still elusive pathophysiology of FSGS and of other glomerulopathies.