1 Department of Nephrology, Veterans General Hospital-Taipei, National Yang-Ming University, Taipei, Taiwan and 2 Departments of Nephrology and Medicine, Monash Medical Centre, Monash University, Clayton, Victoria, Australia
Correspondence and offprint requests to: Dr Hui Y. Lan, Department of Nephrology, Monash Medical Centre, Clayton Road, Clayton, Victoria 3168, Australia.
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Abstract |
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Methods. This study examined glomerular epithelial myofibroblast transdifferentiation (GEMT) in the formation and evolution of glomerular crescents in two distinct rat models of glomerulonephritis: 5/6 nephrectomy and antiglomerular basement membrane (GBM) disease.
Results. Early in the course of both disease models, and prior to crescent formation, immunohistochemistry staining and in-situ hybridization demonstrated de novo expression of alpha-smooth-muscle actin (-SMA), a marker of smooth muscle cells and myofibroblasts, by glomerular parietal epithelial cells (GPEC). The expression of
-SMA by GPEC was accompanied by a loss of E-cadherin staining, a marker of epithelial cells. At this early stage of GEMT, ultrastructural studies identified the presence of characteristic actin microfilaments and dense bodies within GPEC which retained a normal epithelial morphology with apical-basal polarity and microvilli. A late stage of transdifferentiation was seen in fibrocellular crescents. In this case, GPEC attached to intact segments of the capsular basement membrane contained large bundles of actin microfilaments throughout the cell, and this was accompanied by a loss of polarity, microvilli, and tight junctions. There was a significant correlation between the presence of
-SMA+ GPEC and glomerular crescent formation. Cellular crescents contained small numbers of
-SMA+ myofibroblasts. These cells become the dominant population in fibrocellular crescents, which was associated with marked local proliferation. Relatively few
-SMA+ myofibroblasts remained in fibrotic/organizing crescents. Most cells within cellular and fibrocellular crescents expressed transforming growth factor-ß (TGF-ß) and basic fibroblast growth factor (FGF-2), suggesting that these growth factors may regulate this GEMT process during the evolution of glomerular crescents.
Conclusions. This study provides the first phenotypic and morphological evidence that glomerular epithelial-myofibroblast transdifferentiation participates in the formation and evolution of glomerular crescents.
Keywords: glomerular crescents; glomerular epithelial cells; glomerulonephritis; myofibroblasts; transdifferentiation
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Introduction |
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Although fibroblasts play a crucial role in the fibrous organization of cellular crescents, the nature of these cells and their origins is not clear. The expression of -smooth-muscle actin (
-SMA) has been used as a marker of myofibroblasts, a subset of fibroblasts, which are a major source of collagen synthesis [1214]. Indeed, increasing numbers of interstitial
-SMA+ myofibroblasts predicts progressive renal dysfunction in human and experimental glomerulonephritis [1520]. However, the potential role of
-SMA+ myofibroblasts in the formation and progression of glomerular crescents has not been examined.
It has been postulated that the accumulation of fibroblasts in glomerular crescents arises from the entry of periglomerular fibroblasts through the ruptured CBM [5,911]. However, the growing appreciation of the ability of epithelial cells to undergo transdifferentiation into mesenchymal cells, and -SMA+ myofibroblasts in particular, has led us to re-evaluate the possible origins of fibroblast-like cells within glomerular crescents.
The aim of the current study was therefore to examine the origins and contribution of myofibroblasts in the formation and evolution of glomerular crescents. This was performed in two contrasting experimental models of crescentic glomerulonephritis; non-immune renal ablation and immune-induced antiglomerular basement membrane (GBM) glomerulonephritis.
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Subjects and methods |
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Immune model of crescentic glomerulonephritis
Male SpragueDawley rats (150200 g) were obtained from the Monash Animal Services, Melbourne. Experimental crescentic glomerulonephritis was induced as previously described [22]. Briefly, animals were immunized s.c. with 5 mg normal rabbit IgG in Freund's complete adjuvant and injected i.v. with 10 ml/kg body weight rabbit anti-rat GBM serum (12.5 mg IgG/ml) 5 days later (termed day 0). Groups of six animals were killed on days 1, 7, 14, 21, and 35. In addition, a group of six normal animals was used as a control.
Histopathology
Kidney tissues for histological examination were fixed in 10% formalin and 4 µm paraffin sections were stained with haematoxylin and eosin or periodic acidSchiff reagent (PAS). Glomerular crescent formation was scored as previously described [6,11]. Briefly, the percentage of glomeruli with crescent formation was assessed by examining 50100 glomeruli per animal on PAS-stained paraffin-sections, and glomerular crescents were classified into three categories as follow: (i) cellular crescents containing three or more layers of cells, with most cells having a round morphology; (ii) fibrocellular crescents, defined as cellular crescents containing fibroblasts and some collagen; and (iii) fibrous/sclerotic crescents having fibrous appearance with few cells. The integrity of the basement membrane of Bowman's capsule was assessed on PAS-stained paraffin-sections.
Antibodies
Monoclonal antibodies (mAb) used were: 1A4, which specifically recognized the smooth-muscle -actin isoform expressed by rat, mouse and human (Sigma Chemical Co., St Louis, MO); PC10, mouse anti-proliferating cell nuclear antigen (PCNA) which is expressed by cells in G1, S, and G2 phases of the cell cycle (Dakopatts, Glostrup, Denmark); ED1, mouse anti-rat macrophages [23]; OX-7, mouse anti-rat Thy-1 antigen [24]; mouse anti-bovine FGF-2 which cross-reacts with rat FGF-2 but does not recognize bovine or rat FGF-1 (Upstate Biotechnology Inc., Lake Placid, NY); mouse anti-rat E-cadherin (Transduction Laboratories, Lexington, KY); rabbit anti-human transforming growth factor bet
-1 (TGF-ß1) which cross-reacts with rat TGF-ß1 but does not react with other TGF-ß subclasses (Santa Cruz, CA). In addition, peroxidase-conjugated goat anti-mouse IgG, colloidal gold conjugated goat anti-mouse IgG, alkaline phosphatase-conjugated goat anti-mouse IgG, mouse peroxidase anti-peroxidase complexes (PAP) and mouse alkaline phosphatase anti-alkaline phosphatase complexes (APAAP) were purchased from Dakopatts.
Immunohistochemistry
Double immunohistochemistry was performed using a microwave-based two-colour staining technique [25]. Briefly, sections were dewaxed, pre-incubated for 20 min in 10% FCS and 10% normal goat serum to block non-specific binding, incubated with the anti--SMA or anti-E-cadherin mAb for 60 min and washed three times in phosphate-buffered saline (PBS). After inactivating endogenous peroxidase in 0.3% H2O2 in methanol, sections were labelled sequentially with peroxidase-conjugated goat anti-mouse IgG and PAP, followed by development with diaminobenzidine to produce a brown colour. To block antibody cross-reactivity and facilitate antigen retrieval [25], sections were treated with 2x5 min of microwave oven heating in 10 mM sodium citrate pH 6.0 at 2450 MHz and 800 W power. Sections were then pre-incubated as above, incubated with the PC-10 (1:1000) or anti-
-SMA mAb for 60 min, followed by alkaline-phosphatase-conjugated goat anti-mouse IgG and APAAP, and finally developed with Fast Blue BB Base (Sigma Chemical Co.), counterstained with PAS without haematoxylin, and coverslipped in an aqueous mounting medium. Negative controls consisted of substitution of one or both of the primary antibodies with an equivalent concentration of an irrelevant murine monoclonal antibody (73.5, anti-human CD45R). All procedures were performed at room temperature.
Triple immunostaining was performed as described previously [26]. First, sections were stained with the anti-FGF-2 mAb using a three-layer PAP method to produce a brown colour. Second, sections were microwaved and then stained with the ED1 mAb using a three-layer APAAP method to produce a blue colour. Third, sections were microwaved again and then stained with the PC-10 mAb followed by colloidal gold-conjugated goat anti-mouse IgG and silver enhancement to produce a black colour.
Immunostaining for the Thy-1 antigen using the OX-7 mAb was performed on ethanol-fixed, snap frozen tissues using a three-layer PAP method as described above.
Quantitation of immunohistochemical staining
The total number of nucleated cells and the number of -SMA+, PCNA+,
-SMA+PCNA+, and
-SMA+PCNA- cells within glomerular crescents were scored in double-stained sections as previously described [6,26]. At least 10 glomerular crescent cross-sections of cellular, fibrocellular, and fibrous crescents (if present) were scored for each animal in high-power fields (x40). In addition, glomerular parietal epithelial cells were examined by scoring cross-sections of Bowman's space in glomeruli without crescent formation. The total number of nucleated cells and the number of nucleated
-SMA+, PCNA+,
-SMA+PCNA+, and
-SMA+PCNA- cells were scored in at least 50 Bowman's space cross-sections per animal. Data from groups of six animals is expressed as the mean±standard error of the mean (SEM) per Bowman's space cross-section or per crescent cross-section. For all morphological analyses, the observer was blinded to the animal group.
Probes
A 1068 base pair cDNA fragment of rat vascular smooth-muscle -actin [27], and a 980 base pair cDNA fragment of rat transforming growth factor-ß1 [28], were amplified by the polymerase chain reaction and cloned using the pMOSBlue T-vector kit (Amersham International, Buckinghamshire, UK). A 180 base pair cDNA fragment of rat FGF-2 cloned into pGEM3Zf was a gift from Dr Miriam Ford, University of Melbourne. Both sense and anti-sense digoxigenin (DIG)-labelled cRNA probes were prepared using an RNA polymerase kit (Boehringer Mannheim GmbH, Mannheim, Germany). Probes were precipitated and incorporation of DIG was determined by dot blotting.
In situ hybridization
In situ hybridization was performed on 4 µm paraffin sections of formalin-fixed tissue using a microwave-based protocol [26,29]. After dewaxing, sections were treated with a microwave oven for 2x5 min as described above, incubated with 0.2 M HCl for 15 min, followed by 1% Triton X-I00 for 15 min, and then digested for 20 min with 10 µg/ml Proteinase-K at 37°C (Boehringer Mannheim). Sections were then washed in 2xSSC, prehybridized, and then hybridized with 0.3 ng/µl DIG-labelled sense or anti-sense -SMA, TGF-ß, or FGF-2 cRNA probe overnight at 37°C in a hybridization buffer containing 50% deionized formamide, 4xSSC, 2xDenhardt's solution, 1 mg/ml salmon sperm DNA, and 1 mg/ml yeast tRNA. Sections were finally washed in 0.1xSSC at 37°C and the hybridized probe detected using sheep anti-DIG antibody (Fab) conjugated with alkaline phosphatase and colour development with NBT/X-phosphate (Boehringer Mannheim). No signal was seen with the sense riboprobe labelled to the same specific activity.
As another specificity control, some sections were microwave treated after hybridization with the anti-sense -SMA cRNA probe and then stained with the anti-
-SMA mAb using the three-layer APAAP method as described above. All sections were counterstained with PAS minus haematoxylin and mounted in an aqueous medium.
In addition, some sections hybridized with the anti-sense TGF-ß1 cRNA probe were subsequently stained with the anti--SMA mAb using a threelayer APAAP method to produce a blue colour.
Electron microscopy
Small tissue blocks obtained from the renal cortex were fixed in 2.5% glutaraldehyde in PBS (pH 7.4) for 4 h, washed in PBS, post-fixed with 1% osmium tetroxide, dehydrated in graded alcohol, and embedded in Epon 812. Ultrathin sections were stained with uranyl acetate and lead citrate and examined in under a Zeiss electron microscope (EMIOC).
Statistical analysis
Differences in the number of labelled cells counted in tissue sections were analysed by one-way analysis of variance (ANOVA) from the Complete Statistical Analysis program (CSS, Statsoft, Tulsa, IL, USA). In addition, the number of labelled cells scored in tissue sections was correlated with crescent formation using the Pearson single correlation coefficient.
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Results |
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One possible source of -SMA+ cells in crescent formation is the migration of `activated' mesangial cells from the glomerular tuft. However, immunostaining using the OX-7 mAb, a mesangial cell marker, found no evidence of mesangial cells within cellular or fibrocellular crescents (data not shown).
The relationship between -SMA expression by GPEC and the development of crescent formation is shown in Figures 4 and 5
and Table 1
. The percentage of non-crescentic glomeruli containing GEPC with
-SMA staining increased steadily from 25% of glomeruli at week 3 to over 60% by week 17 in the remnant kidney (Figure 4a
). This paralleled glomerular crescent formation (Figure 4a
). A parallel relationship between the percentage of non-crescentic glomeruli with
-SMA staining of GEPC and glomerular crescent formation was also seen in rat anti-GBM glomerulonephritis (Figure 5a
). When assessing all non-crescentic glomeruli, the percentage of GEPC expressing
-SMA gave a highly significant correlation with the absolute number of cellular, fibrocellular and fibrous crescents in both disease models (Table 1
). In addition, the percentage of GEPC expressing
-SMA in all non-crescentic glomeruli gave a highly significant correlation with the number of
-SMA+ cells in cellular, fibrocellular, and fibrous crescents (Table 1
).
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Myofibroblasts in the evolution of glomerular crescent formation
Most crescents present in the early stages of both disease models were of a cellular phenotype. With time, there was a progressive increase in the percentage of crescents with a fibrocellular or fibrous phenotype, and this was associated with an increase in the percentage of crescent cells expressing -SMA (Figures 4b,c and 5b,c
). In both disease models,
-SMA+ cells were detected within cellular crescents (Figures 1d, 2c and 7
), which accounted for 3035% of total crescent cells. Fibrocellular crescents contained a large number of
-SMA+ cells, approximately threefold more than in cellular crescents (Figure 7
), and many of the
-SMA+ cells showed an elongated morphology (Figures 1e and 2d,e
). This increase in
-SMA+ cells was associated with extensive disruption of the CBM. Indeed,
-SMA+ cells accounted for 7080% of total cells within fibrocellular crescents. However, more than 80% of the
-SMA+ cells seen in fibrocellular crescents disappeared and were replaced by collagen in fibrous crescents (Figures 1f, 2f and 7
).
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Of note, double immunostaining showed that approximately 60% of -SMA+ cells within cellular and fibrocellular crescents were proliferating on the basis of PCNA expression (Figures 2c,e
).
Expression of TGF-ß1 and FGF-2 in the evolution of glomerular crescents
Weak expression of TGF-ß1 and FGF-2 was seen in glomerular cells and GPEC in normal rat kidney by in situ hybridization and immunostaining (not shown). There was a marked increase in both TGF-ß1 and FGF-2 expression by GPEC in association with glomerular crescent formation (Figures 3df). Combined in situ hybridization and immunostaining showed that many elongated
-SMA+ myofibroblasts in cellular and fibrocellular crescents expressed TGF-ß1 mRNA (Figure 3d
). Strong expression of FGF-2 mRNA by GPEC was evident in early cellular crescents (Figure 3e
). The use of triple immunostaining showed that FGF-2 mRNA was expressed by many PCNA+ elongated, fibroblast-like cells within fibrocellular crescents (Figure 3f
). In addition, ED1+ macrophages within fibrocellular crescents expressed FGF-2 protein (Figure 3f
).
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Discussion |
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Prior to and during the early stages of crescent formation, parietal epithelial cells were found to express -SMA mRNA and protein and lose expression of the epithelial marker, E-cadherin. These cells in the early stage of transdifferentiation were firmly attached to a largely intact CBM, and maintained a clear cuboidal epithelial morphology. This phenotypic evidence of GEMT was supported by ultrastructural studies showing actin microfilaments in GEPC that had a clear epithelial morphology and polarity. A late stage of transdifferentiation of GEPC was seen within cellular, and more particularly fibrocellular crescents, in which cells still adhering to the CBM lost apical-basal polarity, cellcell junctions, became elongated, and showed large bundles of microfilaments and many dense bodies throughout the cytoplasm.
Cellular crescents consist of epithelial cells, macrophages, or a mixture of the two cell types. In addition, the current study has shown that -SMA+ myofibroblasts are also a component of cellular crescents. We postulate that the
-SMA myofibroblasts present in cellular crescents are derived from glomerular epithelial cells by transdifferentiation. This is supported by the temporal relationship between the appearance of
-SMA+ GEPC in glomeruli prior to, or at the time of, the onset of glomerular crescent formation and the highly significant correlation between
-SMA+ GPEC and the formation and evolution of glomerular crescents. Cellular crescents showed either no disruption, or local areas of partial disruption of the CBM, suggesting that the
-SMA+ myofibroblasts in these crescents are derived by transdifferentiation and proliferation of glomerular cells, rather than by migration of periglomerular myofibroblasts into Bowman's space. While it can be argued that
-SMA+ myofibroblasts attached to the CBM are transdifferentiated parietal epithelial cells, it is possible that other
-SMA+ myofibroblasts within cellular crescents (i.e. not attached to the CBM) could be transformed mesangial or visceral epithelial cells migrating from the glomerular tuft into Bowman's space.
The transdifferentiation described in the current study is not unique to glomerular parietal epithelial cells. Indeed, there are a number of defined circumstances under which epithelial cells can transdifferentiate into mesenchymal cells. For example, all kidney cells, except for the collecting duct cells, are derived from the metanephrogenic mesenchyme [30,31]. In vitro, suspension of definitive epithelia from a number of tissues, including tubular epithelial cell lines, in three-dimensional collagen type I gels promotes epithelialmesenchymal transdifferentiation by turning off cytokeratin expression and turning on mesenchymal gene programs, inducing expression of fibroblast specific protein-1 [3136]. In addition to phenotypic changes, transformed epithelial cells exhibit morphological changes such as a loss of cellcell adhesion specialization and apicalbasal polarity, and become elongated and very invasive with a new front endback end mesenchymal polarity [31,36]. Similar morphological changes were observed in the current study.
Myofibroblasts were found to play an important role in the fibrous organization and progression of glomerular crescents, consistent with the importance given to -SMA myofibroblasts in glomerulosclerosis and interstitial fibrosis [1520]. There was a significant correlation between the number of
-SMA+ cells and the progression of crescents from a cellular to a fibrocellular phenotype. The presence of many
-SMA+ myofibroblasts in fibrocellular crescents is consistent with the active deposition of collagen leading to the development of acellular fibrous crescents. However, the origin of
-SMA+ myofibroblasts in fibrocellular crescents remains uncertain. Some of these
-SMA+ myofibroblasts may be derived by transdifferentiation of glomerular cells, but they may also arise from migration of periglomerular myofibroblasts into Bowman's space through the extensively ruptured CBM evident in fibrocellular crescents. In either case, the high levels of PCNA expression in the
-SMA+ cells indicate that local myofibroblast proliferation is an important mechanism in their accumulation within Bowman's space. This is consistent with the high levels of
-SMA+ myofibroblast proliferation described in experimental interstitial fibrosis [17,37].
Several possible mechanisms may promote the process of GEMT. First, TGF-ß and fibroblast growth factors have been shown to induce synthesis of collagen types I, II, IV and V and FSP-1 by epithelial cell cells [32,38]. TGF-ß and FGF-2 have also been shown to promote epithelialmesenchymal transdifferentiation in vitro [3943]. In the present study, marked upregulation of TGF-ß1 and FGF-2 by glomerular cells was associated with GEMT and glomerular crescent formation and progression, consistent with a potential role of these growth factors in the transdifferentiation process and subsequent proliferation of myofibroblasts within crescents. The up-regulation of FGF-2 and TGF-ß expression has previously been reported in these disease models [24,44]. Second, the association between localized partial disruption of the CBM and expression of -SMA by GPEC suggests that direct interactions between the epithelial cells and the periglomerular extracellular matrix environment promotes induction of the transdifferentiation process. This is supported by in vitro studies showing that laminin A, a basement membrane component, plays a key role in initiating polarity of tubular epithelial cells [45]. Indeed, activation of the mesenchymal gene programme can be achieved in definitive epithelia by simple modifications of the extracellular matrix [31,35,36,46]. Therefore, partial disruption of the CBM allowing direct epithelial cell contact with the interstitial matrix may be crucial in the activation of the mesenchymal gene programme. This is consistent with our recent observation that disruption of the tubular basement membrane is associated with tubular epithelialmyofibroblast transdifferentiation in the development of interstitial fibrosis [47]. Third, the loss of epithelial cell adhesion molecule, E-cadherin, may also promote the transdifferentiation process. The addition of antibodies to E-cadherin causes disaggregation of the MDCK kidney epithelial cell line and their reversion to fibroblast-like cells [48,49]. Similarly, inhibition of E-cadherin expression by the use of anti-sense oligonucleotides causes epithelial cells to lose tight-junctions and became fibroblast-like [50]. Therefore, turning off expression of epithelial-specific genes is also required in the epithelialmesenchymal transdifferentiation process. The present study showed that the gain of
-SMA expression by GPEC was associated with the loss of E-cadherin expression, providing in vivo evidence that the loss of adhesion molecules that maintain epithelial tight junctions is involved in the epithelialmyofibroblast transdifferentiation process.
A role for transdifferentiation in fibrogenesis has been described in a number of pathological situations. In liver fibrosis, lipocytes can transdifferentiate into myofibroblasts with expression of fibrogenic genes [51,52]. A similar transdifferentiation process has been described in pulmonary fibrogenesis [13]. We have also provided evidence that tubular epithelial cells are pro-fibrogenic cells capable of transdifferentiation into -SMA+ myofibroblasts during the progression of tubulointerstitial fibrosis in rat remnant kidney model [47]. The present study extends these findings by: (i) providing more extensive phenotypic evidence for the process of epithelialmyofibroblast transdifferentiation in a pathological process, (ii) providing evidence that epithelialmyofibroblast transdifferentiation is an early event in a localized fibrotic process, and (iii) arguing that epithelialmyofibroblast transdifferentiation is of general importance in renal fibrosis, since the same transdifferentiation process was observed in both immune and non-immune models of crescentic glomerulonephritis.
In summary, this study provides, for the first time, evidence that glomerular epithelialmyofibroblast transdifferentiation participates in the development and progression of crescent formation. This process, which may in part be promoted by local production of TGF-ß and FGF-2, provides a new approach to understanding the mechanisms of glomerular crescent formation and evolution.
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Acknowledgments |
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References |
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